猫疱疹病毒Ⅰ型gIgE基因缺失毒株的构建及生物学特性分析

The construction and the biological characteristics of feline herpesvirusⅠgIgE gene deletion strain

[目的]为了开发一种能够预防感染、阻止潜伏期建立的猫疱疹病毒Ⅰ型(FHV-1)弱毒疫苗,利用同源重组以及CRISPR/Cas9技术将FHV-1的gIgE基因替换为红色荧光蛋白基因(RFP)。[方法]构建针对gIgE基因的3条sgRNA及表达Cas9蛋白的质粒,将含有sgRNA及Cas9蛋白的质粒与含有RFP的转移载体共转染到猫肾细胞(F81),通过噬斑纯化以及有限稀释方法进行病毒纯化,以重组病毒感染F81细胞连续10代验证其遗传稳定性,通过测定重组病毒以及亲本病毒H07的病毒滴度、一步生长曲线以及判断噬斑大小评价其生物学特性。[结果]成功构建了FHV-1 gIgE基因缺失毒株并将其命名为FHV-ΔgIgE-RFP;提取重组病毒DNA,通过PCR验证gIgE基因无目的条带,表明重组病毒纯化成功,再通过PCR验证RFP基因有目的条带,表明重组病毒成功插入外源基因。病毒滴度测定结果显示,与亲本病毒相比重组病毒的病毒滴度下降了90%;在F 1—F 10连续传代过程中RFP荧光表达且无减弱现象,表明重组病毒可稳定表达外源基因;测定FHV-ΔgIgE-RFP以及H07的生长曲线,重组病毒的生长趋势与亲本病毒大致相同,表明插入外源基因并不会改变病毒本身的生长特性;噬斑观察及染色结果显示,在病毒感染细胞前、后期,重组病毒的噬斑都小于亲本病毒,表明缺失gIgE基因后病毒毒力下降。[结论]成功构建并筛选出FHV-1 gIgE基因缺失致弱毒株,为开发疱疹病毒弱毒活载体疫苗奠定了基础。

[Objectives]To develop a attenuated feline herpesvirusⅠ(FHV-1)vaccine that could prevent infection and prevent latency establishment,the gIgE gene of FHV-1 was replaced with a red fluorescent protein gene(RFP)using homologous recombination and CRISPR/Cas9 technology.[Methods]Three sgRNA for gIgE gene and plasmids expressing Cas9 protein were constructed,and the plasmids containing sgRNA and Cas9 protein and the transfer vector containing RFP were co-transfected into cat kidney cells(F81),and the virus was purified by plaque purification and limited dilution method.F81 cells were infected with recombinant virus for 10 successive generations to verify its genetic stability,and its biological characteristics of recombinant virus and parent virus H07 were evaluated by measuring the virus titer,one-step growth curve and judging the size of plaque.[Results]The FHV-1 gIgE gene deletion strain was successfully constructed and named FHV-ΔgIgE-RFP;the recombinant DNA was extracted and PCR was used to verify that gIgE gene had no target band,indicating that the recombinant virus was successfully purified.Then PCR was used to verify that RFP gene had target band,indicating that the recombinant virus had successfully inserted foreign genes.The virus titer of recombinant virus decreased by 90%compared with that of parent virus;the fluorescence expression of RFP was observed in the continuous passage of F 1-F 10,which showed that the recombinant virus could stably express foreign genes;the growth curves of FHV-ΔgIgE-RFP and H07 were determined,the growth trend of recombinant virus was roughly the same as that of parent virus,indicating that the insertion of foreign genes did not change the growth characteristics of the virus itself;the plaque observation and staining results showed that the plaque of recombinant virus was smaller than that of parent virus in both early and late stages of virus infection,indicating that the virulence of the virus decreased after the deletion of gIgE gene.[Conclusions]The FHV-1 gIgE gene deletion attenuated strain was successfully constructed and screened in this study,which laid a foundation for the development of herpes virus attenuated live vector vaccine.