汉坦病毒84Fli株S和M片段基因克隆、序列分析及在真核细胞中表达的研究

目的 克隆汉坦病毒84Fli株S，M片段，测定这些基因的核苷酸序列，用瞬时表达了解S和M片段在真核细胞中的表达。方法 以我国分离的汉坦病毒84Fli株感染的Vero-E6细胞提取总RNA，用逆转录-聚合酶链反应（RT-PCR）技术扩增其S及M片段的全长cDNA，然后将84Fli株的2，M片段cDNA基因分别克隆入pCR2.1-TOPO载体中，随机挑选其中的3个克隆测定核酸序列，确定汉坦病毒84Fli株S和M片段核苷酸序列，根据S和M片段核苷酸序列设计引物，PCR扩增S及M片段的编码区，构建了真核表达质粒pcDNA3/84SC ey 实验室/84MC。用质脂体法把质粒转入COS7细胞，瞬时表达NP及G1和G2糖蛋白，用免疫荧光（IFA）、Western blot和免疫沉淀方法检测核蛋白及糖蛋白的表达。结果 汉坦病毒84Fli株的基因组S片段长度为1688个核苷酸，编码430个氨基酸，汉坦病毒84Fli株的基因组M片段长度为3616个核苷酸，编码1136个氨基酸，用免疫荧光（IFA）检测S和M片段在COS细胞中的瞬时表达为阳性。Western blot结果显示，存在有相对分子质量为50000左右的核蛋白表达，免疫沉淀法显示有核蛋白、G1和G2糖蛋白的表达。结论 84Fli株病毒和其他汉滩病毒在核苷酸序列上虽有差异。但在氨基酸水平上的同源性仍很高，成功地在COS7细胞中瞬时表达了汉滩病毒的核蛋白及糖蛋白。

Molecular cloning,nucleotides sequence and transient expression of S and M genome segment of hantavirus strain 84Fli

Objective To sequence,analyze and express the nucleotide sequences of S and M segments of hantavirus strain 84Fli.Methods S and M segments of hantavirus 84Fli strain were amplified by RT-PCR,the PCR products were cloned into plasmid pCR2.1-TOPOr.Three clones of each segment which have been sequenced were randomly selected. The coding region of S and M segments were amplified by PCR,and cloned into expressing vector pcDNA3.0. Transient expression of nucleocapsid protein,G1 and G2 glycoproteins in COS7 cells were performed by Lipofectin transfection.The expression of NP,G1 and G2 have been conformed by using immunofluorescence,Western blot and immuno-precipitation methods.Results The full length S and M segments cDNA have been amplified and cloned. The S and M segments of hantavirus strain 84Fli contained {1 688} and {3 616} nucleotides respectively. Conclusion Deduced amino acid sequences of NP and glycoproteins revealed high homologue to other Hantaan viruses. NP,G1 and G2 proteins of 84Fli can be transiently expressed in COS7 cells.