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上海市農業遺傳育種重點實驗室

來源: 發布時間:2008-11-09 13:10:00 瀏覽次數: 【字體:

ANIMAL GENETIC ENGINEERING RESEARCH SECTION, SHANGHAI KEY LABROARTORY OF AGRICULTRUAL GENETIC AND BREEDING


1. 
研究方向
根據上海都市農業發展要求和畜牧獸醫科技服務都市公共衛生工作的需要,利用現代生物技術進行動物及人畜共患病免疫診斷制劑的研制。同時追蹤國內外動物生物技術的最新進展,結合現代免疫、生物信息及動物轉基因克隆技術,選育和開發抗病動物品種
1. Research orientation
Zoonotic diseases and new types of animal vaccines, diagnosis reagents and recombinant proteins for enhancing animal growth or immunology. Research on animal transgene and cloning were also carried out.
2. 在研項目進展
2. progress of current research project 
2.1動物基因工程重組蛋白微球注射劑緩控釋技術的研發
(1) 建立重組牛生長激素基因工程大腸桿菌高密度發酵工藝,發酵菌體密度OD550達到60以上;目標蛋白表達量在25%左右。
(2) 確立包含體提取及變復性工藝,包含體純度在80%左右,復性重組牛生長激素純度達90%以上,生物活性檢測表明,可使去垂體大鼠每天增重1-1.5g。
(3) 建立重組牛生長激素原料凍干工藝,采用聚乙二醇(PEG)為賦形劑效果最好。
(4) 以PLGA50/50,1.4萬作為微球骨架材料,S/O/W和W/O/W兩種方法載藥量均可達到10%以上,包封率可達95%以上。體外釋放試驗表明S/O/W制備的微球突釋小,釋放較W/O/W均勻,可緩釋兩周左右。但兩者體外釋放累積量不超過50%。
(5) 采用W/O/W 和S/O/W時穩定劑如糖類、表面活性劑對牛生長激素的穩定性均無明顯提高。而采用S/O/O時,對牛生長激素的穩定性均有一定的提高,其中以甘露醇的效果最好,使90%的蛋白可以復性;有機試劑二甲亞砜、丙酮對蛋白活性的影響較其他有機試劑小。
(6)采用5%蜂蠟作為增稠劑,以PEG作為致孔劑,制備的重組牛生長激素油混懸制劑首日釋放量為20.3%,且能持續釋放至31天。
(7)重組牛生長激素油混懸制劑去垂體大鼠動物試驗表明此種油混懸注射劑在體內至少能緩釋31天,其有望發展成為新型長效制劑。
2.1 Study on the injectable microsphere dose of animal recombinant protein
(1) .High cell density fermentation of recombinant bovine growth hormone E.coli. had finished, cell density is about OD550=60, the rate of recombinant protein to total cell protein is about 25%. 
(2) Recombinant bovine growth hormone (rBGH) had been successfully extracted from host cell in inclusion bodies. The technology to denature and renature rBGH had finished. The renaturation ratio is about 40%,and the purity is about 85%. Injection experiment of hypophysectomized rats proved that we have obtained r-pST with high native bioactivity.
(3) The lyophylization technology of recombinant bovine growth hormone have been finished and found the protein power has 93% activity by adding PEG as protection reagent.
(4)  The carrying ability of S/O/W and W/O/W using PLGA50/50 are at least 10%, packing ratio is more than 90%. In vitro tests indicate that the release of the microglobule prepared by S/O/W is more gradually than that prepared by W/O/W, but the total release rate is less than 50%.
(5) Stabilizing agent such as saccharide or surfacent has no effect on the stability of BST when prepared by S/O/W and W/O/W, but could improve the stability of BST prepared by S/O/O. DMSO and acetone have less maleficence on the protein than other organic solvent.
(6) The percentage of the medicine release of the recombined bovine somatotropin (rbST) oil suspension, with beeswax as dope and PEG as poriform, was 20.3% on the first day, and then released successively for 31 days.
(7) The study on the release effect of recombined bovine somatotropin (rbST) oil suspension with male hypophysectomized rats indicates that the oil suspension can release successively in vivo for about 31 days and it has the potential to be developed successfully as a new long active rbST formulation.
2.2 豬源性戊型肝炎阻斷技術研究
豬戊型肝炎病毒(HEV)主要有1,2,3,4四個基因型,此前,中國大陸僅發現基因1型和4型。本研究從上海郊區的5個豬場收集了65份豬糞便樣品,通過擴增HEV ORF2部分區域的150nt基因片段進行HEV RNA RT-PCR檢測,結果發現15份樣品為陽性,進化樹分析顯示其中8株為基因4型,7株HEV為基因3型,此基因3型與美國的豬US-SW HEV毒株同源性較高,這是迄今為止在中國大陸首次發現豬的HEV基因3型。隨后采用相同方法對分布在上海9個區36個豬場的414份豬糞便樣品進行豬HEV流行病學調查,結果顯示111份為HEV RNA陽性,不同區的HEV感染率為8.3% to 41.7%。對其中的32份陽性樣品的150nt基因片段進行測序,進化樹分析顯示10株為基因4型,22株為基因3型,此研究結果提示基因3型HEV已在上海各區豬場普遍流行。由于大陸地區至今尚未有人感染HEV基因3型的報道,因此本研究分離到的豬基因3型是否具有人畜共患性及其來源需要進一步研究。
2.2 The effective prevention of HEV infection in swine
HEV isolates have been classified tentatively into four major genotypes, genotype 1, genotype 2, genotype 3 and genotype 4. Only strains of HEV genotype 1 and genotype 4 have been isolated so far in china mainland. In the present study, 65 swine fecal specimens were collected from five pig farms located in different Shanghai suburbs. RT-PCR and nested PCR were undertaken using partial nucleotide sequences of Open Reading Frame 2 (ORF2) of HEV to detect HEV RNA. Genetic analysis was based on alignments of an amplified 150-nt ORF2 sequence. RT-PCR revealed 15 HEV positive samples among 65-pig fecal specimens examined. Phylogenetic analysis of the amplified sequences indicated seven HEV strains belonged to genotype 3 and eight strains to genotype 4. The genotype 3 isolate was most closely related to one swine HEV strain, US-SW, which was isolated from pigs in the United States. This is the first time that genotype 3 hepatitis E virus has been identified on the Chinese mainland. To estimate accurately the prevalence of the HEV infection in shanghai , 414 swine feces from 36 different pig farms located all 9 different districts of Shanghai suburb were detected for HEV RNA by using the same method. The result of RT-PCR revealed that 111 HEV positive samples were found among 414-pig fecal specimens. The prevalence of HEV infection in different districts was varied from 8.3% to 41.7%. Among the 111 HEV positive samples, 32 samples were sequenced. Phylogenetic analysis of the amplified sequences indicated that 10 isolates belonged to genotype 4 and the other 22 isolates belonged to genotype 3. The result indicated that genotype 3 HEV has been widespread in pig farms of shanghai suburb. To our knowledge, the human infection of genotype 3 in china mainland has not been reported,so the zoonotic potential and the source HEV genotype 3 in shanghai need further study.  
2.3 口蹄疫注苗與感染牛(血清)鑒別診斷試劑盒中試生產研究
(1)確定的檢測抗原制備工藝可用于該抗原蛋白的中試生產;項目已完成該指標
(2)使用確定的工藝生產的NSP-ELISA試劑盒保存期為6個月;本項目組裝的試劑盒保存期可達10個月,但規定可使用的期限為6個月,以保證檢測結果可靠性。
(3)修正的臨界值對注苗與感染牛(血清)的檢測特異性高于90%,本研究組裝的試劑盒對陰、陽性樣品檢測特異性達91%以上;
(4)完成試劑盒2萬頭份檢測量的中試生產,抽樣檢查試劑盒的檢測誤差不高于10%(與UBI公司產品比較檢測);項目完成近5萬頭份檢測量的抗原生產,抽樣檢測誤差低于10%。
(5)完成試劑盒1萬頭份檢測量的推廣應用,使用報告證明試劑盒可用于口蹄疫注苗與感染牛(血清)的鑒別診斷;本項目已完成18000余份樣品檢測工作,并且使用報告證明可用于臨床FMDV鑒別診斷。
(6)提交試劑盒制造規程和半成品、成品檢驗標準;已撰寫了生物制品申報規程,準備提交。
(7)提交農業部新獸藥(生物制品)申報材料。在進行中
與國內外同類技術比較:研制的試劑盒與國際同類產品性能相當,達與國際先進水平(見查新檢索報告)
成果應用前景和范圍、存在問題及改進意見:產品可用于畜牧養殖場FMDV野毒感染檢測,凈化畜群;進出口活畜檢驗檢疫,防止攜帶FMDV野毒動物進入我國;消費市場檢測,防止攜帶FMDV的畜產品進入流通。據農業部統計,豬存欄4.8億頭(2006上半年),牛存欄14157.5萬頭(2005年),羊存欄達37265.9萬只(2005年),總計近10億頭。按照我國口蹄疫防制計劃和建立無規定疫病區的要求,口蹄疫易感動物的免疫接種密度必須達到80%,即注苗動物總數為8億頭(只),其中牛11326萬頭,按10%的抽檢比例計算,每年應檢測1132萬頭。按本項目所研制的試劑盒每頭份銷售價格為5元計算,則一年的產值約為7140萬元(人民幣)
2.3 Studies on serological detection methods in differentiating of bovine vaccinated with those of infected. 
(1) The technology of antigen preparation can be used in the pilot scale production
(2) The test for NSP-ELISA kit preservation is done for more than ten months, and six month preservation was the suggestion, so that the detection result is reliable.
(3) The detection specificity of immunized bovines and infected ones is higher than 90% to the revised critical value; the detection specificity of the kit is higher than 91% fot detecting of the negative and positive sample.
(4) More than 20000 doses of detection kit have finished by pilot scale production; the relative detecting accuracy of the kit is less than 10% comparing with the control of UBI co., we have detected more than 50,000 cattle serum using the kit, the relative accuracy of sample drawing and detection is lower than 10%.  
(5) Application of the kit for 10,000 bovines serum detection, and the using report displays that the kit can be used for the differential diagnosis of immunized bovines and infected ones; our project have detected 18,000 samples, and the report of detection displays that the kit can be used for the clinical differential diagnosis of FMDV. 
(6) The procedure of the kit production and the test criterion of the semi-product and final product will be; we have prepared the procedure of biological product, we will refer it.
(7) We are referring the materials of the new veterinary drugs to the Ministry of Agriculture.
Comparing to the same trade: 
Our technology has achieved the advanced level of the world.
The outlook of application and the problems: 
The kit can be used to detect the FMDV street strain of the domestic animal; it can be used in the quarantine of the importing livestock; it also can be used to detect the FMDV in the livestock circulation. According to the statistics of the Ministry of Agriculture, the number of breeding pigs in the farm is 4.8×108(2006 .1-6), the number of breeding cattle in the farms is 14157.5×104 in the year of 2005, the number of breeding sheep in the farms is 37265.9×104 in 2005, the total number of the three kind of domestic animal is nearly to 10×108. According to the plan of FMDV prevention and cure, the inoculation rate of the susceptible animal must be 80%, so the total number of inoculated animal is 8×108, including 11326×104 cattle. According to the sampling inspection rate of 10%, 1132×104 cattle should be inspected. Our kit can be sold 5 yuan/cattle, so the value of output will be 7140×104 yuan per year(RMB).
2.4豬流感監測診斷技術研究
(1) SIV血清學檢測方法建立:
利用原核系統成功表達了豬流感病毒NP蛋白;SDS-PAGE電泳檢測其分子量為60ku,經western blot分析,表達產物被SIV抗血清能特異性識別。
完成了重組NP蛋白小量發酵工藝條件的優化,用表達產物作瓊脂擴散試驗進行活性鑒定,證實該融合表達的蛋白具有生物學活性,且只與SIV血清有免疫反應,而與其他幾種豬病毒血清不發生反應,可作為檢測用抗原。
完成了表達蛋白小量純化條件摸索,采用試劑盒純化,獲得高純度重組蛋白。經過對抗原包被濃度、ELISA板的封閉方法、樣品稀釋方法、二抗、底物的選擇、結果判定等進行試驗和篩選,確定ELISA檢測試劑盒基本組成和操作。
進行了試劑盒敏感性、特異性、重復性及符合率試驗,結果,豬流感重組NP抗原ELISA檢測法比HI、AGP方法敏感性高;與美國IDEXX公司SIV抗體檢測試劑盒比較,陽性血清中的檢出率即為相對靈敏度為91.6%,兩者對樣品檢測的符合率為94%,兩種系統的檢測結果無顯著差異(P>0.05)。試劑盒批內重復試驗、批間重復試驗無顯著性差異(P>0.05);撰寫了申報規程草案,并通過初審。
(2)豬流感病毒分型檢測技術研究
SIV 各型通用NP基因通用引物設計篩選:下載Genebank各種亞型NP基因模板,H1、H3、H5、H9、N1、N2序列模板,采用Aligment軟件進行類比分析,篩選保守區域,設計引物,按照擴增片段大小、引物GC含量百分比,再通過模擬PCR(Spcr)篩選與主要血清亞型(H1/H3、H5/H9)基因模板PCR擴增產物情況,篩選到理論上符合條件的引物。
HA分型多重PCR分型檢測體系初步建立 根據NP/H1/H3亞型及NP/H5/H9亞型引物擴增片段大小不同,反應條件的差異,將各引物配比組合,形成不同的配組,再用混合模板(H1/H3;H5/H9)進行擴增,驗證引物組合擴增單一模板、混合模板的檢測效果,獲得針對NP/H1/H3、NP/H5/H9、N1/N2模板的兩體系多重引物組合。
根據試驗篩選到的引物組合,組建多重RT-PCR試劑盒,試劑盒含有從RNA 進行RT及PCR過程所需各組分。
對試劑盒靈敏性和特異性檢測:將RNA模板按比例稀釋,分別用建立的體系做RT-PCR檢測,分析本試劑盒檢測靈敏度,結果:H1/H3組合對H1模板最低檢出量為0.1ng,對H3模板最低檢測量為2ng。而擴增PRRS、PPV、PRV、PCR病毒核酸,結果均為陰性,表明試劑盒特異性好,與幾種常見豬病原無交叉反應,可用于SIV檢測。
2.4 Detection and diagnosis of swine influenza virus
(1) Studies on SIV serology detection method:
The NP protein of SIV has been successfully expressed in the prokaryotic system; the protein’ molecular weight is 60 kd which is displayed through the SDS PAGE. The protein can be recognized by the SIV antiserum in the western blot.
