汉滩病毒 A9 株M基因片段在重组痘苗病毒中的表达及鉴定

EXPRESSION OF HANTAAN VIRUS A9 M GENOME SEGMENT BY USING A VACCINIA VIRUS RECOMBINANT

为发展国产化肾综合征出血热（ＨＦＲＳ）病毒基因工程疫苗，选择了汉滩病毒Ａ９株Ｍ基因片段为目的基因，构建转染质粒ｐＪＳＢＡ９Ｍ。以携带Ｌａｃ基因的重组病毒为亲本，使表达载体ｐＪＳＢ－Ａ９Ｍ上的Ｍ片段与痘苗病毒内的Ｌａｃ基因重组，将Ｌａｃ置换成Ａ９Ｍ片段。用蓝白斑法筛选重组痘苗病毒，经ＰＣＲ扩增证实Ａ９Ｍ片段重组入痘苗病毒基因组内。重组痘苗病毒感染的ＶｅｒｏＥ６细胞，用抗糖蛋白单克隆抗体（ＨＣＯ２、１１Ｅ１０、３Ｄ５、１６Ｄ２）间接免疫荧光法检测，呈阳性反应；放射免疫沉淀法（ＲＩＰ）证实，表达产物可与抗Ｇ２的糖蛋白单克隆抗体出现特异性沉淀带；间接免疫荧光染色法检测表明，重组病毒免疫Ｂａｌｂ／ｃ小鼠的血清与Ａ９株感染的ＶｅｒｏＥ６细胞有较好的特异性反应（１：３２０），说明Ａ９Ｍ片段在痘苗中表达成功。

In order to develope genetically engineered Chinese vaccine of HFRS,the hantaan virus A9 M genome segment of Chinese isolate was chosen as the target gene.The plasmid pJSBA9M,containing the M clone segment,was constructed and controlled by the promoter P11,and also was proved by restriction enzyme digestion.This expression plasmid pJSBA9M was transfected using lipofectin reagent into Vero cells,which were superinfected with the vaccinia virus containing the Lac Z gene in the TK region.The recombinant vaccinia viruses were isolated by plaque assay of transfected-cell lysate on Vero cells with 1% low-melting-point agarose overlay containing 300μg of X-gal per ml.White plaques were removed and subjected to six round plaque purification.From 16 white plaques,only one was identified as the recombinant vaccinia virus by PCR.In addition,immunofluorescent assay(IFA)and Radio-immunoprecipitation(RIP)were used to identify the products of the recombinant vaccinia virus.Furthermore,the neutralizing antibody against hantaan virus with the titer of 1:320 in sera of mice inoculated with the recombinant vaccinia virus was detected.All the above results proved the expression of A9M segment by the recombinant vaccinia virus, and it has great potential to be used as the vaccine against HFRS.