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目的 将我国登革 2型病毒的prM(D2 prM)基因导入甲病毒载体 (pSfV) ,研究该基因及其编码蛋白介导的抗病毒作用。方法 首先将扩增的病毒prM基因导入pSfV的Sp6启动子下游并进行核苷酸序列测定。用SpeⅠ酶将pSfⅤ prM重组DNA线形化 ,再将其体外转录成 5′末端含帽子结构的重组RNA。然后通过电穿孔法将此重组RNA转染BHK 2 1/ 13细胞。采用X Gal原位染色和免疫荧光法对转染细胞的表达产物进行分析。结果 通过碱基序列测定证明病毒的prM基因已插入到甲病毒载体中 ,而且其重组DNA的转录物经RNaseA消化证实为RNA。重组RNA的细胞转染率达 90 %以上。prM蛋白表达细胞约占 80 %。结论 由pSfV prMRNA转染哺乳动物细胞所产生的蛋白可与登革 2型病毒多克隆抗体起特异反应 ,证明体外转录的重组RNA含有登革 2型病毒特异序列。
OBJECTIVE: To introduce the prM (D2 prM) gene of Dengue 2 virus of China into alphavirus vector (pSfV) to study its antiviral activity mediated by its encoded protein. Methods The amplified prM gene was introduced into the downstream of Sp6 promoter of pSfV and the nucleotide sequence was determined. The pSfV prM recombinant DNA was linearized with SpeI enzyme and transcribed in vitro into a 5 ’-capped recombinant capped RNA. This recombinant RNA was then transfected into BHK 2 1/13 cells by electroporation. The expression products of the transfected cells were analyzed by X Gal in situ staining and immunofluorescence. As a result, it was confirmed by base-sequence analysis that the prM gene of the virus was inserted into the alphavirus vector and the transcript of its recombinant DNA was confirmed to be RNA by RNaseA digestion. Recombinant RNA transfection rate of more than 90% of cells. Approximately 80% of prM protein-expressing cells. CONCLUSIONS: The protein produced by mammalian cells transfected with pSfV prMRNA can react specifically with the dengue 2 polyclonal antibody, demonstrating that the in vitro transcribed recombinant RNA contains the dengue 2 virus-specific sequence.