摘要
试验旨在构建能高效表达非洲猪瘟病毒(African swine fever virus,ASFV)p54蛋白的重组伪狂犬病病毒(Pseudorabies virus,PRV)。通过无缝克隆构建含有PRV TK基因同源臂和绿色荧光蛋白(GFP)报告基因的PRV通用转移载体pCAGIG-TK_((l+r)),参考China/2018/AnhuiXCGQ株基因序列,优化合成E183L基因,并连入通用转移载体,构建重组中间转移质粒pCAGIG-TK_((l+r))-p54;重组质粒ScaⅠ酶切线性化后经脂质体介导转染BHK-21细胞,6 h后感染0.1个感染复数(MOI)PRV变异株,出现病变后通过空斑挑选和PCR鉴定纯化重组PRV,进一步通过Western blotting和间接免疫荧光试验(IFA)检测外源蛋白表达,并对重组病毒的遗传稳定性和增殖特性进行研究。结果显示,转染重组质粒pCAGIG-TK_((l+r))-p54的BHK-21细胞24 h后可观察到绿色荧光,说明重组质粒成功转入BHK-21细胞;纯化后的重组病毒表达绿色荧光蛋白且含有ASFV E183L基因,Western blotting和IFA结果证实重组PRV在BHK-21细胞中能表达p54外源蛋白,在26 ku处呈现特异性条带,且能与ASFV阳性血清发生特异性反应,免疫原性较好;重组病毒在BHK-21细胞上连续传代20次均能检测到ASFV E183L基因,遗传稳定性较好;一步生长曲线显示,重组病毒与亲本病毒的增殖特性差异不大,且二者的最高病毒滴度分别为10^(7.34)/0.1 mL和10^(7.61)/0.1 mL,外源片段的插入不影响重组病毒在细胞上的增殖能力。本研究成功获得了表达ASFV p54蛋白的重组病毒rPRV-p54,为进一步研究p54蛋白的免疫原性及开发非洲猪瘟多基因重组PRV载体疫苗奠定了基础。
The objective of this study was to construct a recombinant Pseudorabies virus(PRV)with high expression of the p54 protein of African swine fever virus(ASFV).A universal transfer vector pCAGIG-TK_((l+r))containing homologous arm of PRV TK gene and green fluorescent protein(GFP)reporter gene was constructed by seamless cloning.The E183L gene was optimized and synthesized according to the gene sequence of China/2018/AnhuiXCGQ strain,and inserted into the universal transfer vector to construct the recombinant intermediate transfer plasmid pCAGIG-TK_((l+r))-p54.The recombinant plasmid was linearized by ScaⅠenzyme and then transfected into BHK-21 cells by lipofectamine.6 h later,0.1 MOI of PRV was infected.After the lesion appeared,the recombinant PRV was purified by plaque selection and PCR.Furthermore,the expression of foreign protein was detected by Western blotting and indirect immunofluorescence assay(IFA),and the genetic stability and proliferation characteristics of the recombinant virus were studied.The results showed that green fluorescence could be observed in BHK-21 cells transfected with pCAGIG-TK_((l+r))-p54 after 24 h,indicating that the recombinant plasmid was successfully transferred into BHK-21 cells.The purified recombinant virus expressed green fluorescent protein and contained ASFV E183L gene.Western blotting and IFA results showed that the recombinant PRV could express the p54 protein in BHK-21 cells,which showed a specific band at 26 ku,and had a specific reaction with ASFV positive serum,showing good immunogenicity.The ASFV E183L gene could be detected in BHK-21 cells after 20 successive passages,showing good genetic stability.One-step growth curve showed that there was no significant difference in the proliferation characteristics between the recombinant virus and the parental virus,and their highest viral titers were 10^(7.34)/0.1 mL and 10^(7.61)/0.1 mL,respectively.The insertion of foreign fragments did not affect the proliferation ability of the recombinant virus in cells.In this study,rec
作者
何兴林
邹忠
龚文孝
张宇飞
李成飞
徐婷
陈焕春
金梅林
HE Xinglin;ZOU Zhong;GONG Wenxiao;ZHANG Yufei;LI Chengfei;XU Ting;CHEN Huanchun;JIN Meilin(State Key Laboratory of Agricultural Microbiology,College of Veterinary Medicine,Huazhong Agricultural University,Wuhan 430070,China;Wuhan Keqian Biological Limited Company,Wuhan 430070,China)
出处
《中国畜牧兽医》
CAS
北大核心
2021年第11期4182-4191,共10页
China Animal Husbandry & Veterinary Medicine
基金
湖北省技术创新专项重大项目“非洲猪瘟防控关键技术研究与示范”(2019ABA089)。
