猪细环病毒2型衣壳蛋白的原核表达及纯化
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摘要
本试验通过在大肠杆菌中表达衣壳蛋白,确定最优表达条件并对其进行纯化。用PCR法扩增猪细环病毒2型(TTV2)ORF1抗原基因,定向克隆至质粒pGEX-4T-1中,获得重组质粒pGEX-4T-1-ORF1。经双酶切鉴定及测序正确后转化到BL21(DE3)工程菌中,获得衣壳蛋白大肠杆菌表达株。IPTG诱导表达,表达的重组融合蛋白经SDS-PAGE及Western blotting鉴定,并对影响重组蛋白表达的3个因素,即诱导时间、诱导温度和IPTG浓度进行优化,确定最优表达条件。重组表达质粒PCR及双酶切鉴定结果显示衣壳蛋白原核表达载体构建正确。重组融合蛋白分子质量约80ku,主要以包涵体形式存在。37℃诱导5h表达量最多,IPTG浓度对表达量无明显影响。蛋白质串联肽谱分析检测氨基酸序列与GenBank中公布的TTV2 ORF1基因序列翻译的氨基酸序列相对应,且可被鼠抗GST单克隆抗体特异性识别。该融合蛋白首次用KCl染色切胶回收法对表达的融合蛋白进行纯化,纯化效果较好,性价比高。结果表明,成功构建了pGEX-4T-1-ORF1重组质粒,衣壳蛋白得到了高效表达及纯化,为间接ELISA的建立及单克隆抗体的研制提供了抗原。
The study was aimed to highly express Cap protein of porcine torque teno virus type 2in E.coli,and determine the optimal expression conditions and purify it.ORF1gene was amplified by PCR and cloned into the prokaryotic expression vector pGEX-4T-1to construct recombinant plasmid pGEX-4T-1-ORF1,which was identified by restriction and sequence analysis,then transformed into E.coli BL21(DE3)and expressed under the induction of IPTG.The expression product was identified by SDS-PAGE and Western blotting.The induction time,induction temperature and IPTG concentrations were optimized. Both PCR and restriction analysis proved that the recombinant plasmid was constructed correctly.The best induction condition was 37℃for 5h,whereas IPTG concentration had no significant impact on protein expression.The recombinant fusion protein pGEX-4T-1-ORF1was approximately 80ku in a form of inclusion body,which was recognized specially by mouse anti-GST monoclonal antibody.It was the first time to use cutting the gel slice's method for purification of TTV2Cap protein,which was cost-effective and convenient.Recombinant plasmid pGEX-4T-1-ORF1was constructed and the Cap protein was highly expressed in E.coli which provided antigen for indirect ELISA and preparation of monoclonal antibodies.
The study was aimed to highly express Cap protein of porcine torque teno virus type 2in E.coli,and determine the optimal expression conditions and purify it.ORF1gene was amplified by PCR and cloned into the prokaryotic expression vector pGEX-4T-1to construct recombinant plasmid pGEX-4T-1-ORF1,which was identified by restriction and sequence analysis,then transformed into E.coli BL21(DE3)and expressed under the induction of IPTG.The expression product was identified by SDS-PAGE and Western blotting.The induction time,induction temperature and IPTG concentrations were optimized. Both PCR and restriction analysis proved that the recombinant plasmid was constructed correctly.The best induction condition was 37℃for 5h,whereas IPTG concentration had no significant impact on protein expression.The recombinant fusion protein pGEX-4T-1-ORF1was approximately 80ku in a form of inclusion body,which was recognized specially by mouse anti-GST monoclonal antibody.It was the first time to use cutting the gel slice's method for purification of TTV2Cap protein,which was cost-effective and convenient.Recombinant plasmid pGEX-4T-1-ORF1was constructed and the Cap protein was highly expressed in E.coli which provided antigen for indirect ELISA and preparation of monoclonal antibodies.
引文
1 刘建波,郭龙军,危艳武,等.猪细环病毒(TTV)与猪圆环病毒混合感染的检测及TTV遗传变异分析[J].中国预防兽医学报,2011,33(1):6~10.
