博尔纳病病毒X基因真核表达载体的构建及鉴定

Construction and identification of the eukaryotic expression vector of Borna disease virus X gene

目的构建博尔纳病病毒(Borna disease virus,BDV)X蛋白真核表达载体,为BDV X蛋白的功能研究奠定基础。方法从BDV持续感染的人少突胶质瘤细胞(oligodentrocyte,OL)的总RNA中,通过逆转录和PCR获得BDV X基因的完整序列,将其连接到pc DNA6-myc-His(A)载体上,转化至E.coli感受态细菌后,提取质粒筛选阳性克隆,通过PCR法和DNA测序进行鉴定,并转染至OL细胞,通过Western blot方法验证重组蛋白的表达。结果测序鉴定目的基因序列正确插入至pc DNA6-myc-His(A)载体的酶切位点,PCR扩增的基因片段大小与目的基因相同,Western blot证明转染至OL细胞后可表达BDV X-His融合蛋白。结论成功构建了博尔纳病病毒X基因真核表达载体pc DNA6-myc-His(A)-BDV X。

Objective To construct the pcDNA6-myc-His（A） eukaryotic expression vector of Borna disease virus X gene （BDV X）, and provide theoretical basis for subsequent researches. Methods The target DNA of BDV X was acquired by RT-PCR and inserted into the pcDNA6-myc-His（A） vector. The eukaryotic expression vector was extracted for screening the positive clones. The constructed eukaryotic expression vector was confirmed by PCR and DNA sequencing. The activity of the cukaryotic expression vector was verified by Western blotting. Results PCR and DNA sequencing showed that the eukaryotie expression vector pcDNA6-myc-His （ A ） -BDV X was constructed and its sequence was the same as X gene of BDV H1766 strain. And the expressed protein of eukaryotic expression vector, was deteced by Western blotting, was 10 kDa. Conclusion The eukaryotic expression vector pcDNA6- myc-His（A）-BDV X was constructed successfully.