【摘要】 目的构建乙肝表面抗原(HBsAg)基因(678bp)的真核表达质粒(pcDNA3-HBsAg),转染小鼠乳肝癌细胞(H22),获得其稳定表达细胞株(H22/HBsAg)。方法用PCR方法从含乙肝病毒(HBV)基因的PUC18-HBV质粒上扩增HBsAg基因序列;经酶切、连接构建pcDNA3-HBsAg;利用改良的阳离子脂质体介导法将其转染入H22细胞,经G418筛选获得阳性克隆细胞株,用RT-PCR法检测阳性细胞株的HBsAg mRNA。结果成功构建了重组HBsAg真核表达载体,并获得了稳定表达HBsAg的细胞株,为进一步的实验研究奠定了基础。更多还原

【Abstract】 Objective To establish an eukaryotic vector encoding Hepatitis B Antigen(HBsAg),pcDNA3-HBsAg,and to screen HBsAg positive clones from mouse hepatic cancer cell line H22.Methods The HBsAg gene was amplified from PUC18-HBV by PCR.pCDNA3-HBsAg was prepared by inserting the fragment into the plasmid pcDNA3.Then the recombinant vector was identified by restriction enzyme and sequencing.Next pcDNA3-HBsAg was transfected into the H22 cell line and the positive clones(H22/HBsAg) were screened with G418.Finally,the expression of HBsAg in H22/HBsAg was detected by RT-PCR.Results The fragment of HBsAg was amplified and pcDNA3-HBsAg was prepared successfully.No errors were found both in the sequence and ORF of the acquired fragment.The expected fragment of HBsAg(678bp) was amplified from H22/HBsAg by RT-PCR.Conclusion:An eukaryotic plasmid encoding HBsAg(pcDNA3-HBsAg) has been constructed and HBsAg was transfected stably into H22 cell.更多还原