杨硕,陈静妮,胡海浩,王雅,闫秀英,简纪常

• 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

摘要(Abstract)：

【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。

关键词(KeyWords)： 草鱼呼肠孤病毒;vp7基因;表达;免疫原性

Abstract:

Keywords:

基金项目(Foundation): 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);; 973项目(2009CB118704)

作者(Author): 杨硕,陈静妮,胡海浩,王雅,闫秀英,简纪常

参考文献(References)：

• [1]YE X,TIAN Y Y,DENG G C,et al.Complete genomic sequence of a reovirus isolated from grass carp in China[J].Virus Research,2012,163(1):275-283.
• [2]YAN X Y,WANG Y,XIONG L F,et al.Phylogenetic analysis of newly isolated grass carp reovirus[J].Springer Plus,2014,3(4):190-199.
• [3]WANG Q,ZENG W W,LIU C,et al.Complete genome sequence of a reovirus isolated from grass carp,indicating different genotypes of GCRV in China[J].Journal of Virology,2012,86(22):12466.
• [4]黄毅昌,雷燕,杨玉滔,等.草鱼出血病分子流行病学及GCRV多样性研究[J].安徽农业科学,2016,44(11):120-125.
• [5]TANG Y F,ZENG W W,WANG Y Y,et al.Comparison of the blood parameters and histopathology between grass carp infected with a virulent and avirulent isolates of genotype II grass carp reovirus[J].Microb Pathog,2020.doi:10.1016/j.micpath.2019.103859.
• [6]FAN Y D,RAO S J,ZENG L B,et al.Identification and genomic characterization of a novel fish reovirus,Hubei grass carp disease reovirus,isolated in 2009 in China[J].Journal of General Virology,2013,94(Pt 10):2266-2277.
• [7]李永刚,曾伟伟,王庆,等.草鱼呼肠孤病毒分子生物学研究进展[J].动物医学进展,2013,3(4):97-103.
• [8]张盛权.注射草鱼出血病疫苗免疫效果的观察报告[J].中国水产,2006(7):85-86.
• [9]JIANG Y L.Hemorrhagic disease of grass carp:Status of outbreaks,diagnosis,surveillance,and research[J].The Israeli Journal of Aquaculture-Bamidgeh,2009,61(3):188-192.
• [10]FANG Q,SENG E K,DING Q Q,et al.Characterization of infectious particles of grass carp reovirus by treatment with proteases[J].Arch Virology,2008,153(2):675-682.
• [11]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.
• [12]HE Y,YANG Q,XU H X,et al.Prokaryotic expression and purification of grass carp reovirus capsid protein VP7 and its vaccine potential[J].African Journal of Microbiology Research,2011,5(13):1643-1648.
• [13]LUO S X,YAN L M,ZHANG X H,et al.Yeast surface display of capsid protein VP7 of grass carp reovirus:Fundamental investigation for the development of vaccine against hemorrhagic disease[J].J Microbiol Biotechnol,2015,25(12):2135-2145.
• [14]ZHU B,LIU G L,GONG Y X,et al.Protective immunity of grass carp immunized with DNA vaccine encoding the vp7 gene of grass carp reovirus using carbon nanotubes as a carrier molecule[J].Fish Shellfish Immunol,2015,42(2):325-34.
• [15]韦先超,杨泽晓,王印,等.草鱼出血病病毒VP7蛋白基因的人工合成与原核表达[J].海洋与湖沼,2013,44(3):645-650.
• [16]HAO K,CHEN X H,QI X Z,et al.Display of GCRVvp7 protein on the surface of Escherichia coli and its immunoprotective effects in grass carp(Ctenopharyngodon idella)[J].Fish Shellfish Immunology,2018,72:199-209.
• [17]郝贵杰,潘晓艺,姚嘉赟,等.草鱼呼肠孤病毒衣壳蛋白VP7基因真核表达载体p CI-VP7的构建及鉴定[J].水产学报,2010,34(5):807-813.
• [18]闫秀英,郭定利,郑树城,等.草鱼呼肠孤病毒096vp4基因生物信息学分析及其在肾细胞中的表达[J].广东海洋大学学报,2015,35(4):56-62.
• [19]YAN X Y,XIE J G,LI J,et al.Screening and analysis on the protein interaction of the protein VP7 in grass carp reovirus[J].Virus Genes,2015,50(3):425-33.
• [20]YAN X Y,XIONG L F,LI J,et al.GCRV 096 VP6protein and its impacts on GCRV replication with different genotypes in CIK cells[J].Aquaculture and Fisheries,2018,3(5):184-190.
• [21]郑树城,谢吉国,王雅,等.草鱼呼肠孤病毒096 vp7基因生物信息学分析及其酵母表达载体的构建[J].广东海洋大学学报,2014,34(6):31-37.
• [22]KULESHOV M V,JONES M R,ROUILLARD A D,et al.Enrichr:a comprehensive gene set enrichment analysis web server 2016 update[J].Nucleic Acids Research,2016,44(W1):W90-97.
• [23]WANG T,LI J L,LV L Q.Quantitative in vivo and in vitro characterization of co-infection by two genetically distant grass carp reoviruses[J].Journal of General Virology,2013,94(Pt 6):1301-1309.
• [24]HE L B,ZHANG A D,PEI Y Y,et al.Differences in responses of grass carp to different types of grass carp reovirus(GCRV)and the mechanism of hemorrhage revealed by transcriptome sequencing[J].BMCGenomics,2017,18(1):452.
• [25]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):67-71.
• [26]陈丛琳,孙小云,廖兰杰,等.利用抗草鱼Ig M的单链抗体分析草鱼呼肠孤病毒的免疫原性[J].中国科学：生命科学,2012,42(12):986-992.
• [27]刘世旭,王庆,常藕琴,等.基因Ⅰ型草鱼呼肠孤病毒VP7蛋白合成肽抗体的制备及应用[J].南方农业学报,2018,49(9):1849-1857.
• [28]MA J,ZENG L B,FAN Y D,et al.Significant inhibition of two different genotypes of grass carp reovirus in vitro using multiole shRNAs expression vectors[J].Virus Research,2014,189:47-55.