 We have optimised the minor fermentation technology of the fused NP protein. The protein has the biological activity in the agar diffusion reaction, and the SIV serum can only recognize the protein, the other virus’ serum cannot recognize the protein, so the protein can be used to detect the SIV.
 We have finished the optimization of the minor purification technology of the NP protein, and we harvested the pure protein. We have ascertained the composition and procedure of the ELISA detection kit through coating antigen, method of blocking, the assessment of the result, and the choosing of the substrate and the antibody against the anti-serum.
  We have done the experiment of the kit’s sensitivity, specificity, repeatability, and coincidence. The result displays that the method of NP protein Elisa detection is more sensitive than the method of HI and AGP. The positive serum’ detection rate of the kit is 91.6%, compare to the SIV antibody detection kit of the American company IEXX, the coincidence rate of the two kits is 94%, the two system don’t have a significant deviation (p> 0.05). There is not a significant deviation in an experiment and the different times’ experiments (p> 0.05). We have finished the regulations of the kit, and it has been qualified in the first inspection.
(2) The genotyping diagnosis of the SIV:
 The design of the NP gene universal primer of the deferent type of SIV:
The deferent subtype NP gene templates, and the H1, H3, H5, H9, N1, N2 gene sequences have been downloaded from Genbank. We designed the optical primers by analyzing in the software of Aligment, screening the conservative region, screening the length of the PCR product, comparing the percentage of GC, screening in the simulation PCR, and the PCR amplification of the subtype H1/H3、H5/H9.
(3) The preliminary construction of the HA multi-PCR genotyping system:
 According to the length of NP/H1/H3, NP/H5/H9 amplification product and the condition of PCR, we mix the deferent primers and templates in deferent ratio to amplify the product. We verified the detection result of single template and multi-templates, obtained two systems of multi-primers groups for NP/H1/H3, NP/H5/H9, and N1/N2 templates.
According to the screened primer groups, we construct the multi-RT-PCR kit, which contains the composition for the reverse transcription and the PCR.
The sensitivity and the specificity of the kit:
We used the constructed kit to do the RT-PCR to detect the RNA template, which was diluted in proportion. The result displayed that the minimum value of the subtype H1 template can be detected is 0.1 ng by the H1/H3 primer group, and the minimum value of the subtype H3 template is 2ng. We used the kit to detect the PRRS, PPV, and PRV, and the result is negative. It displays that the specificity of the kit is well, it don’t have the cross-reaction with the universal swine’s pathogen, so it can be used to detect the SIV.
2.5豬種種質資源長期保存技術研究
(1) 豬精子冷凍技術的研究:篩選并研制出高效、低毒(無毒)的冷凍保存液,進一步優化了冷凍程序;建立了豬精子冷凍保存的實用化技術--精液大管(5ml)冷凍技術,克服了傳統顆粒、細管法有的局限,實現一支冷凍精液輸精一頭母豬,填補了這一技術在國內的研究、使用空白,使豬精液冷凍保存技術在地方豬種種質資源長期保存中更具可操作性。凍精解凍后精子活力達0.4級以上,受胎率達70%以上。
(2) 豬卵母細胞玻璃化冷凍保存:GMP法是一種冷凍豬卵母細胞的有效方法,與普通麥管法相比,該法能顯著提高豬MII期卵母細胞凍后存活率(34.5% vs 63.3%)。對卵母細胞的冷凍保存、體外受精、凍卵的超微結構和細胞骨架變化等進行了系統的研究,表明冷凍過程中細胞骨架的破壞是影響豬卵母細胞(胚胎)凍后復蘇的主要原因。本實驗首次報道豬GV期卵母細胞冷凍保存后,經體外受精獲得桑椹胚發育;在國內率先報道冷凍豬卵母細胞內細胞骨架的變化,
(3) 豬胚胎冷凍保存的研究:研究冷凍保存液、胚胎包裝方法、凍前胚胎預處理技術等對豬胚胎凍后存活力的影響;比較程序法、OPS法(開放式拉長麥管)和GMP法(玻璃微細管)的冷凍保存效果;建立了豬胚胎玻璃化冷凍技術。在我國首次獲得豬胚胎超低溫(–196℃)冷凍后代。GMP法玻璃化冷凍法產仔率30%(3/10)。
在完成上述研究的基礎上,成功保存了3個地方品種(系)的冷凍精子和冷凍胚胎。
2.5Research on techniques of long-term conservation for pig breeds
(1) Study on the cryopreservation of boar spermatozoa: According to a series of experiments, one kind of high efficiency and low-noxious(innocuity) cryopreservation solution was developed; the techniques of 5mL maxi-straws for the freezing of boar spermatozoa, a practical method, which overcomed the limit of conventional pellet and straw methods and successfully realized using only one 5mL maxi-straw in each insemination.of sow. The techniques padded an item of domestic blank in the area of sperm freezing, which made the cryopreservation of boar spermatozoa become more maneuverability in the long-term conservation of pig breeds. The sperm motility after warming could reach 0.4 and the pregnant rate could get more than 70%.
(2) Cryopreservation of porcine oocytes by vitrification: GMP(glass micropipette) is an effective method for vitrification of porcine oocytes. Compared with conventional straw method, this method could significantly improved the survival rate(34.5% vs 63.3%) of porcine MII stage oocytes after warming. The systemic research on the cryopreservation, in vitro fertilization and the ultrastructure & cytoskeletal of vitrified oocytes showed that the damage of cytoskeletal structure was one of the key reasons that caused the low efficiency of the cryopreservation of porcine oocytes. In our experiment, we first got the morula development after in vitro fertilization porcine vitrified GV stage oocytes in the world and first reported the cytoskeletal change of porcine vitrified oocyes at home. 
(3) Cryopreservation of porcine embryos: Studies on the effects of different cryopreservation solution, vitrified carriers, and different pre-treatments to the viability of porcine embryos; Comparison the different freezing methods on the effect of the embryo cryopreservation, such as the programmed freezing with straw, OPS(open pulled straw) method and GMP method; Establish a reasonable technique of porcine embryo vitrification method and got the first example with alive piglets by vitrification in China. The litter rate of GMP vitrification method was 30%.
Based on those research of porcine breed resources, we successfully cryopreserved three kinds of local pig sperms and embryos.
2.6大腸桿菌熱敏性腸毒素作為疫苗粘膜免疫佐劑功能研究
利用體外PCR擴增、重疊延伸方法結合,獲得熱敏性腸毒素6個突變體:三個位點單點突變體3個、3個雙位點突變體。分別克隆到T載體中,進行測序,以確定突變位點的正確性。將連接到T載體上的外源基因亞克隆到原核表達載體,經PCR鑒定和序列分析證實,成功獲得了6個LT表達突變體。
分別采用不同的表達條件:溫度、培養基、宿主表達菌、不同誘導時間等表達重組蛋白,篩選最佳表達條件,SDS-PAGE電泳證表達了兩種不同分子量的蛋白LTA、LTB。
FPLC純化LT突變體表達蛋白,經用HIStag抗體和抗CTB抗體進行Western檢測,結果兩條蛋白帶均可與抗His-tag抗體產生免疫學反應,而LTB還可與具有相似免疫原性的抗CTB抗體發生反應。
利用patent-mouse小鼠毒性試驗、ADP-核糖轉移酶活性試驗篩選毒性、酶活性也降低的LT突變體,目前已成功篩選到具有較低毒性的LT突變體,為后續免疫佐劑功能研究奠定了基礎。
2.6 Adjuvant function of E. coli. Heat labile enterotoxin through mucosal immunization.
Six heat-labile enterotoxin mutants by PCR amplification and overlap extension were obtainede, which include 3 single site mutation and 3 mutants of two site mutation. The mutate gene were cloned into the T-vector, and sequence it to verify the mutante site mutation. Then the mutate gene were sub cloned into pET vector separately, and abstained 6 mutants of LT. The positive clones were verified by sequencing and PCR. 
 The condition of expression was optimized, which included the temperature, the nutrient medium, the expression bacteria, and the induction time of the experiment. The two deferent molecular weight proteins LTA and LTB were displayed in the SDS-PAGE.
 The mutant protein LT was purified by FPLC. The two proteins both can react with the anti-His tag antibody in the western blotting, and the LTB protein can react with the anti-CTB antibody.
  We have screened the low virulent LT mutant by the patent-mouse toxicity experiment and the ADP-ribosyltransferase activity experiment; it has established the base for the research of the immunoadjuvant function
2.7豬流感及新城疫病毒檢測方法研究
(1) SIV多重PCR技術研究及標準制訂研究進展
病毒增殖培養:將保存的SIV 亞型流感病毒毒種取出,接種雞胚進行增殖,收獲病毒后,HA/HI測定病毒效價及血清亞型。
引物設計:采用Primier Primer 5.0 設計了一系列引物組合,針對流感病毒的NP基因,分型檢測的H1、H3亞型引物組合。
核酸提取及單一PCR體系建立與引物對篩選:采用Trizol方法提取病毒基因組RNA,分別采用H1、H3亞型病毒RNA篩選NP基因引物組合。再分別用不同亞型引物擴增模板,篩選型特異性引物對。已篩選到NP基因2對,H1亞型特異性引物對3對,H3亞型特異性引物對3對。且擴增片段大小不同,可用于多重PCR體系組合。
對不同引物組合進行了初步篩選,已經獲得較好的H1/H3亞型組合,N1/N2組合,并組裝了鑒別診斷試劑盒。對試劑盒靈敏性特異性檢測表明,可用于SIV分型檢測。
 起草了上海市地方標準申報書,并獲得上海市質量技術監督局批準,制訂上海市地方標準“豬流感病毒及H1/H3亞型多重RT-PCR檢測方法”,目前正撰寫送審稿。
(2) 出口家禽新城疫病毒檢測方法研究
通過對大量不同毒力新城疫病毒F基因核苷酸序列分析,根據毒力和序列間的相關規律,已分別設計與合成了兩組引物。
已完成對大批量臨床樣品整理及病毒分離工作, 建立了PCR-ELISA檢測新城疫病毒方法,并獲得上海市質量技術監督局批準,與上海出入境檢驗檢疫局合作制定上海市地方標準地方標準。
2.7 Detection method of swine influenza virus and Newcastle disease virus
 (1) Study on multi-PCR detection method of SIV and the standard of the method
   The viral multiplication cultivation: different subtype influenza virus were inoculated into the chicken embryos, and detected the titer of the virus and the subtype of the serum by the HA/HI.
   The design of the primers: We designed a series of primer groups, included that the primers for the NP gene and the primers for the subtype H1, H3.
RNA extractions, construction of single PCR system and screening of the primers: genome RNA of the virus was extracted by Trizol reagent, and screened the NP primer pairs of the subtype H1, H3 virus. Then we screened the specific primer pairs by the deferent amplification template of the subtype.
Specific H1/H3 and NP primers pairs were obtained, and a serious primer mixture were gained to construct the diagnosis kits. It is proved that it can be use to detect the SIV by the sensitivity and specificity experiment.
After the method was established, local standard on multiplex RT-PCR for detection and subtyping SIV was drafted, and the Shanghai Municipal Bureau of Quality and Technical Supervision licensed it. Now we are soliciting the suggestion and writing the illustration of the standard.
(2) The research of the detection of Necastle Disease Virus of the exporting poultry:
Two pairs of primers have designed and synthesized by analysing the deferent NDV strain’ s F gene sequence.
The disposal of the lots of clinical samples and the isolation of the NDV have finished by using 9-day old SPF chichen embryos.
We have established the NDV PCR-ELISA detection method, and the Shanghai Municipal Bureau of Quality and Technical Supervision has licensed it. We cooperated with the Entry-Exit Inspection and Quarantine Bureau to institute the local standard.
2.8 豬核移植胚DNA甲基化改變與“印跡”基因表達特征研究
(1)建立了豬核移植供體細胞培養技術。
首先選定合適的豬核移植所需的體細胞。通過比較成纖維細胞、卵丘細胞、顆粒細胞、輸卵管上皮細胞等體細胞。表明成纖維細胞、卵丘細胞是較合適的供體細胞,取材方便,形成重構胚后囊胚發育率較高。
(2)改進了豬卵母細胞去核方法。
豬卵母細胞去核是影響豬體細胞核移植的關鍵技術之一。本實驗室對傳統的McGrath-Solter、Willadsen去核法技術進行了改進,建立了二步擠壓去核法,在豬體外成熟卵母細胞上的去核操作表明,無論在去核操作成功率(73.9%)、還是在去核效率(81.2%)都顯著好于McGrath-Solter去核法(42.5%、67.4%,p<0.05)。
(3)建立了豬體細胞核移植重構胚的融合、激活和核移植重構胚體外培養方法。
