草鱼呼肠孤病毒HZ08株外衣壳蛋白VP4的节段克隆表达和免疫学分析
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摘要
目的原核表达草鱼呼肠孤病毒(GCRV)HZ08株S6基因特定保守区域编码的外衣壳蛋白VP4并探究其免疫原性,为草鱼出血病(GCHD)防治提供新思路。方法将VP4基因特定保守区域克隆到原核表达质粒p ET-32a(+)中,诱导表达并用镍柱进行纯化,用纯化的目的蛋白免疫SD大鼠获取免疫血清;应用ELISA检测实验组(重组蛋白皮下免疫大鼠)和对照组(PBS免疫大鼠)血清Ig G抗体滴度及亚类的水平;应用间接免疫荧光实验(IFA)检测r VP4抗血清对病毒的识别情况。结果PCR、双酶切及DNA测序结果均表明p ET32a-VP4重组质粒构建成功;SDSPAGE结果表明目的基因在大肠杆菌BL21/DE3中获得高效表达,融合蛋白分子量约为43 k Da,且在37℃、1mmol/L IPTG条件下诱导表达效果最优;经亲和层析获得了高纯度蛋白;免疫大鼠6周后血清Ig G抗体滴度高达1∶819 200,Ig G1、Ig G2a水平差异无统计学意义(P>0.05);IFA结果显示抗血清组特异性荧光显著,而对照组未见荧光。结论 GCRV-HZ08 VP4具有良好的免疫原性,可诱导大鼠产生Th1/Th2混合型免疫反应,且r VP4抗血清可识别该病毒,为VP4作为疫苗候选分子提供了依据。
Objective To study the prokaryotic expression of outer capsid protein VP4 encoded by grass carp reovirus(GCRV) HZ08 strain conserved regions of S6 gene and the immunogenicity of the recombinant protein, and provide new ideas for prevention and control of the grass Carp hemorrhagic disease(GCHD). Method VP4 gene was cloned to prokaryotic vector p ET32a(+) and transformed into E.coli BL21 / DE3. The recombinant VP4(r VP4) protein was purified by Ni2+ affinity chromatography column, and was then used to immune rats for antiserum preparation. Titer and isotype of rat anti-r VP4 Ig G was tested and analyzed by ELISA. The antiserum was then identified by immunofluorescence.Result PCR, double enzyme digestion and DNA sequencing results showed that recombinant plasmid p ET32a-VP4 was successfully constructed. SDS-PAGE results showed that the molecular weight of the fusion protein was about 43 k Da. The optimal condition for the expression in E.coli BL21 / DE3 was at 37℃ and with 1 mmol / L IPTG. The titer of rat antir GCRV-HZ08 VP4 Ig G was up to 1 ∶819 200 at 6 weeks after the immunization, and Ig G1, Ig G2 a levels showed no statistical difference(P >0.05) from 2 to 6 week. Immunofluorescence result showed specific staining in test group, while not in the negative control group. Conclusion GCRV-HZ08 VP4, with good immunogenicity, could induce combined Th1 / Th2 immune response in rats and anti-r VP4 serum could recognize GCRV. This study provided evidence for the utilizing of VP4 as an effective vaccine candidate for grass carp hemorrhagic disease.
