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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S ,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及pcDNA3 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)、Westernblot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 16 88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 36 16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。Westernblot结果显示 ,存在有相对分子质量为 5 0 0 0 0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
Objective To clone S and M fragments of hantavirus 84Fli strain and determine the nucleotide sequence of these genes. Express the expression of S and M fragments in eukaryotic cells by transient expression. Methods Total RNA was extracted from Vero E6 cells infected with Hantavirus 84Fli strain isolated in China. The full-length cDNA of S and M fragments was amplified by reverse transcription-polymerase chain reaction (RT PCR). Then, the cDNA fragments of S and M fragments of 84Fli were cloned into pCR2 1 TOPO vector, and three of them were selected randomly to determine the nucleotide sequences of S and M fragments of Hantavirus 84Fli strain. Based on the nucleotide sequences of S and M fragments, primers were designed and the coding regions of S and M fragments were amplified by PCR. The eukaryotic expression plasmids pcDNA3 84SC and pcDNA3 84MC were constructed. Plasmid was transfected into COS7 cells by liposome method to transiently express NP and G1 and G2 glycoproteins. Nuclei and glycoproteins were detected by immunofluorescence (IFA), Western blot and immunoprecipitation. Results The genomic S fragment of Hantavirus 84Fli strain was 1688 nucleotides in length and encoded 430 amino acids. The genomic M fragment of Hantaan virus 84Fli strain is 36 16 nucleotides in length and encodes 1136 amino acids. Immunofluorescence (IFA) was used to detect transient positive expression of S and M fragments in COS cells. Westernblot results showed that there was a nuclear protein with a relative molecular mass of about 50000, and immunoprecipitation showed the expression of nucleoprotein, G1 and G2 glycoproteins. Conclusion Although there are differences in nucleotide sequence between 84Fli and other Hantaan viruses, the amino acid homology is still high.