細胞融合液組成為:0.3mol/L甘露醇,0.4mol/LMgSO4, 0.5mol/L CaCL2和3% BSA;融合條件為:100 V/mm DC,30μs刺激一次;電刺激后的重構卵在NCSU-23(加4 mg/mL BSA)培養液中培養30min。                   
激活和核移植重構胚體外培養方法:將重構胚置于10μmol/L鈣離子載體A23187的NCSU-23培養液中培養5min 或1個直流脈沖(125 V/mm,70μs),隨后置于NCSU-23(含2mmol/L 6-DAMP 和4 mg/mL BSA)培養液中培養5h,培養條件39℃,5%CO2。
核移植重構胚的體外發育囊胚率最高可達11.7%。
(4) 豬核移植胚DNA甲基化改變與“印跡”基因表達特征研究
在獲得各階段正常受精胚、體外受精胚、孤雌激活胚及體細胞核移植重組胚的基礎上,采用免疫熒光技術研究體細胞克隆胚各時期DNA甲基化的變化;采用定量RT-PCR
(real-time)技術比較U2afbp-rs基因的表達。表明,體細胞核移植重組胚體外發育過程中DNA甲基化、U2afbp-rs基因的表達發生了顯著的變化。
該研究初步闡明了豬體細胞核移植重組胚早期發育過程中死亡率高的分子生物學機理,從而為提高體細胞克隆豬成功率奠定基礎。
2.8 DNA methylation and the expressing characteristics of “imprinting gene” in porcine nuclear transfer reconstituted embryos
(1)The techniques of the culture of donor cells for nuclear transfer
First the different donor cells was examined to establish a preliminary procedure for porcine cloning. Fibroblast cells(FC), oviduct epithelial cells(OEC), granulose cells(GC) and cumulus cells(CC) were taken in this experiment. The FC and CC were considered as the better donor cells because of its convenient sources and high blastocyst developmental rate of reconstructed embryos.
(2)Improve of the enucleated method of porcine oocytes
Enucleated method is one of the key techniques for animal cloning. The results show that the modified Willadsen’s method in our lab for enucleation, two step-squeezing method is significantly better than McGrath-Solter in the enucleated rates(73.9%, 42.5%, P<0.05) and the rates of absolutely removing nuclei matter(81.2%, 67.4%, P<0.05). 
(3)The methods of electronic fusion, activation and the in vitro culture of reconstructed embryos have been successfully established in our lab.
The composing of the electronic fusion were 0.3mol/L D-mannitol, 0.4mol/LMgSO4, 0.5mol/L CaCL2and 3% BSA. The condition of the fusion were 100 V/mm DC, 30μs and only one times. The electronic activated embryos were cultured in the medium of NCSU23(4 mg/mL BSA) for 30 min. ………………
The method of activation and the in vitro culture of reconstructed embryos were: put the reconstructed embryos into the medium of NCSU23 contained with 10μmol/L A23187 for 5 min and one time of 125 V/mm and 70μs DC, then the embryos were transferred into the NCSU23(with 2mmol/L 6-DAMP and 4 mg/mL BSA) for 5 min.
The blastocyst developmental rate of reconstructed embryos could got 11.7% in vitro.
(4)DNA methylation and the expressing characteristics of “imprinting gene” in porcine nuclear transfer reconstituted embryos
In this experiment, different stage embryos from normal fertilized, in vitro fertilized, parthenogenetically activated and nuclear transferred embryos were got; The Immunocytochemistry with an antibody that specifically recognizes methylated H3/K9 was adopted to investigate the change of DNA methylation of different developmental nuclear transferred embryos; The expression of the U2afbp-ra gene was compared by a real-time PCR techniques. The results showed that the DNA methylation and the genic expression of the U2afbp-ra changed significantly with the development of reconstructed embryos cultured in vitro.
This study partly clarified the molecular biological mechanism of the high mortality rate of reconstructed embryos in the early development, and layed a foundation for improving the efficiency of porcine nuclear transfer.
2.9 豬流感病毒表位疫苗研究
根據流感病毒HA、NA基因核酸序列與蛋白結構預測,采用在線軟件篩選疏水區域,選擇不同亞型HA、NA抗原位點1-2個,串聯形成表位基因片段。經PCR擴增后酶切連接到原核表達載體中,并通過誘導表達獲得了重組蛋白,SDS檢測蛋白表達分子量21.0 kd。經Western 檢測,該蛋白可與SIV  H1、H3亞型血清發生免疫反應,通過變性條件純化,獲得了純化蛋白,正在進行大量表達與蛋白活性分析試驗。
通過在N、C端分別連接免疫佐劑分子,構建了表位基因片段與分子免疫佐劑基因串聯表達載體,并成功獲得原核表達。期望通過分子內佐劑作用來提高表位疫苗免疫效果,通過SDS-PAGE及Western blot檢測分析了表達產物免疫活性,證實了表達蛋白的生物學活性,目前正進動物免疫試驗。
2.9 Study on epitope vaccine of swine influenza virus
 We analyzed the HA, NA gene sequence, anticipated the protein structure, and screened the hydrophobic region in the online software. After that, we selected 1-2 antigen sites of the subtype gene, and constructed the tandem epitope gene. The epitope gene was cloned into the prokaryotic expression vector by PCR and enzymolysis. The protein was expressed, and its molecular weight is 21.0 kd. The protein can react with the serum of the SIV subtype H1/H3.We purified the protein under the denaturation condition, now we are massively purifying the protein, and analyzing the activity of the protein.
We constructed the tandem epitope and immunoadjuvant gene vector by ligating the immunoadjuvant gene to the end of N’and C’, and it was successfully expressed. We hope that the internal molecular immunoadjuvant can enhance the immunizing potency of the epitope vaccine. The protein is active which is proved by the SDS-PAGE and western blot. Now we are massively purifying the protein so that the animal immunity test can be carried out in the next step
2.10豬體細胞克隆技術的建立和優化
探討了體細胞的組織來源及培養代數對豬核移植重構胚發育的影響。體外成熟培養40-44h的豬卵母細胞去核后,將經血清饑餓(0.5%FBS)培養2-9d、0.1mg/L Aphidicolin(APD)培養+0.5% FBS培養2-9d或一般培養法(10%FBS)培養的卵丘細胞、顆粒細胞、輸卵管上皮細胞和耳皮成纖維細胞,直接注射到去核的卵母細胞質中,或注射到卵周隙中,再經電融合(100V/mm, 30μs,電脈沖1次)構建重構胚。重構胚以鈣離子載體A23817 或電脈沖結合6-DMAP 激活處理,體外培養6天。耳皮成纖維細胞和顆粒細胞經0.1 mg/L APD + 0.5% FBS培養處理后的重組胚卵裂率,均高于血清饑餓和一般培養處理的同種供體細胞(P<0.01)。卵丘細胞、顆粒細胞經0.1 mg/L APD + 0.5% FBS處理后進行核移植的分裂率和發育率均高于輸卵管上皮細胞和耳皮成纖維細胞(P<0.05)。以豬顆粒細胞為核供體時,電融合法的重構胚分裂率顯著高于胞質內注入法(P<0.05),但囊胚發育率無顯著差異(P>0.05)。培養3代和6代的豬顆粒細胞以及培養6代和10代的耳皮成纖維細胞,其具有正常二倍染色體的細胞比例均無顯著差異(P>0.05);以這2種細胞不同培養代數做供體進行核移植時,各代之間核移胚的體外分裂率、囊胚發育率無顯著差異(P>0.05)。這些結果表明:(1)豬耳皮成纖維細胞和顆粒細胞經培養傳代所建立起來的細胞系相對比較穩定;(2)0.1 mg/L APD預培養處理供體細胞能提高豬體細胞核移植的效果,血清饑餓培養則無明顯效果;(3)豬顆粒細胞和耳皮成纖維細胞等均可做供核細胞,核移植后都能得到體細胞克隆的囊胚,但前者的效果略優于后者,且其核移植效果不受供核細胞培養代數的影響;(4)電融合核移植胚胎的發育率高于胞質內直接注入法,但兩者的總體效率相近。
在完善體細胞核移植重構胚構建方法的基礎上,課題組從10月份開始,在巴馬小型豬、上海白豬上進行重構胚的移植,目前正在觀察移植效果,以決定下一步的研究工作重點。
2.10 The establishment and optimization of porcine cell cloning technique
This study was undertaken systematically to examine effects of different donor cells and passages on the development of nuclear transfer porcine embryos, so as to establish a preliminary procedure for porcine cloning. Porcine oocytes obtained from ovaries at slaughter were matured in vitro for 40-44 h and then enuleated in manipulation medium containing 5μg/mL cytochalasin B Fibroblast cells(FC), oviduct epithelial cells(OEC), granulose cells(GC) and cumulus cells(CC) after 3~9passages in TCM+10%FBS for2~9 days or continue culturing in 10%PBS for 2~9 days, then, transferred into enucleated oocytes by microinjection or electronic fusion(100V/min, 30μs and 1 pulses). Reconstituted embryos were activated with a combination of calcium ionophore A23187 or electric pulse and 6-DMAP, and cultured for 6 days, to evaluate their cleavage and embryonic development. The cleavage rate of reconstructed embryos with FC and GC pretreated with 0.1μg/mL APD+0.5%PBS were significantly higher than that of serum starvation group and control group(P<0.01). There were significantly difference in the cleavage rate and embryonic development among embryos derived from GC, CC, and FC, OEC pretreated with 0.1μg/mL APD+0.5%PBS. The cleavage rate of embryos reconstructed with GC by electrofusion was significantly higher than that by microinjection(P<0.05), but no difference was found in their proportion developing to blastocysts. 75% to 85% of GC at 3 and 6 passages, and FC at 6 and 10 passages had normal karyotype, which did not show significant difference among them(P>0.05) When GC at G3, G4, G5 and G6 of passages were as donor cells, the cleavage rate and blastocyst rate was similar, moreover, FC at G6, G7, G8 and G9 of passages also resulted in a similar cleavage rate and blastocyst development. These results indicate that: (1) FC and GC can be cultured up to 9 passages and keep relatively stable karyotepe; (2) Using 0.1μg/mL APD to treat donar cells to nuclear transfer can improve the efficiency of somatic cell muclear transfer in buffalo but serum starvation is inefficient in our system; (3) Both of FC and GCcells can be used as the donor karyoplasts for nuclear transfer, and their efficiency are not influenced by the culture passages; (4) The development of reconstructed embryos by electrofusion is high than that by microinjection, but there is no difference in the total efficiency between the two methods.
Based on the efficient nuclear transfer system, our research group operated the reconstructed embryo transfer in the Bama minipigs and Shanghai White pigs from last October. Now we are observing the result of nuclear transfer and decide the emphases in our next research work
2.11重組畢赤酵母高密度發酵表達豬α干擾素的研究
本項目已經完成。在單因素試驗的基礎上,利用Plackett-Burman實驗設計優化重組畢赤酵母產豬IFNα的生長和誘導條件,采用ANN-PR-控制方式建立了重組畢赤酵母在5L發酵罐中高密度分泌表達重組豬α型干擾素的工藝。                   
 and high density fermentation
a2.11 Experssion conditions of porcine IFN- for genetically engineered Pichia Pastoris
This project has been finished. The growth phase and induce phase conditions of the genetically engineered Pichia Pastoris have been established according to Plackett-Burman which based on single factor experiment. We have established the expression technology of  in Pichia Pastoris using ANN-PR controlling
arecombinant porcine IFN- method.
2.12 
重組豬α干擾素的本體動物實驗
重組α豬干擾素的發酵液通過離心、透析、過濾等步驟進行了處理。采用WISH細胞-VSV系統,以病變抑制法檢測了重組豬α干擾素的生物學活性,效價為1.2×106IU/mL,利用BCA法檢測了重組α豬干擾素的蛋白含量,含量為1.6mg/ml。現已初步選定甘露醇、甘氨酸、牛血清白蛋白、右旋糖苷作為重組α豬干擾素的凍干配方,已進行了共熔點的測定,下一步將對以上配方進行篩選。
2.12 
aThe animal experiment of porcine recombinant IFN-
Porcine recombinant (PoIFNα) fermentation has been dealt with according to centrifugal,
aIFN-   hasadialysis and filtration . The biology activity of porcine recombinant IFN- been detected using WISH-VSV system and cytopathic effect method. The anti-virus activity of PoIFNα is1.2×106IU/mL. The production of PoIFNα concentration is 1.6mg/ml using the method of BCA. Four lyoprotective protocol have been tested to select the optimum lyophilized formation of PoIFNα. Their common melting point has been detected.
2.13
新疆阿克蘇地區肉牛的改良與提高
課題組已于2006年7月、8月,贈送黑白花牛、南德溫牛精液各1000支給新疆維吾爾自治區溫宿縣畜禽品種改良站,按照課題要求在當地有計劃地進行雜交改良工作。
2.13The improvement of the meat cattle in Xinjiang Akesu region
Our research group has sent 1000 numbers of freezing sperm of Holstein bull and South Devon cattle to the Wensu county rescue station of Xinjiang Uygur Autonomous Region for free. Now we are designedly processing the improve work according to the request of the task.

