外分泌型的乙型肝炎病毒核心抗原核酸疫苗的构建与体外表达

目的:构建含有组织型纤溶酶原激活剂(tPA)信号肽的乙型肝炎病毒核心抗原(HBcAg)核酸疫苗。方法将HBcAg基因用PCR方法扩增并分别引入相应的限制性内切酶酶切位点,然后克隆入pJW4303载体中获得相应的核酸疫苗,经筛选鉴定后,用上述核酸疫苗与野生型HBcAg核酸疫苗及空载体质粒pJW4303分别用脂质体瞬时转染293T细胞,应用蛋白印迹法检测核心抗原的表达。结果成功构建以pJW4303为载体的含tPA信号肽的HBcAg核酸疫苗,体外表达证实含tPA信号肽的HBcAg在293T细胞胞内和胞外均能表达,含tPA信号肽的HBcAg核酸疫苗的核心抗原表达水平较高。结论以pJW4303为载体的含tPA信号肽的HBcAg核酸疫苗能够将细胞内的HBcAg分泌到细胞外,为进一步研究其免疫原性打下了基础。

Construction and in Vitro Expression of Genetic Vaccine Encoding Hepatitis B Virus Core Antigen Fused to Tissue Plasminogen Activator Signal Sequence

Objective: To construct and express nucleic acid vaccine encoding hepatitis B virus core antigen(HBcAg) fused to tissue plasminogen activator signal sequence(tPA) in vitro.Methods: Restriction sites were introduced to HBcAg cDNA using polymerase chain reaction(PCR).The gene product was cloned to pJW4303 to construct nucleic acid vaccine and all constructs were identified by sequenc-ing,and 293T cells were transiently transfected with pJW4303/HBc/tPA,pJW4303/HBc and pJW4303 blank vector.The HBcAg protein was measured by western blot.Results: pJW4303/HBc/tPA nucleic acid vaccine was constructed successfully and expressed HBcAg in lysates and supernatants in 293t cells in vitro.Expression level of pJW4303/HBcAg/tPA was higher than that of pJW4303/HBc.Conclusion: HbcAg encoded by nucleic acid vaccine pJW4303/HBc/tPA and the expressed product in 293t cells could be secreted out of the cells.This research is the foundation for the further study of the pJW4303/HBc/tPA nucleic acid vaccine.