摘要
目的构建携带人IFN5α基因的X基因缺陷的HBVDNA真核表达质粒,并在真核细胞中表达。方法在pBR322BHBV质粒(含adrⅠ型HBVDNA)中插入人工合成片段破坏其HBV-X基因区,再与IFN5α片段连接,最后克隆入真核表达载体pcDNA3,获得携带人IFN5α基因的X基因缺陷的HBVDNA真核表达质粒pcDNA3-KN-F1F2-IFN-5α。同时构建两组对照质粒,即连接完整HBVDNA的真核表达质粒和连接X基因缺陷的HBVDNA真核表达质粒,将上述3种质粒以脂质体法同时转染HepG2细胞,G418筛选出稳定表达系统后选用荧光定量PCR法、ELISA法、RTPCR法检测3种包装病毒的复制表达以及IFN5α的表达水平。结果获得目的真核表达质粒pcDNA3-KN-F1F2-IFN-5α;目的质粒转染HepG2细胞后其包装病毒的复制表达水平均较两个对照组降低;插入的IFN-5α片段可以在mRNA以及蛋白质水平表达。结论成功构建携带人IFN-5α基因的X基因缺陷的HBVDNA真核表达质粒,并能在HepG2真核细胞中表达。
Objective To construct an X-gene defect and IFN gene contained HBV expression plasmid and explore its expression effect in HepG2 cell line. Methods By employing pBR322-B-HBV plasmid which contained type adrⅠHBV DNA, we destroyed HBV X-gene by inserting mutation and connected the defect HBV with IFN sequence. After recombination, it was introduced into a mammalian plasmid and was constructed as the objective plasmid. At the same time, two control groups were constructed. One was pcDNA3-ES-HBV2, which contained the full length of HBV DNA. The other was pcDNA3-KN-F1F2, which contained the X-gene defect HBV DNA. Then we transfected HepG2 cells with all the plasmid. After the cells were screened by G418, the X-gene defect and IFN gene contained HBV(KN-F1F2-IFN) and IFN protein from culture medium were detected by fluorescence- quantitative-PCR and ELISA respectively. Results pcDNA3-KN-F1F2-IFN, as the objective plasmid, was constructed successfully; contrary with the control virus, the amounts of both HBV and HBsAg were lower; both IFN mRNA and IFN protein were detected.Conclusions An X-gene defect and IFN gene contained HBV expression plasmid was constructed successfully and it can be expressed in HepG2 cell line.
出处
《中华传染病杂志》
CAS
CSCD
北大核心
2005年第2期99-103,共5页
Chinese Journal of Infectious Diseases