2 张青占,周艳君,龙进学,等.2011年—2012年中国部分地区猪细环病毒在不同猪群中的流行病学调查及其与PCV2、PPV4或PRRSV的共感染分析[J].中国预防兽医学报,2013,35(7):550 ~554.
3 李淞,朱玲,周远成.猪细环病毒2型ORF1基因的原核表达及其多克隆抗体的制备[J].中国兽医科学,2013,43(11):1162~1166.
4 南文金,娄高明,黄健强.华南地区猪输血传播病毒流行病学调查与分析[J].河南农业科学,2011,40(12):145~148.
5 南文金,娄高明,胡鸿惠.猪细环病毒流行病学及致病性研究进展[J].中国畜牧兽医,2012,39(11):186~190.
6 洪绍锋,刘磊,刘欢.广西部分地区猪细环病毒的分子流行病学调查[J].中国兽医科学,2013,43(3):325~330.
7 高慎阳,查恩辉,王珅.一种“高性价比”切胶纯化原核表达蛋白的方法[J].中国农学通报,2010,36(22):24~26.
8 Biagini P,Gallian P,Attoui H,et al.Genetic analysis of fulllength genomes and subgenomic sequences of TT virus-like mini virus human isolates[J].Journal of General Virology,2001,82 (2):379~383.
9 Ellis J A.Effect of coinfection with genogroup 1porcine torque teno virus on porcine circovirus type 2-associated postweaning multisystemic wasting syndrome in gnotobiotic pigs[J].Am J Vet Res,2008,69(12):1608~1614.
10 Hijikata M,Iwata K,Ohta Y,et al.Genotypes of TT virus(TTV)compared between liver disease patients and healthy individuals using a new PCR system capable of differentiating 1aand1 btypes from others[J].Archives of Virology,1999,144(12):2345~2354.
11 Huang Y W,Harrall K K,Dryman B A,et al.Expression of the putative ORF1capsid protein of Torque teno sus virus 2(TTSuV2)and development of Western blot and ELISA serodiagnostic assays:Correlation between TTSuV2viral load and IgG antibody level in pigs[J].Virus Research,2011,158(1):79~88.
12 Kekarainen T,Sibila M,Segalés J.Prevalence of swine Torque teno virus in post-weaning multisystemic wasting syndrome(PMWS)-affected and non-PMWS-affected pigs in Spain[J].Journal of General Virology,2006,87(4):833~837.
13 Leary X R,Erker J C,Chalmers M L,et al.Improved detection systems for TT virus reveal high prevalence in humans,non-humans primates and farm animals[J].J Gen Virol,1999,80(8):2115~2120.
14 Maggi F,Bendinelli M.Immunobiology of the Torque teno viruses and other anelloviruses[M].TT Viruses.Springer Berlin Heidelberg,2009.
15 Martínez-GuinóL,Kekarainen T,Segalés J.Evidence of Torque teno virus(TTV)vertical transmission in swine[J].Theriogenology,2009,71(9):1390~1395.
16 Mushahwar I K,Erker J C.Molecular and biophysical characterization of TT virus:Evidence for a new virus family infecting humans[J].Proc Nati Acad,1999,96(6):3177~3182.
17 Nishizawa T,Okamoto H,Konishi K,et al.A novel DNA virus(TTV)associated with elevated transaminase levels in posttransfusion hepatitis of unknown etiology[J].Biochemical and Biophysical Research Communications,1997,241(1):92~97.
18 Okamoto H,Nishizawa T,Kato N,et al.Molecular cloning and characterization of a novel DNA virus(TTV)associated with posttranfusion hepatitis of unknown etiology[J].Hepatol Res,1998,10(1):1~16.
19 Okamoto H,Takahashi M,Nishizawa T,et al.Genomic characterization of TT viruses(TTVs)in pigs,cats and dogs and their relatedness with species-specific TTVs in primates and tupaias[J].Journal of General Virology,2002,85(6):1291~1297.
20 Teixeira T F,Dezen D,Cibulski S P,et al.Torque teno sus virus(TTSuV)in tissues of pigs and its relation with the occurrence of postweaning multisystemic wasting syndrome[J].Virus Genes,2013,47(2):276~281.
