乙型脑炎病毒C蛋白编码基因重组质粒构建与表达

目的构建流行性乙型脑炎病毒(JEV)C蛋白编码基因重组子并鉴定。方法以JEV SA14-14-2株总RNA为模板,应用反转录-聚合酶链反应(RT-PCR)方法扩增JEV C蛋白编码基因,克隆至pMD19-TSimple载体测序。为便于分析JEV C蛋白编码基因重组子在哺乳动物细胞中的表达,在JEV C蛋白编码基因5′端附加FLAG序列,并亚克隆至pcDNA 3.1(+)载体中,构建重组子pJc并经酶切及DNA测序分析。脂质体法将pJc转染中华仓鼠卵巢(CHO)细胞。免疫荧光检测转染的CHO细胞中JEV C蛋白分布与表达。结果 pJC经BamH I/EcoR I酶切释出的插入子片段(414bp)分别与预期结果相符合。所编码的融合蛋白主要分布于胞质,少量分布于胞膜。结论 pJC成功构建,转染的CHO细胞可表达JEV C蛋白。

Construction and expression of recombinant encoding C protein derived from Japanese encephalitis virus

Objective To construct and express the recombinant encoding C protein derived from Japanese encephalitis virus(JEV).Methods Gene encoding C was amplified by RT-PCR technique from JEV SA14-14-2 strain total RNA.JEV C protein gene was obtained with restriction endonuclease BamH I/EcoR I from the prokaryotic expression vector named after pMD19-T simple,and subcloned into pcDNA3.1(+) eukaryotic vector,named after pJC.The recombinant was confirmed by restriction enzymes analysis and DNA sequencing,then was transfected into China hamster ovary(CHO) cells by Lipofectamine 2000.Distribution and expression of the C protein encoded by pJC in transfecd CHO cells were detected by immunofluorescence.Results DNA fragments(414bps)of the inserts released from pJC with BamHI/EcoRI restriction endonuclease were the expected theoretic results.The expression of above C protein was mainly distributed in endochylema of transfected CHO cells,and not much in membrane of transfected CHO cells.Conclusion The recombinant pJC was constructed and transfected into CHO cells successfully,and transfected CHO cells can express JEV C protein.