表达猪流行性腹泻病毒纤突蛋白的重组水泡性口炎病毒构建和鉴定  

Construction and identification of recombinant vesicular stomatitis virus expressing porcine epidemic diarrhea virus S protein

作　　者：柯勇 肖贤 毕波 赵秋华 方心葵[1,2] 孙涛 KE Yong;XIAO Xian;BI Bo;ZHAO Qiuhua;FANG Xinkui;SUN Tao(School of Agriculture and Biology,Shanghai Jiao Tong University,Shanghai 200240,China;Shanghai City Key Laboratory of Veterinary Science,Shanghai 200240,China;Shanghai Pig Breeding Farm,Shanghai 201408,China;Animal Disease Prevention and Control of Minhang District,Shanghai 201109,China)

机构地区：[1]上海交通大学农业与生物学院,上海200240 [2]上海市兽医生物技术重点实验室,上海200240 [3]上海市种猪场,上海201408 [4]上海市闵行区动物疫病预防控制中心,上海201109

出　　处：《畜牧与兽医》2019年第2期76-82,共7页Animal Husbandry & Veterinary Medicine

基　　金：上海市闵行区产学研合作计划项目(2016MH250);2015年度科技创新行动计划(15391901600)

摘　　要：为表达猪流行性腹泻病病毒(porcine epidemic diarrhea,PEDV)纤突蛋白,利用Mega7基于PEDV纤突蛋白氨基酸序列进行遗传进化分析,选择新发流行毒株纤突蛋白基因,以重组水泡性口炎病毒(rVSV_(MT))为载体,在rVSV_(MT)基因组G和L间插入目的基因,通过反向遗传学技术拯救VSV_(MT)-S△19重组病毒,Western blot和RT-PCR鉴定重组病毒是否成功表达PEDV纤突蛋白,采用一步生长曲线鉴定VSV_(MT)-S△19在Vero细胞中的生长特性,并与弗氏佐剂混合后肌肉注射小鼠探究其在动物体内的免疫效果。遗传进化分析发现,插入rVSV_(MT)的纤突蛋白基因序列PEDV/CHN/SH-2012-5属于PEDV新发流行毒株GⅡ型; RT-PCR分析重组病毒表达纤突蛋白基因无突变; Western blot鉴定显示出PEDV纤突蛋白和VSV病毒特征条带;一步生长曲线表明重组病毒VSV_(MT)-S△19体外复制滴度可达108TCID50/m L,与VSV_(MT)-GFP无显著性差异(P> 0. 05);小鼠动物试验结果表明,重组病毒成功激发PEDV中和抗体产生,与对照PBS接种组差异显著(P<0. 05)。研究表明,利用重组水泡性口炎病毒(rVSV_(MT))为载体表达GⅡ型PEDV纤突蛋白能有效激发机体产生高水平中和抗体,为PEDV亚单位疫苗的研制提供了理论和实践依据。In order to express the porcine epidemic diarrhea( PEDV) Spike protein,the evolutionary genetic analysis of the amino acid sequence of PEDV Spike protein by Mega 7 was used to screen out the new epidemic strain of PEDV. The recombinant vesicular stomatitis virus( r VSVMT) was used as a vector to insert the target gene between the G and L of the rVSVMTgenome. The VSVMT-S△19recombinant virus was rescued by reverse genetics,and Western and RT-PCR identification indicated that the recombinant virus successfully expressed the PEDV Spike protein. The growth characteristics of VSVMT-S△19in the Vero cells were determined by a one-step growth curve. Then,VSVMT-S△19was mixed with Freund’s adjuvant,and the mice were intramuscularly injected with the mixture to investigate their immune effects in the rodents. The results of genetic evolution analysis indicated that the Spike protein gene sequence PEDV/CHN/SH-2012-5 inserted into r VSVMT belonged to a new epidemic strain of PEDV GII-b. The RT-PCR result showed that the recombinant virus expressed the Spike protein gene without mutation. The Western-blot result exhibited the characteristic bands the PEDV Spike protein and the VSV virus. The one-step growth curve indicated that the in vitro replication titer of the recombinant virus reached 108TCID50/m L. The results of the experiments on the mice showed that the recombinant virus successfully stimulated the production of PEDV neutralizing antibody. In summary,the expression of GIItype PEDV Spike protein by recombinant vesicular stomatitis virus( rVSVMT) effectively stimulated the body to produce high levels of neutralizing antibodies,which provided a theoretical and practical basis for development of the subunit vaccine of a porcine epidemic diarrhea virus.

关 键 词：水泡性口炎病毒 猪流行性腹泻病 S蛋白 

分 类 号：S852.6[农业科学—基础兽医学]