摘要
目的构建含有乙型肝炎病毒(HBV)X基因的真核表达载体,并对其表达进行鉴定。方法用聚合酶链反应(PCR)方法扩增X基因序列,对pcDNA6(+)载体及X基因PCR产物双酶切并转化大肠杆菌DH5α,筛选阳性克隆,并对其进行双酶切和测序鉴定,构建HBV X基因真核表达载体pcDNA6(+)-HBx。以多聚乙烯酰胺(PEI)法将其转染到HepG2细胞,Western Blot鉴定目的蛋白。结果 pcDNA6-HBx酶切后,琼脂糖电泳可见到HBx基因片段,经序列测定其含有完整的X基因片段;Western Blot结果显示pcDNA6(+)-HBx能在HepG2细胞中表达X蛋白。结论成功构建了真核表达载体pcDNA6(+)-HBx,并能在HepG2细胞中表达X蛋白,为进一步研究HBV X基因与慢性乙型肝炎及肝癌的关系提供便利条件。
Objective To establish hepatitis B virus（HBV）X gene eukaryotic expression vector and identify its expression.Methods Polymerase chain reaction（PCR）was used to amplify X gene sequence,and the pcDNA6（＋）vector and X gene PCR product double enzyme digestion transformed into Escherichia coli DH5 alpha.The positive clones were screened,and the double enzyme digestion and DNA sequencing were used to establish HBV X gene eukaryotic expression vector pcDNA6（＋）-HBx.By polyetherimide（PEI）method,the transformation into HepG2 cells was performed,and Western blot identification of target protein was carried out.Results After pcDNA6-HBx enzyme digestion,agarose gel electrophoresis found HBx gene fragment,and sequencing results showed containing the complete X gene fragment.Western blot results showed that the pcDNA6（＋）-HBx in HepG2 cells expressed X protein.Conclusions The eukaryotic expression vector pcDNA6（＋）-HBx is established successfully,and there is X protein expression in HepG2 cells.It provides convenience for the further study of HBV X gene with chronic hepatitis and hepatocellular carcinoma.
出处
《检验医学》
CAS
2012年第11期921-924,共4页
Laboratory Medicine
关键词
乙型肝炎病毒
X基因
真核表达
Hepatitis B virus
X gene
Eukaryotic expression
