论文部分内容阅读
目的构建流行性乙型脑炎病毒(JEV)非结构4A(NS4A)蛋白编码基因重组子并鉴定。方法以JEV SA14-14-2株Total RNA为模板,运用RT-PCR方法扩增JEV NS4A蛋白编码基因,克隆至pMD19-T Simple载体并测序。为便于分析JEV NS4A蛋白编码基因重组子在哺乳动物细胞中的表达,在JEV NS4A蛋白编码基因5’端附加FLAG序列,亚克隆至pcDNA3.1(+)载体中,构建重组子pJNS4A并作酶切及DNA测序分析;采用脂质体法将pJNS4A转染中华仓鼠卵巢(CHO)细胞,采用免疫荧光法检测转染的CHO细胞中JEV NS4A蛋白分布与表达。结果重组质粒pJNS4A经BamHⅠ/EcoRⅠ酶切释出的插入子在830bp左右,与JEV NS4A蛋白编码基因和FLAG基因序列之和(834bp)相一致。JEV NS4A蛋白编码基因重组质粒转染的CHO细胞可见绿色荧光标记,主要分布在胞膜。结论 pJNS4A构建成功,转染的CHO细胞可稳定表达JEV NS4A蛋白。
Objective To construct and identify the recombinant non-structural 4A (NS4A) gene of Japanese encephalitis virus (JEV). Methods The total RNA of JEV strain SA14-14-2 was used as template to amplify the gene encoding JEV NS4A protein by RT-PCR and cloned into pMD19-T Simple vector and sequenced. To facilitate the analysis of the expression of the JEV NS4A protein-encoding gene recombinant in mammalian cells, a FLAG sequence was attached to the 5 ’end of the gene encoding JEV NS4A protein and subcloned into the pcDNA3.1 (+) vector to construct recombinant pJNS4A The expression of JEV NS4A protein in CHO cells transfected with pJNS4A was detected by immunofluorescence. Results The inserted fragment of pJNS4A was about 830bp by BamHⅠ / EcoRⅠ, which was consistent with the sum of the sequence of JEV NS4A protein and FLAG gene (834bp). CHO cells transfected with JEV NS4A protein-encoding gene were green fluorescently labeled and mainly distributed in the cell membrane. Conclusion pJNS4A was successfully constructed and transfected CHO cells stably expressed JEV NS4A protein.