 

 

 


豬IFNα在畢赤酵母中分泌表達條件
的優化及高密度發酵
藍勝芝1,2于瑞嵩2,3,俞明月1,2,董世娟2,3,曹祥榮1,李震2,3*
(1).南京師范大學生命科學學院,江 南京210097)
2).上海市農業遺傳育種重點實驗室.上海20l106)
3).上海市農業科學學院畜牧獸醫研究所,上海20l106)
摘要  從發酵時間、接種量、pH值、誘導劑量等方面對重組基因工程(畢赤酵母甲醇利用緩慢型)的搖瓶發酵條件進行了優化,進一步探索了酵母菌表達豬IFNα的發酵工藝,包括種細胞密度對初始發酵期的影響,補加甘油、甲醇速率條件的控制等.結果表明,搖瓶發酵時,誘導最佳時間為96 h,甲醇最適濃度為8 g/L,發酵pH范圍為6.4~9.0.最適接種量2:1.在分批發酵、接種量為10ck且種子細胞光密度(0D600)為5~6時,最有利于細胞的高密度培養,在補料發酵時,根據溶氧控制甘油流加速率與時機,細胞干重達到11 8.75g/L,在48h重組豬IFN-α表達量達到1.304 g/ L .
關鍵詞  豬IFNα,畢赤酵母,搖瓶發酵,高密度發酵
Studies on Expression Conditions of PoIFNα and High?density Fermentationfor Genetically Engineered Pichia Pastoris
Lan Shengzhi 1,2,Yu Ruisong2,3,Yu Mingyue 1,2,Dong Shijuan2,3 ,Cao Xiangrong1 ,Li Zhen2,3* 
(1),School of Life Sciences,Nanjing Normal University,Nanjing 2 1 0097,China
2).Shanghai Key Laborator3. Agricultural Breeding,Shanghai 201 106.China  
3).Institute of Shanghai Key Laboratory Livestock Breeding and Veterinarian,Shanghai 201106.China)
Abstract:The optimum fermentation conditions of genetically engineered Pichia pastoris in shake-flask cultivation and in fed-batch fermentation are reported respectively in this paper.Experimental results revealed that the fermentation period induced with methanol is 96 h.The optimum methanol is 8 g/L. pH range is 6。4-9.0 and the seed inoculum amount is 2:1 in shake-flask fermentation.Although the seed inoculum amount increases,the target protein concentration decreases. The 10% inoculum and 5 -6 OD600 of seed are benefit to the high density cultivation.With glycerol feeding strategy based on DO leve1.The cell dry weight of broth reaches 118.7g/L and the IFN concentration is 1.034g/L by methanol inducement for 48 hours at the fed-batch fermentation phase.
Key words:PoIFNα,pichia pastoris,shake-flask fermentation,high-density fermentation
原載于:《南京師大學報》 2006,29(3):76~80


