SFTS病毒非结构蛋白重组质粒的构建及其体液免疫原性研究

Construction of the recombinant plasmids encoding nonstructural proteins of SFTS virus and immunogenicity study

目的 研究SFTS病毒（SFTS virus,SFTSV）的非结构蛋白（Nonstructural proteins,NSs）重组质粒的体液免疫原性.方法 利用聚合酶链反应法（Polymerase chain reaction,PCR）构建真核表达质粒pJW4303-NSs、pJW4303-NSs-opt、pJW4303-NSs-Flag;引入Nhe Ⅰ酶切位点及tPA（Tissue plasminogen activator,tPA）信号肽,构建质粒pJW4303-tPA-NSs-opt.将质粒转染至HEK 293T细胞,蛋白质印迹法（Western blot,WB）验证NSs表达.NSs相关质粒免疫BALB/c小鼠,采用酶联免疫吸附法（Enzyme-linked immunosorbent assay,ELISA）检测SFTSV NSs特异性IgG抗体.结果 成功构建了重组NSs质粒,WB验证了NSs可在HEK293T细胞内表达.pJW4303-NSs、pJW4303-NSs-opt、pJW4303-tPA-NSs-opt重组质粒均可诱导小鼠体内产生特异性IgG,其中单优化型质粒pJW4303-NSs-opt的免疫原性较好,在第6、8周的IgG水平显著高于野生型NSs质粒,差异具有统计学意义.结论 本研究构建的SFTSV的NSs重组优化型质粒具有良好体液免疫原性,其中单优化型效果最佳.

Objective To study the construction and humoral immunogenicity of the recombinant plasmids encoding nonstructural proteins（NSs） of severe fever with thrombocytopenia syndrome（SFTS） virus （SFTSV）.Methods Recombinant plasmids encoding NSs gene and optimized NSs gene of SFTSV were constructed by polymerase chain reaction （PCR） and cloned into eukaryotic expression vector pJW4303,which were named pJW4303-NSs and pJW4303-NSs-opt,respectively.Then,the plasmid pJW4303-NSs was tagged with Flag,named pJW4303-NSs-Flag.Meanwhile,Nhe Ⅰ restrict site and tissue plasminogen activator （tPA） signal sequence were inserted to construct bi-optimized recombinant plasmid named pJW4303-tPA-NSs-opt.All plasmids were identified by sequencing.The transient expression of NSs was confirmed by Western blotting in human embryonic kidney 293T cells.The NSs-specific IgG antibodies in BALB/c mice which were immunized by intramuscular injection with electroporation were examined by enzyme-linked immunosorbent assay （ELISA）.Results The recombinant plasmids pJW4303-NSs,pJW4303-NSs-opt,pJW4303-tPA-NSs-opt and pJW4303-NSs-Flag were successfully constructed.The expression of NSs was confirmed in lysates and supernatants of 293T cells.The NSs-specific IgG responses of all three recombinant plasmids were detected by ELISA in BALB/c mice.It was found that optimized recombinant plasmid pJW4303-NSs-opt elicited higher levels of the NSs-specific IgG than that of pJW4303-NSs in week 6,8 which induced stronger immune response.Conclusions The recombinant plasmids encoding SFTSV NSs possess the satisfied immunogenicity.In addition,the plasmid pJW4303-NSs-opt could induce the strongest humoral immune response.