Objective To study the prokaryotic expression of outer capsid protein VP4 encoded by grass carp reovirus(GCRV) HZ08 strain conserved regions of S6 gene and the immunogenicity of the recombinant protein, and provide new ideas for prevention and control of the grass Carp hemorrhagic disease(GCHD). Method VP4 gene was cloned to prokaryotic vector p ET32a(+) and transformed into E.coli BL21 / DE3. The recombinant VP4(r VP4) protein was purified by Ni2+ affinity chromatography column, and was then used to immune rats for antiserum preparation. Titer and isotype of rat anti-r VP4 Ig G was tested and analyzed by ELISA. The antiserum was then identified by immunofluorescence.Result PCR, double enzyme digestion and DNA sequencing results showed that recombinant plasmid p ET32a-VP4 was successfully constructed. SDS-PAGE results showed that the molecular weight of the fusion protein was about 43 k Da. The optimal condition for the expression in E.coli BL21 / DE3 was at 37℃ and with 1 mmol / L IPTG. The titer of rat antir GCRV-HZ08 VP4 Ig G was up to 1 ∶819 200 at 6 weeks after the immunization, and Ig G1, Ig G2 a levels showed no statistical difference(P >0.05) from 2 to 6 week. Immunofluorescence result showed specific staining in test group, while not in the negative control group. Conclusion GCRV-HZ08 VP4, with good immunogenicity, could induce combined Th1 / Th2 immune response in rats and anti-r VP4 serum could recognize GCRV. This study provided evidence for the utilizing of VP4 as an effective vaccine candidate for grass carp hemorrhagic disease.
引文
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[17]He YX,Yang Q,Xu HX,et al.Prokaryotic expression and purification of grass carp reovirus capsid protein VP7 and its vaccine potential[J].African Journal of Microbiology Research Vol,2011,5(13):1643-1648.
[18]Lu L,Xu H,He Y,et al.Protection of grass carp,Ctenopharyngon idellus(Valenciennes),through oral administration of a subunit vaccine against reovirus[J].Journal of Fish Diseases,2011,34(12):939-942.
[19]刘林,徐诗英,李婧慧,等.草鱼出血病病毒VP6蛋白的原核表达、纯化及免疫效果[J].水产学报,2012,36(3):429-435.Liu L,Xu SY,Li JH,et al.Prokaryotic expression,purification and immune efficacy of VP6proteinof grasscarpreovirus[J].Journal of Fisheries of China,2012,36(3):429-435.
[20]徐诗英,刘林,李婧慧,等.草鱼呼肠孤病毒VP7基因核酸疫苗的构建及免疫效果[J].水产学报,2011,35(11):1694-1700.Xu SY,Liu L,Li JH,et al.Construction and immune efficacy studies on DNA vaccine with VP7 gene of grass carp reovirus[J].Journal of Fisheries of China,2011,35(11):1694-1700.
[2]孙恒昌,陈庭金,黄艳,等.鱼用疫苗研究进展[J].热带医学杂志,2014,14(12):1651-1656.Sun HC,Chen TJ,Huang Y,et al.Advances in fish vaccine research[J].J Trop Med,2014,14(12):1651-1656.
[3]Essbauer S,Ahne W.Viruses of low vertebrates[J].J Vet Med B Infect Dis Vet Public Health,2001,48(6):403-475.
[4]方勤,朱作言.水生呼肠孤病毒研究进展[J].中国病毒学,2003,18(1):82-86.Fang Q,Zhu ZY.Advances in aquareovirus research[J].Virologica Sinica,2003,18(1):82-86.
[5]迟妍妍.南方养殖草鱼呼肠孤病毒分子特性的分析及VP4重组蛋白免疫效果初步研究[D].上海:上海海洋大学,2011.Chi YY.Molecular characterization of GCRV in Southern China and immune effect of the VP4 recombinant protein[D].Shanghai:Shanghai Ocean University,2011.
[6]刘林.草鱼出血病病毒vp6核酸疫苗和亚单位疫苗的免疫效果研究[D].苏州:苏州大学,2012.Liu L.Evaluation of immune efficacy for GCRV vp6 DNAvaccine and subunit vaccine[D].Suzhou:Suzhou University,2012.
[7]王杭军,叶星,田园园,等.草鱼呼肠孤病毒GCRV-GD108株VP5蛋白功能及免疫原性分析[J].水产学报,2013,37(1):109-116.Wang HJ,Ye X,Tian YY,et al.Analysis of function and immunogenicity of GCRV-GD108 VP5[J].Journal of Fisheries of China,2013,37(1):109-116.
