作　　者： (张锦鹏）; (张冠文）; (宣国云）; (孟强）; (谢文宇）; (高宏）; (姜东伯）; (杨琨）;

机构地区： 第四军医大学基础部免疫学教研室,陕西西安710032 第四军医大学学员旅,陕西西安710032

出　　处： 《转化医学电子杂志》 2017年第8期36-40,共5页

摘　　要： 目的:利用p VAX1真核表达载体构建汉滩病毒糖蛋白Gn及Gc与人溶酶体相关膜蛋白LAMP分子的重组真核表达载体,鉴定其在人真核细胞系He La中的表达.大量抽提去内毒素质粒,皮下注射Balb/c小鼠后初步对目标载体作为基因疫苗进行安全性评价.方法:利用PCR的方法分别以汉滩病毒编码糖蛋白M片段的c DNA为模板,扩增目的基因Gn和Gc,构建重组真核表达载体p VAX-Gn、p VAX-LAMP/Gn、p VAX-Gc和p VAX-LAMP/Gc,并进行酶切鉴定和Sanger测序.同时构建带有EGFP标签的四种重组质粒,LipofectamineTM2000脂质体将重组质粒瞬时转染He La细胞,通过荧光显微镜观察结合免疫荧光检测目的蛋白的表达.抽提高纯度去内毒素的重组质粒,采用皮下注射免疫小鼠,并取重要脏器进行组织学分析.结果:双酶切鉴定和测序结果证实目的基因Gn、LAMP/Gn、Gc和LAMP/Gc成功克隆入真核载体p VAX 1,基因与原序列一致.荧光显微镜观察连有EGFP的表达载体转染的He La细胞,可见明显的绿色荧光,瞬时转染后特异性抗体免疫荧光检测到目的蛋白的表达.安全性评价结果显示Balb/c小鼠耐受性良好,多次注射后无明显组织形态学改变.结论:成功构建了重组嵌合载体p VAX-Gn、p VAX-LAMP/Gn、p VAX-Gc和p VAX-LAMP/Gc,并在真核细胞系中表达出目的蛋白,免疫小鼠显示了良好的安全性,为后期对汉滩病毒的靶向基因疫苗的研究奠定了基础. AIM: To construct recombinant eukaryotic expression vector of p VAX-Gn、p VAX-LAMP/Gn、p VAX-Gc and p VAXLAMP/Gc through p VAX1,identify the expression of chimeric protein in Hela cells and evaluate the safety of targeting vector.METHODS: The whole Gn and Gc gene were amplified from the HTNV c DNA by polymerase chain reaction. And then Gn,Gc and LAMP1 chimeric constructs were constructed and termed p VAX-Gn,p VAX-LAMP/Gn,p VAX-Gc and p VAX-LAMP/Gc. Then,the restriction enzyme analysis and Sanger sequencing were performed.The recombinant vectors were transiently transfected into He La cells with LipofectamineTM2000 transfection reagent. The expression of chimeric protein was detected through immunofluorescence assay. Meanwhile,p VAX-Gc-EGFP and p VAX-LAMP/Gc-EGFP recombinant plasmids were constructed and transfected into He La cells by liposome. The expression of the target protein was observed by fluorescence microscopy. The mice were immunized with the recombinant plasmids,which were extracted endotoxin free. Pathological analysis was performed on the important organs of mice. RESULTS: According to the double restriction enzyme digestion and sequence analysis,the target gene Gn,LAMP/Gn,Gc and LAMP/Gc were successfully cloned into vector p VAX1 and they were consistent with the original sequence. Immunofluorescence showed target protein was expressed in transiently transfected He La cells. The transfected He La cells were observed by fluorescence microscopy, and the expression vector containing EGFP showed green fluorescence. Balb/c mice were used as animal models to evaluate the safety of the vaccine. The results showed that the mice were well tolerated and there was no pathological change after repeated injections. CONCLUSION: The recombinant chimeric vectors were successfully constructed and expressed in eukaryotic cells,showing good safety. It lays foundation for the further study of Hantavirus targeted gene vaccine.

关 键 词： 汉滩病毒包膜蛋白 分子 载体构建 蛋白表达鉴定 安全性评价