乙型肝炎病毒核心蛋白突变体干扰HBV包装的实验研究

目的：构建乙型肝炎病毒（HBV）核心蛋白突变体基因的真核表达载体，转染HepG2细胞，观察其表达及干扰HBV颗粒包装的显性负调节作用。方法：采用PCR从质粒pHBVadrl-A1中扩增HBVadr1-A1中扩增HBV C基因和S基因，分别克隆到pGEM-T载体上，构建成pGEM-T-C和pGEM-T-S，进而构建成pGEM-T-CS载体，用HindⅢ切出克隆基因片段与pcDNA3.1连接，经PCR鉴定后构建成真核表达载体pcDNA3.1^ -CS,DNA测序显示基因融合正确，表达载体转染HepG2细胞，经G418筛选得到高拷贝转化子，逆转录－聚合酶链反应（RT－PCR）检测重组蛋白体外表达，用HBV阳性血清感染HepG2细胞，72h后提提细胞内HBV DNA，斑点杂交法分析各组DNA，结果：核心蛋白突变体在HepG2细胞内得到表达，且表达了该重组蛋白的HepG2细胞内HBV DNA量不同程度地低于对照组，表明重组蛋白具有抗HBV包装的DN突变体作用，结论：HBV核心蛋白与表面蛋白融合基因的真核表达载体pcDNA3.1-CS能够体外表达核心蛋白突变体，该突变体具有干扰HBV颗粒包装的显性负调节作用。

Effect of viral core protein mutant on HBV encapsidation

Objective To observe a recombinant mutant of HBV core protein for dominant negative gene therapy against HBV encapsidation in vitro. Methods C gene and S gene of HBV were acquired through PCR and subcloned into pGEM T to construct pGEM T C and pGEM T S respectively. After digestion and ligation of these two plasmids, pGEM T CS was constructed. The cloned gene was inserted into pcDNA3.1 + to construct pcDNA3.1 + CS, which was identified by DNA sequencing. The recombinant plasmids were transformed into HepG2 cells, and screened with G418. The resistant HepG2 cell clones were chosen to test the expression of core surface protein by RT PCR, and the expressing HepG2 clones were cultured with 10% HBV DNA positive human serum for 72 hours. The intracellular HBV particles were extracted and the DNA was subjected to dot hybridization. Results The analysis showed that the HepG2 cells expressing mutant C protein had capabilities to resist HBV invasion in varied degrees. The mutant C protein had a dominant negative role in the encapsidation of HBV compared with the naive part of core protein. Conclusions The production of recombinant mutant core protein has a potential value for gene therapy against HBV infection.