[8]李永刚,曾伟伟,王庆,等.草鱼呼肠孤病毒HZ08株S10编码蛋白多克隆抗体制备及其特性分析[J].水产学报,2014,38(3):449-456.Li YG,Zeng WW,Wang Q,et al.Preparation and characteristics of polyclonal antibody against S10encoded protein of grass carp reovirus HZ08 strain[J].Journal of Fisheries of China,2014,38(3):449-456.
[9]王方华,李安兴.草鱼病毒性出血病研究进展[J].南方水产,2006,2(3):66-71.Wang FH,Li AX.Advances in research of hemorrhage of grass carp[J].South China Fisheries Science,2006,2(3):66-71.
[10]Zeng WW,Wang Q,Wang YY,et al.A one-step molecular biology method for simple and rapid detection of grass carp Ctenopharyngodon idella reovirus(GCRV)HZ08 strain[J].JFish Biol,2013,82(5):1545-1555.
[11]Wang Q,Zeng W,Liu C,et al.Complete Genome Sequence of a Reovirus Isolated from Grass Carp,Indicating Different Genotypes of GCRV in China[J].J Virol,2012,86(22):12466.
[12]Freire JM,Santos NC,Veiga AS,et al.Rethinking the capsid proteins of enveloped viruses:multifunctionality from genome packaging to genometransfection[J].FEBS J,2015,282(12):2267-2278.
[13]曾伟伟,王庆,张乐生,等.草鱼呼肠孤病毒HZ08株VP4蛋白的原核表达、纯化及多克隆抗体的制备[J].中国生物制品学杂志,2011,24(12):1482-1485.Zeng WW,Wang Q,Zhang LS,et al.Prokaryotic Expression and purification of VP4 protein of grass carp reovirus HZ08strain and preparation of polyclonal antibody[J].Chin JBiologicals,2011,24(12):1482-1485.
[14]Azouzou N,Petrofsky M,Yong LS,et al.Mycobacterium avium infection in mice is associated with time-related expression of Th1 and Th2 CD4 T-lymphocyte response[J].Immunology,1997,91(3):414-420.
[15]Fang Q,Seng EK,Dai W,et al.Construction and coexpression of grass carp reovirus VP6 protein and enhanced green fluorescence protein in the insect cells[J].Virologica Sinica,2007,22(5):397-404.
[16]He YX,Xu HX,Yang Q,et al.The use of an in vitro microneutralization assay to evaluate thepotential of recombinant VP5 protein as an antigen for vaccinating against grass carp reovirus[J].Virol J,2011,8(1):132.
[17]He YX,Yang Q,Xu HX,et al.Prokaryotic expression and purification of grass carp reovirus capsid protein VP7 and its vaccine potential[J].African Journal of Microbiology Research Vol,2011,5(13):1643-1648.
[18]Lu L,Xu H,He Y,et al.Protection of grass carp,Ctenopharyngon idellus(Valenciennes),through oral administration of a subunit vaccine against reovirus[J].Journal of Fish Diseases,2011,34(12):939-942.
[19]刘林,徐诗英,李婧慧,等.草鱼出血病病毒VP6蛋白的原核表达、纯化及免疫效果[J].水产学报,2012,36(3):429-435.Liu L,Xu SY,Li JH,et al.Prokaryotic expression,purification and immune efficacy of VP6proteinof grasscarpreovirus[J].Journal of Fisheries of China,2012,36(3):429-435.
[20]徐诗英,刘林,李婧慧,等.草鱼呼肠孤病毒VP7基因核酸疫苗的构建及免疫效果[J].水产学报,2011,35(11):1694-1700.Xu SY,Liu L,Li JH,et al.Construction and immune efficacy studies on DNA vaccine with VP7 gene of grass carp reovirus[J].Journal of Fisheries of China,2011,35(11):1694-1700.