尿素濃度梯度復性重組牛白細胞介素-2
于瑞嵩1,2 ,黃建珍2,3,李震1,2
(1). 上海市農業科學院畜牧獸醫研究所,上海201106;
2). 上海市農業遺傳育種重點實驗室,上海201106;
 3). 江西農業大學動物科技學院,南昌330045)
摘要:尿素濃度梯度已成功用于多種重組蛋白的復性,對尿素濃度梯度復性rblL-2進行研究,結果表明:調整變性rblL-2的濃度在4 mg/mL左右,添加DTT使其達到10 mmol/L,4℃攪拌過夜,4℃ 分別對4、3、2、1 mol/L尿素,50 mmol/L Tris?Cl,pH 8.5透析12 h。然后再對pH 8.5 的PBS透析12 h,可成功對rblL-2復性,復性率達70%左右。生物活性測定表明得到了具有較高生物活性的rblL-2。
關鍵詞:rblL-2;復性;尿素濃度梯度
Renaturation of rblL-2 by dialysis against urea concentration gradient
YU Rui-song1,2 , HUANG Jian-zhen2,3 , Li Zhen 1,2
(1).AnimalHusbandry andVeterinaryResearch Institute.SAAS,Shanghai 201106,China;
2). Shanghai Municipal Key Laboratory of Agri-Genetics and Breeding,Shanghai 201106.China;
3). College of Animal Science,Jiangxi agriculture University,Nanchang 330045,China)
Abstract:Several kinds of recombinant protein had been successfully renatured by dialysis against urea gradient.The study on the renaturation of rbIL-2 hy urea gradient method indicated that adjusting the denatured protein(rhIL-2) concentration around 4mg/mL,adding DTT up to 10 mmol/L , and stirring the solution overnight at 4℃ ,and then dialyzing the denatured mlution against 4,3,2 and 1 mmol/L urea with 50mmol/L  Tris’HCI at pH 8.5 in turn for 12 hours at 4℃ could successfully  renature the rblL-2.and the renaturation rate reached about 70%.The determination of  biological activity showed that the renatured rbIL-2 had a rather high biological activity(1.2×104 IU/mL).
Key words:rbIL-2;Renaturation;Urea concentration gradient


原載于《上海農業學報》 2006,22(4):33-36

 

 


重組牛白細胞介素-2對豬瘟及豬繁殖呼吸綜合征疫苗免疫抗體的影響
李 震1,2周宗清1,2 于瑞嵩1,2 黃建珍3 李祥瑞3孫景元3 趙 磊3 
(1上海市農業科學院畜牧獸醫研究所 201106)
(2上海市農業遺傳育種重點實驗室 動物遺傳工程分室 201106)
(3南京農業大學 動物醫學院 南京210095)
摘要:為確定重組牛白細胞介素-2(rbIL-2)對豬瘟及豬藍耳病(豬繁殖與呼吸綜合征)疫苗的免疫增強作用,分別利用杜長上雜交肉豬或巴馬小型豬(斷奶初期)進行了豬瘟及豬藍耳病弱毒疫苗接種時的牛IL-2注射試驗。實驗證明連續5天注射牛IL-2制品可有效提高豬瘟和豬藍耳病疫苗的免疫抗體水平。對于豬藍耳病所進行的試驗證明:rbIL-2制品可使初免后正常免疫劑量組抗體水平提高164%,二免時可使疫苗劑量減半組抗體水平提高54%。利用豬瘟疫苗所進行試驗的結果顯示:rbIL-2制品對于豬瘟疫苗初次免疫作用不明顯;在豬瘟疫苗二免時對疫苗劑量減半組具有良好的免疫增強作用,可使二免后10-20天的免疫抗體水平提高25%~52%,而且對免疫抗體高水平的維持具有良好作用。
關鍵詞:免疫 牛白細胞介素-2 豬瘟 豬繁殖呼吸綜合征 
Effect of Recombinant Bovine Interleukin-2 on Swine Sera Antibodies against Viruses of Classical Swine Fever and Swine Reproductive and Respiratory Syndrom
Li Zhen1,2 Zhou Zongqing1 Yu Ruisong1,2 Huang Jianzhen3 Li Xiangrui3 Sun Jingyuan3  Zhao Lei3
1.Institute of Animal Sciences and Veterinary Medicine, Shanghai Acedemy of Agricultrual Sciences  Shanghai 201106
2.Animal Genetic Division, Shanghai Municipal Laboratory of Agricultural Genetic and Breeding  Shanghai 201106
3.College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095
Abstract: To confirm the immunology enhancing effect of recombinant bovine interleukin-2 (rboIL-2) on vaccination of classical swine fever (CSF) and swine reproductive and respiratory symdrom(PRRS), pigs were immunized with the vaccines and at the same time injected with rboIL-2. The experiment showed that 5d injection of rboIL-2 could raised the sera antibody levels of the two vaccines. rboIL-2 could increase the sera antibody levels by 164% and 54% at the first and second PRRS vaccination. Intead of imposing an effect on the first CSF vaccination, rboIL-2 could enhance the 2nd CSF vaccination, increasing antibody levels by 25% at the 10th day and 52% at 20th day of the 2nd CSF vaccination.

Key words: Immunology; bovine interleukin-2; classical swine fever; swine reproductive and respiratory syndrome


原載于: 《西北農林科技大學學報》(2006)第34卷。

Study on renaturation of recombinant
bovine interleukin-2 expressed in Escherichia coli
Huang Jian-zhen 1,3,YU Rui-song1,2 
,SHEN Qiu-gu3, LI Xiang-rui4 , LI-Zhen1,2
(1), Animal Husbandryand Veterinary Research Institute,Shanghai Academy of Agricultural Science ,Shanghai 201106,China ;
 2), Shanghai Municipal Key Laboratory of Agri-Genetics and Breeding, Shanghai 201106,
China; 
3), College of Animal Science, Jiangxi Agricultural University, Nanchang 330045, China; 
4), Faculty of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095, China )
Abstract:Recombinant bovine interleukin-2 forms insoluble inclusion body when over expressed in Escherichiacoli,and must be denatured and renatured in vitro be fore acquiring biological activity.We systematically studied such conditions of renaturing  rBolL-2 as pH,protein concentration,DTT concentration,ratio of oxidizer(GSSG) to reducer(GSH),and additions of Cu2+,L-Arginine and Guandine-HCl of low concentration as well as renaturation methods.The results showed that adding 20mmol/L DDT to denaturation solution could improve the efficiency of the subsequent rBolL-2 renaturation,and the refolding rate of 3.5mg/mL  rBolL-2 protein solution could reach 50% by means of urea gradient renaturation.In which the bioactivity of 9.6×104 IU/mL could be obtained.
Key wolds:Recombinant bovine interleukin-2(rBoIL-2);Inclusion body;Renaturation
大腸桿菌表達重組牛IL.2復性技術的研究
黃建珍1,3 ,于瑞嵩1,2 ,沈秋姑3,李祥瑞4 ,李震1,2
(1). 上海市農業科學院畜牧獸醫研究所,上海201106;
2).上海市農業遺傳育種重點實驗室,上海201106;
3).江西農業大學動物科技學院,南昌330045;
4).南京農業大學動物醫學院,南京210095)
摘要: 研究了重組牛白介素2復性條件,如pH,蛋白濃度,DTT濃度,氧化劑(GSSG)和還原劑(GSH)的比例,Cu2+及L-Arg,低濃度的鹽酸胍的添加,復性方法等。結果表明,復性液中添加20mmol/mL DTT可以提高rBolL-2的復性效率;利用尿濃度梯度復性法3.5mg/mL 的rBolL-2蛋白溶液復性率可達50%以上,測定活性效價達9.6×104 IU/mL。
關鍵詞:重組牛白細胞介素2;包含體;復性

 


原載于 <上海農業學報> 2006,22(4):27—32


上海地區雞傳染性支氣管炎病毒S2基因的檢測及分析
劉惠莉1,2 ,陸承平1*,朱偉云1
(1).南京農業大學動物醫學院,江蘇南京210095;
2).上海市農業科學院畜牧獸醫研究所  上海市農業遺傳育種重點實驗室,上海201106)
摘要:參考GenBank發表的傳染性支氣管炎病毒(infectious bronchitis virus,IBV)S基因序列,設計合成了1對引物,模擬PCR表明,該引物能擴增現已發表的所有IBV 的S2基因部分核酸序列,擴增片段493 bp。建立的RT-PCR檢驗體系檢測靈敏度達10~50 ELD50,檢出限量為0.1 ng。采集上海地區12個雞場疑似IBV 禽樣品34份進行檢洲,陽性為32份,序列分析表明均為腎變型IBV S2基因片段,S2基因同源性較高(≥99 ),與其他地區腎變型IBV 也有較高同源性,而與腺胃型和呼吸型相比較,序列差異較大。進化分析表明,盡管IBV S2片段變異率明顯低于S1基因,但二者表現相同的進化趨勢,S2基因亦可反映出IBV的分子變異及毒株間的親緣關系。
關鍵詞:雞傳染性支氣管炎;S2基因;進化分析
Molecular Epidem ics of Infectious Bronchitis Virus in Shanghai District andAnalysis of Its S2 Fragment
LIU Hui—li1,2 ,LU Cheng—ping1 ,ZHU Wei—yun1
(1).College of Animal Veterinary Medicine ,Nanjing Agricultural University,Nanjing 210095,China;
2).Animal Husbandry and Veterinary Research Institute, Shanghai Academy of Agricultural Sciences; Shanghai Municipal Key Laboratory of Animal Geneticsand Breeding,Shanghai 
201106.China)
Abstract:A pair of specific primer was designed and synthesized in the conserved region of S2 gene according to the infectious bronchitis virus(IBV) S sequences at GenBank.A 493 bp am plicon was abtained from sim ulating PCR using softwareSPCR3.0.The PCR system was specific with IBV while no reaction with other avian virus tested in the study.10-50 ELD50 of the virus was detected.By using this method to detect the clinical samples,which were collected from l 2 chicken factories.32out of 34 suspicious samples were positive.Further analysis showed all the IBV-positive belonged to the nephropathogenic viruses.The S2 genes of the epidemic viruses share high identities with each other,while has much more difference with the proventriculopathogenic IBV and other viruses causing respiratory  symptom in China.Phylogenic analysis showed though S2 fragments have less divergence among isolates than S1 genes,it shows the same phylogenic tendency with S gene among the viruses.
Key words:avian infectious bronchitis virus;S2 gene;phylogenic analysis

