突变型乙肝病毒核心抗原DNA疫苗的体外表达

Expression of DNA Vaccine Encording Hepatitis B Virus Core Antigen with Mutating Terminal in Vitro

目的:构建突变型核心抗原核酸疫苗,观察该核酸疫苗在体外蛋白的表达。方法:采用基因工程定点突变技术,构建5种突变型核酸疫苗,分别去除乙肝病毒核心抗原N端的第1、2位氨基酸,命名为M12,去除3、4位氨基酸命名为M34以及去除5、6位的氨基酸命名为M56,用上述构建的核酸疫苗与野生型HBc核酸疫苗(pJW4303/HBc)及空载体质粒pJW4303分别用脂质体转染293T细胞,应用蛋白印迹法检测核心蛋白的表达。结果:经过pst1和BglII双酶切和测序鉴定结果突变型核心抗原核酸疫苗构建成功。在去除2个氨基酸的核酸疫苗结果中显示:野生型pJW4303/HBc、M12、及M56体外转染293T细胞后,在细胞上清和裂解中能很好的表达,而M34上清未见表达,仅裂解中可见极少量疑似表达条带;在原有基础上分别去除第3位和第4位氨基酸,命名为M3和M4,结果显示M3上清未见表达,裂解液中可见少量表达,而M4在上清和裂解中均可见明显的表达。结论:去除核心抗原N端第3位的氨基酸(M3)可以明显影响核心抗原的表达,HBcAg氨基端第3位氨基酸对蛋白的表达可能起到重要的作用。

Objective： To study the expression of DNA vaccine encording HBV core antigen with removing N-amino terminal. Methods： We constructed the DNA vaccine encording HBV core antigen with truncation N- amino terminal by site-directed mutagenesis： removed the first and second amino acid, the third and forth amino, the fifth and sixth amino,moreover, the third and forth from N-amino terminal was separately wiped out for the need study. We named the constructed DNA vaccine as M12, M34, M56, M3 and M4. 293T cells were transiently transfected with M12, M34, M56, M3, M4, wild-type HBc DNA vaccine （pJW4303/HBc） and pJW4303 vector. The protein was measured by westem blot. Results： DNA vaccine encording HBV core antigen with truncation N-amino termio was constructed successfully and expressed HBcAg in lysate and supernatant. Although supernantant of transfected 293T cells can not express the HBcAg, but the lysate of with M^4 and M3 can deliver thimbleful suspected and a little expression. However, HBcAg can be detected in both supematant and lysate of 293T cells transfected with M4. Conclusion： Deletion of the third amino acid in the N-terminal may decrease the expression of HBcAg. The third amino acid may play an important role in the expression of HBcAg protein.