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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。
Objective: To construct hepatitis B virus core antigen (HBcAg) DNA vaccine containing tissue plasminogen activator (tPA) signal peptide. Methods The HBcAg gene was amplified by PCR and introduced into the corresponding restriction endonuclease sites, then cloned into pJW4303 vector to obtain the corresponding nucleic acid vaccine. After screening and identification, the HBcAg gene was compared with the wild-type HBcAg nucleic acid Vaccine and empty vector plasmid pJW4303 were transiently transfected 293T cells with liposomes, Western blot detection of core antigen expression. Results HBcAg DNA vaccine containing tPA signal peptide was successfully constructed. The HBcAg containing tPA signal peptide was expressed in both intracellular and extracellular of 293T cells in vitro, and the core antigen of HBcAg nucleic acid vaccine containing tPA signal peptide was expressed Higher level. Conclusion HBcAg nucleic acid vaccine containing tPA signal peptide with pJW4303 as carrier can secrete HBcAg intracellularly to lay a foundation for further study of its immunogenicity.