 

原載于《 中國獸醫學報》2006,26(2)126~132


口蹄疫病毒3AB試劑盒的檢測及比較
潘 潔, 陳 波, 邢繼蘭, 饒 忠, 徐泉興, 尤永進
(上海市農業科學院畜牧獸醫研究所,上海市農業遺傳育種重點實驗室,上海 201106)
摘 要:以重組口蹄疫病毒非結構蛋白3AB 為檢測抗原,研制了口蹄疫病毒非結構蛋白ELISA 檢測試劑盒,該試劑盒可用于判定被檢動物是否感染口蹄疫病毒。重組口蹄疫病毒非結構蛋白3AB 作為檢測抗原包被酶標板反應孔,以辣根過氧化物酶標記的重組蛋白A/ G作為第二抗體,建立了抗非結構蛋白抗體檢測的方法。結果表明,在1746 份免疫豬血清中,3AB-ELISA 對免疫豬血清的特異性為98.68%;在1077 份健康非免疫豬血清中,3AB-ELISA 對健康非免疫豬血清的特異性為98.68%;在36份人工感染豬血清中,3AB-ELISA 對陽性檢出率為100 %。而牛的特異性的各項指標和陽性檢出率分別為94.04%,97.96%和100%。豬、牛口蹄疫病毒非結構蛋白酶聯免疫吸附試驗檢測試劑盒與國外同類試劑盒比較,陽性檢出率高出12.5 個百分點。
關鍵詞: 非結構蛋白3AB ;試劑盒; 檢測
Detection and comparison of diagnostic kits for FMDV-3AB
PAN Jie , CHEN Bo , XING Ji Lan , RAO Zhong , XU Quan xing , YOU Yong jin
( Animal Husbandry and Veterinary Research Institute , Shanghai Academy of A gricutural Sciences ;Shanghai Key Lab of A gricultural Genetics and Breeding , Shanghai 201106 , China)
Abstract :In order to judge if the detected animal is infected with foot and mouth disease virus(FMDV) ,a FMDV- NS 3AB EL ISA diagnostic kit was developed by using the recombinant non-structural protein 3AB as a detecting antigen. The reaction microplate wells were coated with the detecting antigen based on the non- structural protein 3AB , the recombined protein A/ G marked with horseradish peroxidase was used as the second antibody , and the anti-non-structural protein antibody detecting method was established. The specificity of FMDV-NS 3AB EL ISA diagnostic kit was 96. 68 % in 1 746 immune swine serums and 7. 96 % in 1077 normal swine serums ; it s positive detection rate was 100 % for 36 swine serums artificially infected with FMDV. And for bovine serums the above indexes were respectively 94. 04 % , 97. 96 % and 100 %. The positive detection rate of the enzyme-linked immunosorbent assay kit for FMDV-3AB of swine / bovine was 
12. 5 percentage points higher than that of the similar kits abroad.
Key words : Non-structural protein 3AB ; FMDV-NS 3AB EL ISA kit ; Detection


原載于<<上海農業學報>> 2006 ,22 (2) : 20 - 23
 

 

豬胚胎開放式拉長細管玻璃化冷凍保存研究
張德福1,2 ,劉 東1,2 ,吳華麗1,2 ,鄭筱峰3 ,王昭凱4 ,王少兵4
(1)上海農業科學院畜牧獸醫研究所,上海 201106
2)上海市農業遺傳育種重點實驗室動物遺傳工程研究室,上海 201106
3)南京農業大學動物醫學院,南京 210095
4)南京農業大學動物科技學院,南京 210095)
摘 要 從20頭供體母豬獲得的291枚可用胚胎(囊胚/桑葚胚),采用二步法開放式拉長細管(OPS,open pulled straw)玻璃化冷凍技術進行保存,即胚胎首先在冷凍液I(TCM199+20%FBS+10%EG+10%DMSO)中平衡3min,然后立即轉入冷凍液II(TCM199+20%FBS+20%EG+20%DMSO+0.4mol/L SUC)中并在lmin內裝管,直接投入液氮保存;3個月后解凍移植給8頭受體母豬,其中1頭懷孕產仔(8頭活仔),在我國首次獲得豬胚胎超低溫(一196℃)冷凍后代。
關鍵詞 豬,冷凍胚胎,玻璃化冷凍
Study on Vitrification of Porcine Embryos by Open Pulled 
Straw Method
ZHANG De—Fu1,2 , LIU Dong1,2, W U Hua-Li1,2, ZHENG Xiao—Feng3
WANG Zhao—Kai4, WANG Shao—Bing4
(1) Animal Husbandry and Veterinary Research Institute,Shanghai Academy ofAgricultural Scierwes,Shanghai 201106,China
2) DivisionofAnimal Genetic Engineering,ShanghaiMunicipalKey Laboratory ofAgri-Genetics and Breeding,Shanghai 201106,China
3) AnimalMedicine College,Nanjing Agriculture University,Nanjing 252100,China
4 )Animal Sci&Technology College,Nanjing Agriculture University,Nanjing 252100,China)
Abstract  291 embryos(Blastocyst/Morula)from 20 donor SOWS were vitrified by two step method with OPS(open pulled straw) in solution I(TCM199+20%FBS+10%EG+10%DMSO)for 3min, and solution II(TCM199+20%FBS+20%EG+20%DMSO+O、4mol/L SUC)for 1min,stored in liquid nitrogen for 3 months,and transferred into 8 recipient SOWS after warming,one recipient SOW was pregnant and 8 alive piglets were born.This is the first paper to report getting alive piglets by vitrification inChina.
Key words  pig,frozen embryos,vitrification
原載于<生物工程學報>2006, 22, (5):845-849

 


豬體細胞核移植重構胚發育異常及印跡
基因表達特征研究的進展
張德福 1,2,吳華莉1,2 ,劉東1,2 ,陳學進3,陳瑩3,王凱3
(1) 上海市農業科學院畜牧獸醫研究所,上海201106;
2) 上海市農業遺傳育種重點實驗室,上海201106;
3) 上海第二醫科大學,上海市發育生物學重點實驗室,上海200025)
摘 要 由于豬的器官極可能成為人類未來器官的供應源,豬體細胞核移殖及轉基因等方面的研究已成為全球的熱點。近幾年來,豬體細胞核移植研究取得了一定的進展,但總體來看核移植效率還很低(1 ~2%),還需要不斷完善核移植程序及對一些基礎問題的深入研究。本文主要對豬體細胞核移植重構胚發育異常及“印跡” 基因表達特征作一綜述與分析, 旨在為研究者提供一些有益的啟示
關鍵詞 豬;核移植;印跡基因
Abnormal development and expressing characteristics of
‘‘imprinting gene” in reconstituted embryos from
porcine somatic nuclear transfer
ZHANG De—fu1,2,WU Hua—li1,2 ,LIU Dong1,2,CHEN Xue—jin3,
CHEN Yin3,WANG Kai3
(1)Animal Husbandry and Veterinary Research Institute,Shanghai Academy of Agricultural Sciences,Shanghai 201106,China;
2)Shanghai Municipal Key Laboratory of Agri—Genetics and Breeding,Shanghai 201106,China;
3)Shanghai Municipal Key Laboratory of Developmental Biology,Shanghai No.2 Medical University,Shanghai 200025,China)
Abstract Research in the fields of somatic cell nuclear transfer(SCNT)and transgenic cloning in pigs has become global highlight,because porcine organs would probably become the first source of donor organs for human xen0transplantation.Recently,great progress has been made in porcine SCNT.However,the efficiency of nuclear transfer remains very low (1 ~ 2 ).It is necessary to improve the procedure Of nuclear transfer and to further investigation Of some basic problems.In this paper,we mainly focus on the abnormal development and expressing characteristics of “imprinting gene” in reconstituted embryos from porcine somatic nuclear transfer, including its applications and approach of increasing the efficiency of SCNT.
Key words  pigs;nuclear transfer;imprinting gene
原載于<<廣西農業生物科學>>2006,25(增刊):145-149


