丙型肝炎病毒核心蛋白基因真核表达质粒的构建和表达

Construction and Expression of Eukaryotic Expression Plasmid of Core Gene of Hepatitis C Virus

目的:构建丙型肝炎病毒(HCV)核心蛋白真核表达质粒,并在HepG2细胞中稳定表达。方法:采用RT-RCR方法从丙型肝炎患者血清中得到HCVC区的cDNA序列,然后克隆入pcDNA3.0载体,构建真核表达质粒pcDNA-HCV,并进行酶切鉴定和测序鉴定;采用脂质体转染技术将pcDNA-HCV稳定转染于HepG2细胞系,并用G-418进行筛选;采用Western Blotting方法和免疫细胞化学方法检测HepG2细胞中HCV核心蛋白表达情况。结果:从HCV感染者血清中扩增得到的503bp HCV cDNA序列,属于HCV 1型基因C区,并成功构建了真核表达质粒pcDNA-HCV经Western blotting和免疫细胞化学检测,稳定转染的HepG2细胞中有HCV核心蛋白的表达结论:成功构建HCV核心蛋白真核表达质粒pcDNA-HCV。

Objective： To construct the eukaryotic expression plasmid of core gene of hepatitis C virus （HCV） and to express it in HepG2 cells. Methods： The cDNA fragment of HCV core region was synthesized by the reverse-transcriptase-polymerase chain reaction （RT-PCR） from the sera of patients with hepatitis C and cloned into the eukaryotic expression vector pcDNA3.0 to construct the plasmid pcDNA-HCV, which was identified by cutting with restriction endonuclease and sequencing. The plasmid pcDNA-HCV was then transfected into HepG2 cells and screened by G-418. The expression of HCV core protein in HepG2 cells was analyzed by Western Blotting and immunocytochemical technique. Results： 503 bp cDNA fragment of HCV core gene,which was obtained from the sera of patients with hepatitis C, belonged to C region of genotype 1 of HCV. By Western Blotting analysis and immunocytochemical technique,it was found that there was the expression of HCV core protein in HepG2 cells. Conclusions： Recombinant eukaryotic expression plasmid pcDNA-HCV is successfully constructed and stably expresses in HepG2 cells.