蜱传脑炎病毒E基因的重组质粒构建及其在原核细胞的表达

通过表达蜱传脑炎病毒E蛋白,为制备蜱传脑炎快速检测试纸条的奠定基础。根据GenBank数据库中已发表的蜱传脑炎病毒的基因序列,检索并合成蜱传脑炎病毒目的基因E,设计引物,PCR扩增,克隆至原核表达载体pET-30a(+)构建重组质粒,在BL21细菌内表达蜱传脑炎病毒E蛋白。结果表明:经过PCR试验和双酶切试验鉴定,克隆测序正确,通过重组质粒,成功地表达出蜱传脑炎病毒E蛋白。

Construction of recombinant plasmid of E gene of tick-borne encephalitis virus and expression in the prokaryotes

This paper aims to express the E protein of tick-borne encephalitis virus in the prokaryotes,and thereby lay a basis for further study on the development of specific and rapid diagnostic reagent.The target gene E of tick-borne encephalitis virus was acquired from GenBank database.One pair of primers were designed according to the nucleotide sequence of gene E of tick-borne encephalitis virus,and then by PCR amplification,and construct recombinant plasmid.Then the target gene E was cloned into the vector pET-30a(+) and transformed to competent BL21.Positive clones were screened and identified by PCR and enzyme digestion.Finally the E protein of tick-borne encephalitis virus was successfully expressed by the vector pET-30a(+).The results showed that positive clones were identified through PCR,enzyme cutting test and sequencing.The E protein of tick-borne encephalitis was expressed by prokaryotic vector.The target gene was cloned in the recombinant plasmid and the target protein was expressed in E.coli bacteria.