豬卵母細胞冷凍保存研究
張德福1,2 ,朱良成3 ,劉 東1,2 芮 榮3湯琳琳1,2 項智峰3
( 1)上海市農業科學院畜牧獸醫研究所,上海201106;
2)上海市農業遺傳育種重點實驗室/動物遺傳工程研究室,上海201106
3)南京農業大學動物醫學院,南京252100)
摘 要 【目的】本研究試圖通過比較豬卵母細胞超低溫冷凍保存方法、冷凍承載工具、冷凍卵母細胞的類型,從而有效地保存豬卵母細胞;【方法】利用屠宰場卵巢采集的卵母細胞,以臺盼藍染色、二乙酸熒光素(FDA)染色鑒定卵母細胞冷凍后的成活率,以體外成熟和體外受精鑒定卵母細胞冷凍后的發育能力,研究了不同冷凍方法和不同冷凍保護劑對豬卵母細胞的冷凍效果【結果】(1)程序化法冷凍保存中,9%乙二醇(EG),l0%二甲基亞砜(DMSO),10%甘油(Giy)均對豬MII期卵母細胞有冷凍保護作用,極顯著高于對照組(FDA染色成活率33.8%,25.8%,23.5%vs 2.5%,P<0.01),且以9%EG的效果最好,顯著高于另外兩組(33.8%VS 25.8%,2 3.5%,P<0.05)。(2)玻璃微細管(GMP)法是豬卵母細胞超低溫冷凍的較好方法,以普通的麥管(Straw)法進行的程序化冷凍為對照組,GMP管法顯著提高豬卵母細胞的冷凍成活率(分別為63.3%和34.5%,P。(3)在玻璃化冷凍方法中,不同的冷凍液載體對豬卵母細胞冷凍成活率有影響以EFS40為冷凍液,Straw和GMP管作冷凍液載體,卵母細胞的成活率分別為45.0%和65.9%,二者差異顯著(P<0.05)(4)用straw的程序化冷凍法和用GMP管的玻璃化冷凍法對豬GV期卵母細胞冷凍均有效,但二者差異顯著(成活率分別為30.0%和59.7%,P冷凍后繼續培養,分別有2.8%和6.3%的卵母細胞周圍顆粒層發生擴散。(5)冷凍對MII期卵母細胞的發育潛能影響較大,用新鮮精液使其受精,僅有4.9%的受精卵分裂,極顯著低于對照組的49.5%(P;發育至4一細胞期的比例為1.7%,但未能發育至8一細胞期以上胚胎。【結論】選用MⅡ期卵母細胞、以GMP管為冷凍承載工具、應用玻璃化冷凍方法能夠較好地保存豬卵母細胞,為進一步完善豬卵母細胞超低溫冷凍保存技術體系提供了參考依據。
關鍵詞 豬;卵母細胞;冷凍保存;體外受精
Studies on Cryopreservation tudes G of Porcine 0oocyte
ZHANG De-fu1,2,ZHU Liang-cheng3 , LIU Dong1,2,RUI Rong3,TANG Lin-1in1,2 ,
XIANG Zhi-feng3
(1)Animal Husbandary and Veterinary Research Institute, Shanghai Academy of Agricultural Sciences, Shanghai 201106;
2)Division of Animal Genetic Engineering, Shanghai Municipal Key Laboratory of Agri-Genetics and Breeding, Shanghai 201106;
3) Animal Medical College, Nanjing Agriculture University, Nanjing 252100)
Abstract 【Objective】The aim of the present study was to try to cryopreserve porcine oocytes efficiently, and to investigate the effect of cryopreservation method,cryopreservation tool and types of cryoprotectant on pig oocyte survival and its in vitro maturation and cleavage following IVF and IVC.【Method】Experiments in pig oocyte cryopreservation and in vitro fertilization(IVF) were conducted using oocytcs collected from a slaughterhouse.The efects of diferent methods an d diferent cryoprotectant solution on cryopreservation of porcine oocytes were examined.Survival was assessed by trypan blue(TB)staining,fluorescein diacetate(FDA) staining,maturation in vitro and cleavage after IVF.The results were showed as follows:【Results】(1)Cryoprotectant solution (9%EG,IO%DMSO,10%Gly)were efective to cryopreserve pig MII oocytcs when in programmed freezing.The survival rates (FDA dyeing)of pig oocytes after frozen—thawed were very significantly higher than that of control(33.8%,25.8%,23.5%VS 2.5%,P<0.01).Among these three cryoprotectant solution,9%EG Was significantly superior to the other two in survival rate(33.8%VS 25.8%,23.5%,P<0.05).(2)The freezing method by GMP(glass micropipette)was suitable and eficient to cryopreserve  pig oocytes.Compared with programmed freezing method using Straw,GMP method could significantly promote the survival rate of pig oocytes (63.3%and 34.5%,respectively,P<0.05).(3)The vitrification solution carrier had a positive efect on survival rate of pig oocytes.When EFS40 was used as vitrification solution,Straw and GMP led to different survival rates,which were 45.0% and 65.9%,respectively(P<0.05).(4)The programmed freezing method by Straw and vitrification method by G were available in cryopreservation of pig oocytes,but these two methods resulted in different survival rates(30.0%and 59.7%,respectively, P<0.05).(5)Cryopreservation had a great effect on the developmental ability of pig oocytes after IVF and IVC.Vitrified MII oocytes were
fertilized with fresh spermatozoa an d only 4.9% eggs cleaved after 48 culture.which was very significan tly lower than the cleavage rate of control(49.5%,P<0.01).the subsequent developmental rateto4-cell stage was 1.7%,but none to 8-cell stage.【Conclusion】The cryopreservervation of porcine oocytes with MII oocytes.vitrification and GMP were efficient.
Key Words Pig;Oocyte;Cryopreservation;in vitro fertilization( IVF)


原載于<中國農業科學>2006,39(6):1233-1240

 

 

 


體細胞的組織來源及培養代數對豬核移植
重構胚發育的影響
張德福1,2 劉 東1,2 湯琳琳1,2 王英1,2 陳茵3 王凱3  Karl ScheHande4 LIN Cai Lu4
(1)上海市農業科學院畜牧獸醫研究所,上海201106
2)上海市農業遺傳育種重點實驗室,上海201106
3)上海第二醫科大學,上海市發育生物學重點實驗室,上海200025;
4)Animal Breeding Institute,Bonn University,Bonn 53121,Germany)
摘 要  本研究系統探討了體細胞的組織來源及培養代數對豬核移植重構胚發育的影響。體外成熟培養40—44h的豬卵母細胞去核后,將經血清饑餓(0.5%FBS)培養2-9d、0.1mg/L Aphidicolin (APD)培養+0.5% FBS培養2-9d或一般培養法(10%FBS)培養的卵丘細胞、顆粒細胞、輸卵管上皮細胞和耳皮成纖維細胞,直接注射到去核的卵母細胞質中,或注射到卵周隙中,再經電融合(100V/mm,30s,電脈沖1次)構建重構胚。重構胚以鈣離子載體A23817或電脈沖結合6-DMAP激活處理.體外培養6天。耳皮成纖維細胞和顆粒細胞經0.1mg/L APD+0.5% FBS培養處理后的重組胚卵裂率,均高于血清饑餓和一般培養處理的同種供體細胞(P<0.01)。卵丘細胞、顆粒細胞經0.1mg/L APD+0.5% FBS處理后進行核移植的分裂率和發育率均高于輸卵管上皮細胞和耳皮成纖維細胞(P<0.05)。以豬顆粒細胞為核供體時,電融合法的重構胚分裂率顯著高于胞質內注入法(P<0.05),但囊胚發育率無顯著差異(P>0.05)。培養3代和6代的豬顆粒細胞以及培養6代和10代的耳皮成纖維細胞,其具有正常二倍染色體的細胞比例均無顯著差異(P>0.05).以這2種細胞不同培養代數做供體進行核移植時。各代之間核移胚的體外分裂率、囊胚發育率無顯著差異(P>0.05)。這些結果表明:(1)豬耳皮成纖維細胞和顆粒細胞經培養傳代所建立起來的細胞系相對比較穩定;(2)0.1mg/L APD預培養處理供體細胞能提高豬體細胞核移植的效果,血清饑餓培養則無明顯效果;(3)豬顆粒細胞和耳皮成纖維細胞等均可做供核細胞,核移植后都能得到體細胞克隆的囊胚,但前者的效果略優于后者,且其核移植效果不受供核細胞培養代數的影響;(4)電融合核移植胚胎的發育率高于胞質內直接注入法,但兩者的總體效率相近。
關鍵詞 豬體細胞 核移植 細胞周期
EFFECTS OF DⅡ FERENT DONOR CELLS AND PASSAGES ON
THE DEVELOPM ENT OF NUCLEAR
TRANSFER PORCINE EM BRYOS
ZHANG De Fu1,2  LIU Dong1,2  TANG Lin Lin1,2  WANG Ying1,2  CHEN Yin3
WANG Kai3 Karl Schellander4  LIN Cai Lu4
( 1)Anima! Husbandry and Veterinary Research Institute,Shanghai Academy of Agricultural Sciences,Shanghai 201106;
2)Division of Anima! Genetic Engineering,Shanghai Municipal Key Laboratory of Agri—Genetics and Breeding,Shanghai 201106;
3) Shanghai Municipal Key Laboratory of Developmental Biology,Shanghai No.2 Medical University,
Shanghai 200025; Anima! Breeding Institute,Bonn University,Bonn 53121,Germany)
ABSTRACT  This study was undertaken to systematically examine effects of different donor cells and passages on the development of nuclear transfer porcine embryos,so as to establish a preliminary procedure for porcine cloning.Porcine oocytes obtained from ovaries at slaughter were matured in vitro for 40-44h and then enucleated in manipulation medium containing 5μg/mL cytochalasin B.Fibroblast cells(FC),oviduct epithelial cells(OEC),granulosa cells(GC) and cumulus cells(CC)after 3-9 passages in TCM + 10% FBS were treated by serum starvation ( 0.5% FBS for 2-9 days),0.1μg/mL aphidiconlin (APD) for 1 day and 0.5% FBS for 2-9 days or continue culturing in 10% FBS for 2-9 days, then, were transferred into enucleated oocytes by microinjection or electronic fusion(100 V/mm,30μS and 1 pulses).Reconstituted embryos were activated with a combination of calcium ionophore A23187 or electric pulse and 6-DMAP.And cultured for 6 days, to evaluate their cleavage and embryonic development.The cleavage rate of embryos reconstructed with FC and GC pretreated with 0.1g/mL APD +0.5%FBS were significantly higher than that of serum starvation group and control group (P<0.01). There was significantly μdifference in the cleavage rate and embryonic development among embryos derived from GC,CC and FC,OEC pretreated with 0.1mL APD + 0.5%FBS. The cleavage rate of embryos reconstructed with GC by electro fusion was significantly higher than that by microinjection(P<0.05),but no difference was found in their proportion developing to blast ocysts.75% to 85%of GC at 3 and 6 passages, and FC at 6 and 10 passages had normal karyotype which did not show significant difference among them (P>0.05).When GC at G3,G4,G5 and G6 of passages were used as donor cells, the cleavage rate and blastocyst rate was similar. Moreover, FC at G6,G7,G8 and G9 of passages also resulted in a similar cleavage rate and blastocyst development. These results indicate that:(1) FC and GC can be cultured up to 9 passages and keep relatively stable karyotepe; (2)Using0.11μg/mL APD to treat donor cells prior to nuclear transfer can improve the efficiency of somatic cell nuclear transfer in buffalo but serum starvation is inefficient in our system; (3) Both of FC and GC cells can be used as the donor karyoplasts for nuclear transfer, and their efficiency (4) The development of reconstructed embryos by electro fusion is higher than that than microinjection,but there is no difference in the total are not influenced by the culture passages; by electro fusion is higher than that by efficiency between the two methods.
Key words:Pigs.Somatic cells nuclear transfer.cell cycle
原載于<分子細胞生物學報>2006,39(2):145-151

 

