草鱼呼肠孤病毒GCRV 096 VP7蛋白的表达及免疫原性  

Expression and Immunogenicity of VP7 Protein in Grass Carp Reovirus(GCRV) 096 Strain

作　　者：杨硕 陈静妮 胡海浩 王雅 闫秀英[1] 简纪常[1] YANG Shuo;CHEN Jing-ni;HU Hai-hao;WANG Ya;YAN Xiu-ying;JIAN Ji-chang(Guangdong Key Laboratory of Pathogenic Biology and Epidemiology for Aquatic Economic Animals,and Key Laboratory of Control for Diseases of Aquatic Economic Animals of Guangdong Higher Education Institutes,Fisheries College of Guangdong Ocean University,Guangdong Ocean University,Zhanjiang 524088,China)

机构地区：[1]广东海洋大学水产学院//广东省水产经济动物病原生物学及流行病学重点实验室//水产经济动物病害防控广东普通高校重点实验室,广东湛江524088

出　　处：《广东海洋大学学报》2020年第4期1-6,共6页Journal of Guangdong Ocean University

基　　金：国家自然科学基金(31602199);广东省自然科学基金(2015A030313622);湛江市科技计划项目(2015A03027);广东海洋大学校级项目(K15246)和973项目(2009CB118704)。

摘　　要：【目的】研究草鱼呼肠孤病毒(grass carp reovirus, GCRV)096 vp7基因的原核和真核表达及其编码蛋白的免疫原性。【方法】应用RT-PCR技术获得GCRV 096 vp7基因,构建GCRV 096 vp7基因原核表达载体pET-VP7,用原核表达的GCRV 096 VP7蛋白免疫草鱼(Ctenopharyngodon idellus)15 d后,用GCRV 096和GCRV GD108分离株对其攻毒,检测GCRV 096 VP7的免疫原性。构建GCRV 096 vp7基因真核表达载体p EGFP-N3-VP7,分析其在草鱼CIK细胞中的表达。【结果与结论】GCRV 096 vp7基因的开放阅读框ORF (GenBank登录号为JN206665)为831 bp,编码276个氨基酸。成功构建GCRV 096 vp7基因原核表达载体pET-VP7,并在大肠杆菌BL21中诱导表达成功;最优表达条件是0.2 mm/L IPTG、28℃下表达5 h,通过HisTrap HP柱纯化融合蛋白,Western blot分析结果表明,所表达的蛋白为目的蛋白。经免疫及GCRV攻毒后,GCRV 096(Ⅰ型)免疫组vp7基因的表达水平降低(P <0.05),而GCRV GD108(Ⅱ型)免疫组vp7基因的表达无显著差异,表明GCRV 096 VP7蛋白对GCRV096具有良好的免疫效果,但对GCRVGD108无明显的免疫效果。成功构建GCRV096vp7基因真核表达载体pEGFP-N3-VP7,Western blot分析结果表明,GCRV 096 VP7蛋白在草鱼肾(CIK)细胞中成功表达。【Objective】To study the prokaryotic and eukaryotic expression of the vp7 gene of grass carp reovirus(GCRV)096 strain,and the coded protein’s immunogenicity.【Method】The Open Reading Frame(ORF)of GCRV 096 vp7 gene was acquired by RT-PCR.The prokaryotic expression vector pET-VP7 of GCRV 096 vp7 gene was structured.Grass carp(Ctenopharyngodon idellus)was challenged with GCRV 096 or GCRV GD108 after 15 days immunization with the expressed fusion GCRV 096 VP7 protein,and the immunogenicity of GCRV 096 VP7 protein was analyzed.Furthermore,the eukaryotic expression vector pEGFP-N3-VP7 of GCRV 096 vp7 gene was structured,and the expression of GCRV096 vp7 gene in CIK cells was analyzed.【Result and Conclusion】The ORF of GCRV 096 vp7 gene(Gen Bank accession number:JN206665)was 831 bp encoding for a protein with 276 amino acid residues.The prokaryotic expression vector pET-VP7 of the GCRV 096 vp7 gene was constructed successfully,and the GCRV 096 VP7 protein was expressed successfully transformed into Escherichia coli BL21.The optimal expression condition was 0.2 mmol/L IPTG at 28℃for 5 hours.Then the expressed fusion GCRV 096 VP7 protein was purified with the His Trap HP purification column.Western blot result showed that the recombinant VP7 was of expected size.After the grass carp was immunized and challenged,the expression level of GCRV 096 vp7 gene decreased(P<0.05)in the immunized group(GCRV 096,genotype I)compared to the control group;but the expression level of GCRV GD108 vp7 gene had no significant difference between the immunization group(GCRV GD108,genotypeⅡ)and the control group.The results indicated that the recombinant protein had good immunogenicity for GCRV 096 but with no obvious immune effect for GCRV GD108.Moreover,eukaryotic expression vector p EGFP-N3-VP7 of GCRV 096 vp7 gene was structured successfully.The results of western blot showed that GCRV 096 VP7 protein was expressed successfully in CIK cells.

关 键 词：草鱼呼肠孤病毒 VP7基因 表达 免疫原性 

分 类 号：Q78[生物学—分子生物学] Q959.46