2 张青占,周艳君,龙进学,等.2011年—2012年中国部分地区猪细环病毒在不同猪群中的流行病学调查及其与PCV2、PPV4或PRRSV的共感染分析[J].中国预防兽医学报,2013,35(7):550 ~554.
3 李淞,朱玲,周远成.猪细环病毒2型ORF1基因的原核表达及其多克隆抗体的制备[J].中国兽医科学,2013,43(11):1162~1166.
4 南文金,娄高明,黄健强.华南地区猪输血传播病毒流行病学调查与分析[J].河南农业科学,2011,40(12):145~148.
5 南文金,娄高明,胡鸿惠.猪细环病毒流行病学及致病性研究进展[J].中国畜牧兽医,2012,39(11):186~190.
6 洪绍锋,刘磊,刘欢.广西部分地区猪细环病毒的分子流行病学调查[J].中国兽医科学,2013,43(3):325~330.
7 高慎阳,查恩辉,王珅.一种“高性价比”切胶纯化原核表达蛋白的方法[J].中国农学通报,2010,36(22):24~26.
8 Biagini P,Gallian P,Attoui H,et al.Genetic analysis of fulllength genomes and subgenomic sequences of TT virus-like mini virus human isolates[J].Journal of General Virology,2001,82 (2):379~383.
9 Ellis J A.Effect of coinfection with genogroup 1porcine torque teno virus on porcine circovirus type 2-associated postweaning multisystemic wasting syndrome in gnotobiotic pigs[J].Am J Vet Res,2008,69(12):1608~1614.
10 Hijikata M,Iwata K,Ohta Y,et al.Genotypes of TT virus(TTV)compared between liver disease patients and healthy individuals using a new PCR system capable of differentiating 1aand1 btypes from others[J].Archives of Virology,1999,144(12):2345~2354.
11 Huang Y W,Harrall K K,Dryman B A,et al.Expression of the putative ORF1capsid protein of Torque teno sus virus 2(TTSuV2)and development of Western blot and ELISA serodiagnostic assays:Correlation between TTSuV2viral load and IgG antibody level in pigs[J].Virus Research,2011,158(1):79~88.
12 Kekarainen T,Sibila M,Segalés J.Prevalence of swine Torque teno virus in post-weaning multisystemic wasting syndrome(PMWS)-affected and non-PMWS-affected pigs in Spain[J].Journal of General Virology,2006,87(4):833~837.
13 Leary X R,Erker J C,Chalmers M L,et al.Improved detection systems for TT virus reveal high prevalence in humans,non-humans primates and farm animals[J].J Gen Virol,1999,80(8):2115~2120.
14 Maggi F,Bendinelli M.Immunobiology of the Torque teno viruses and other anelloviruses[M].TT Viruses.Springer Berlin Heidelberg,2009.
15 Martínez-GuinóL,Kekarainen T,Segalés J.Evidence of Torque teno virus(TTV)vertical transmission in swine[J].Theriogenology,2009,71(9):1390~1395.
16 Mushahwar I K,Erker J C.Molecular and biophysical characterization of TT virus:Evidence for a new virus family infecting humans[J].Proc Nati Acad,1999,96(6):3177~3182.
17 Nishizawa T,Okamoto H,Konishi K,et al.A novel DNA virus(TTV)associated with elevated transaminase levels in posttransfusion hepatitis of unknown etiology[J].Biochemical and Biophysical Research Communications,1997,241(1):92~97.
18 Okamoto H,Nishizawa T,Kato N,et al.Molecular cloning and characterization of a novel DNA virus(TTV)associated with posttranfusion hepatitis of unknown etiology[J].Hepatol Res,1998,10(1):1~16.
19 Okamoto H,Takahashi M,Nishizawa T,et al.Genomic characterization of TT viruses(TTVs)in pigs,cats and dogs and their relatedness with species-specific TTVs in primates and tupaias[J].Journal of General Virology,2002,85(6):1291~1297.
20 Teixeira T F,Dezen D,Cibulski S P,et al.Torque teno sus virus(TTSuV)in tissues of pigs and its relation with the occurrence of postweaning multisystemic wasting syndrome[J].Virus Genes,2013,47(2):276~281.