豬卵母細胞去核方法的改進

張德福1,2 ,劉 東1,2 ,湯琳琳1,2 ,陳曉宇3,Lin Cailu4 
(1).上海市農業科學院畜牧獸醫研究所,上海201106;
2).上海市農業遺傳育種重點實驗室動物遺傳工程研究室,上海201106;
3).西北農林科技大學動物科技學院,陜西楊凌712100;
4).Animal Breeding Institute,Bonn University,Bonn 53121,Germany)
摘 要 對豬體細胞克隆技術中關鍵步驟之一的去核方法作了較深入的研究。結果表明,由本實驗室改進的Willadsen去核法-二步擠壓去核法,無論在去核操作成功率(73.9% )上還是在去核效率(81.2% )上都明顯好于McGrath—
Soher 去核法(42.5%、67.4%,P<0.05);同時,本試驗還發現,卵母細胞成熟培養36 h后去核組去核效率(89.1% )顯著地高于成熟培養44 h后去核組(55.8%,P<0.05),而核移植重構胚的發育率2個組間無顯著差異。
關鍵詞 豬;卵母細胞;去核;核移植
A Modified Enucleation Method of Porcine Oocytes
ZHANG De—fu 1,2,LIU Dong1,2,TANG Lin—lin1,2 ,CHEN Xiao—yu3,LIN Cai—lu4 
(1).Animal Husbandry and Veterinary Research Institute,Shanghai Academy Agricultural Sciences,Shanghai 201106,China;
2).Laboratory of Animal Genetic Engineering,Shanghai Municipal Key Laboratory of Agri—
genetics and Breeding,Shanghai 201106,China;
3).College of Animal Science and Technology,North—west Sci—tech University of Agriculture and Forestry,Yangling,Shanxi 7121 00,China;
4).Animal Breeding Institute,Bonn University,Bonn 53121,Germany)
Abstract  This experiment was conducted to study enucleated method,which iS one of the key techniques for animal cloning.The results show that the modified Willadsen’ S method for enucleation,two—step—squeezing method is significantly better than McGrath—Soher’S method in the enucleated rates(73.9%,42.5% , P<0.05)and the rates(81.2%,67.4%,P<0.05) of absolutely removing nuclei matter.It was also found that the rates(89.1% )of absolutely removing nuclei matter for 36 h —matured—in—vitro oocytes is significantly higher than that for 44 h (55.8%,P<0.05).
Key words pigs;oocytes;enucleation;nuclear transfer


原載于<中國獸醫學報>2006,26(5):574-577

在研項目一覽表


序號

項目名稱

項目來源

起止日期

承擔者

主持人

參加人數

1

動物基因工程重組蛋白微球注射劑緩控釋技術的研發

市農委科技興農攻關

20042007

動物分室

 

7

2

豬源性戊型肝炎阻斷技術的研究

市科委

20062008

 

動物分室

 

10

3

豬流行性腹瀉減毒活疫苗的研究

中荷國際科技合作

2007-2010

動物分室

 

12

4

豬體細胞克隆技術的建立和優化

市農委科技興農攻關

20052008

動物分室

張德福

 

10

5

地方豬種種質資源長期保存技術研究

市農委科技興農攻關

20032006

動物分室

張德福

 

10

6

豬核移植胚DNA甲基化改變與“印跡”基因表達特征研究

市科委

20042006

動物分室

張德福

 

10

7

豬流感監測、診斷方法研究

 

上海市農委重點攻關

20052007

動物分室

劉惠莉

7

8

豬流感及新城疫病毒檢測技術

上海市科委

2005-2007

動物分室

劉惠莉

10

9

豬流感表位疫苗研究

上海市科委

2005-2007

動物分室

劉惠莉

7

10

大腸桿菌熱敏性腸毒素作為疫苗粘膜免疫佐劑功能的研究

上海市農委

2005.10-2007.12

動物分室

劉惠莉

7

11

口蹄疫注苗與感染牛(血清)鑒別診斷試劑盒中試生產研究

市農委科技興農攻關

20032006.

動物分室

劉惠莉

 

7

12

預防仔豬細菌性腹瀉微生態制劑的研究

市農委科技興農攻關

20032006

動物分室

劉惠莉

   

7

13

口蹄疫免疫抗體ELISA檢測試劑盒研究

市農科院

20042006

動物分室

劉惠莉

5

14

重組畢赤酵母高密度發酵表達豬α干擾素的研究

市農科院青年科技基金

20042006

動物分室

于瑞嵩

7

15

新疆阿克蘇地區肉牛的改良與提高

市科委西部合作項目

2004-2006

動物分室

張德福

5

16

重組豬α干擾素的的本體動物試驗

上海農科院青年基金

20062008

動物分室

董世娟

6

17

豬卵母細胞冷凍保存的研究

上海市農科院青年基金

20032006

動物分室

劉東

7


新立項目一覽表


序號

項目名稱

項目來源

起止日期

承擔者

主持人

經費:萬元

1

豬源性戊型肝炎阻斷技術的研究

市科委

20062008

 

動物分室

 

40

2

重組豬α干擾素的的本體動物試驗

市農科院青年科技基金

20062008

動物分室

董世娟

4

3

豬流行性腹瀉減毒活疫苗的研究

中荷國際合作項目

2007-2010

動物分室

 

140









鑒定與獲獎成果一覽表


序號

成果名稱

成果類別

鑒定時間

獲獎情況

完成單位














論文(或專著)一覽表


作者

論文(或專著)名稱

載體或

機構、會議

發表(出版)

時間

卷、期、頁

(字數)

蘭勝芝 于瑞嵩  俞明月 董世娟曹祥榮 李 震

豬IFNα在畢赤酵母中分泌表達條件的優化及高密度發酵

南京師范大學學報

2006293

76-80

于瑞嵩黃建珍 李震

尿素濃度梯度復性重組牛白細胞介素-2

上海農業學報

 200622(4)

33-36

黃建珍 于瑞嵩 沈秋姑 李祥瑞 李 震

Study on renaturation of recombinant bovine interleukin-2 expressed in Escherichia coli

上海農業學報

2006224

27-32

劉惠莉

陸承平

朱偉云

 

上海地區雞傳染性支氣管炎病毒S2基因克隆與表達

中國獸醫學報

2006262

126132

潘 潔   陳 波

邢繼蘭 饒 忠徐泉興 尤永進

口蹄疫病毒3AB試劑盒的檢測及比較

上海農業學報

2006,22(2)

20-23

張德福 劉 東湯琳琳 王英  陳茵 王凱

體細胞的組織來源及培養代數對豬核移植

重構胚發育的影響

分子細胞生物學報

2006,39(2)

145-151

張德福 劉  東吳華麗鄭筱峰 王昭凱  王少兵

豬胚胎開放式拉長細管玻璃化冷凍保存研究

生物工程學報

 

2006,22(5)

845-849

張德福 朱良成劉   東 芮 榮

湯琳琳 項智峰

豬卵母細胞冷凍保存研究

 

中國農業科學

2006,39(6)

1233-1240

張德福  劉 東

湯琳琳 陳曉宇Lin Cailu

豬卵母細胞去核方法的改進

 

中國獸醫學報

 

200626(5)

 

574-577

張德福 吳華莉劉  東  陳學進

陳 瑩   王  凱

豬體細胞核移植重構胚發育異常及印跡基因表達特征研究的進展

廣西農業生物科學

 

2006,25(增刊)

145-149

項智峰 張金洲 王青華李培慶張德福

點壓去核法對豬體細胞核移胚發育影響

 

農業生物技術學報

 

20061

 

137-138

張德福 劉  東

吳華莉 陳學進

王  凱 陳  瑩

豬作為異種器官移植供體的研究現狀及發展趨勢

豬業科學

 

20067

 

14-18

張德福 張似青

國外對梅山豬的研究現狀及其進展

遺傳育種

2006, 4

51-53

張德福 吳華莉

劉  東

生物技術在地方豬種種質資源保存中的應用

豬業科學

2006,9

46-49

張德福,劉東,吳華麗

豬精液大管(5ml)冷凍技術研究

 

中國畜牧獸醫學會動物繁殖學分會第13屆學術研討會論文集,488-493

20067

 


張德福 吳華劉東

豬體細胞核移植重構胚發育異常及印跡基因表達特征研究的進展

全國動物生物技術學術研討會

2006,10


劉惠莉 邢繼蘭楊秋峰

多重RT-PCR鑒別診斷禽流感病毒及H5H9血清亞型

中國畜牧獸醫學會禽病學分會第XIII次學術研討會

2006 9

172-175

 

 震,周宗清,于瑞嵩,黃建珍,李祥瑞,孫景元,趙 

重組牛白細胞介素-2對豬瘟及豬繁殖呼吸綜合征疫苗免疫抗體的影響

西北農林科技大學學報

2006。第34卷。增刊。

35-39

 震,周宗清,于瑞嵩,黃建珍,李祥瑞,孫景元,趙 

重組牛白細胞介素-2下游處理技術研究及動物試驗

中國科學技術協會2006年會論文集

20069

77-85


學術交流情況(1)[State of academic exchanges(1)

報告人

日期

報告題目

地點

會議名稱

張德福

2006.10

 

生物技術在地方豬種種質資源長期保存中的應用

上海市農業科學院

上海市生物技術學會學術論壇

張德福

2006.7

豬精液大管(5ml)冷凍技術研究

河北保定

中國畜牧獸醫學會動物繁殖學分會第13屆學術研討會

劉惠莉

2006,9

多重RT-PCR鑒別診斷禽流感病毒及H5H9血清亞型

湖南長沙

中國畜牧獸醫學會禽病學分會第XIII次學術研討會

 

2006.8

動物健康養殖與食品安全

陜西楊凌

海峽兩岸畜牧獸醫可持續發展高新技術論壇

 

2006.9

豬戊型肝炎研究進展

上海

中國科學院巴斯德研究所肝炎病毒專題學術研討會

 

2006.9

重組牛白細胞介素-2下游處理技術研究及動物試驗

北京

中國科學技術協會2006年會(分會報告)

 

2006.11

豬戊型肝炎上海流行狀況調查

上海

中國科學院巴斯德研究所肝炎病毒第二次專題學術研討會

學術交流情況(2[State of academic exchanges(2)


報告人

  

  

報告題目

 












研究生培養情況
動物研究分室與南京師范大學生命科學院、南京農業大學、江南大學、四川農業大學、山東農業大學聯合培養碩士研究生19名,與南京師范大學、南京農業大學、四川農業大學共同培養的8位碩士研究生2007年6月以優異的成績通過碩士論文答辯畢業。