重叠PCR合成乙型肝炎病毒核心抗原基因及其在大肠杆菌中表达

SYNTHESIS OF GENE ENCODING HEPATITIS B CORE BY OVERLAPPING PCR AND ITS EXPRESSION IN ESCHERICHIA COLI

目的:构建乙型肝炎病毒核心抗原(HBcAg)基因的融合表达质粒并在原核系统表达,为治疗性疫苗的研制奠定基础。方法:根据大肠杆菌偏爱密码子,利用Synthetic Gene Designer软件对HBcAg基因进行密码子优化,分成24条长约40bp寡核苷酸片段,将重叠PCR合成的HBcAg基因插入到pET30a中,经限制性核酸内切酶酶切鉴定和核酸序列分析无误后转化BL21(DE3)中,IPTG诱导表达,SDS-PAGE电泳分析表达情况。结果:酶切和测序鉴定表明,成功构建了重组质粒pET30a/HBcAg,SDS-PAGE分析显示目的蛋白以包涵体的形式在大肠杆菌中获得高表达,表达量占总菌体蛋白的64.3%。结论:应用重叠PCR合成HBcAg基因并在大肠杆菌中获得了高效表达。

Objective： To construct fused expression vector of Hepatitis B core antigen （HBcAg）gene and to express HBcAg protein in E. coli for future research and manufacture of its therapeutic vaccine. Methods： By selecting the biased codon in E. coli, the HBcAg gene, consisting of 435 bp,was optimized by software of Synthetic Gene Designer and the 24 synthetic fragments were synthesized using overlapping PCR by two steps. Then the complete target fragment was cloned into pET30a vector. After analyzed with restriction endonuclease digestion and DNA sequencing analysis, pET30a/HBcAg was induced with isopropy-β-D-thiogalactoside （IPTG）. Finally, the expression product was identified with SDS-PAGE. Results ：By restriction endonuclease digestion and DNA sequencing analysis completely proved the recombinant plasmid validity. The target protein existed in form of inclusion body was expressed highly in E. coli and amounted to 64.3 % by total protein. Conclusion：The HBcAg gene has been synthesized by overlapping PCR and the recombinant protein has been expressed in E. coli successfully.