HCV NS3与CD40L胞外段融合蛋白表达载体的构建及免疫性

目的 构建带有丙肝病毒NS3与CD40L胞外段融合蛋白的重组乳酸球菌表达载体,并探讨其电转乳酸球菌后的免疫原性.方法 在乳酸球菌表达质粒pMG36e的基础上,应用基因重组技术构建重组表达载体pMG36e-NS3-CD40L.该载体电转乳酸球菌MG1363,口服的方式免疫雄性ICR小鼠.ELISA法检测小鼠外周血中抗NS3蛋白抗体,流式法检测小鼠脾T淋巴细胞亚型,CTL杀伤实验检测小鼠脾淋巴细胞的杀伤活性.结果 构建了乳酸球菌表达载体pMG36e-NS3-CD40L,该载体成功电转入乳酸球菌MG1363.ELISA检测结果显示,该重组乳酸菌口服免疫小鼠后,能诱发小鼠产生高滴度的抗NS3抗原的抗体.流式结果证实,该菌能上调小鼠CD8+T淋巴细胞和CD4+T淋巴细胞比率,且以CD8+T淋巴细胞增加更为显著.CTL杀伤结果显示,该重组乳酸菌能上调小鼠CTL杀伤能力.结论 成功制备了乳酸球菌表达载体pMG36e-NS3-CD40L,该载体转入乳酸球菌并经口服免疫小鼠后,能够诱发高水平的细胞杀伤活性.

Construction of lactobacillus expression vector with HCV NS3 and CD40L extracellular segment fusion pro tein and immunogenicity analysis

Objective To construct the HCV NS3 antigen and CD40L extracellular domain fusion protein lactobacillus ex pression vector and observe its immunogenicity.Methods The lactobacillus vector pMG36e-NS3-CD40L was constructed based on the original vector pMG36e using the gene recombination technique.This vector was transferred into the lactobacillus with electroporation method.The male ICR mice were orally immunized with bacteria.HCV NS3 antibody in the peripheral blood was measured by ELISA method.T lymphocyte subtypes in mice spleen were examined using flow cytometry method,CTL damage detection was used to value the killing activity of mice spleen lymphocytes.Results The lactobacillus vector pMG36e-NS3-CD40L constructed,this vector could induce high titer of antibody against HCV NS3 antigen,flow cytometry re sults confirmed that the plasmid could enhance the mouse CD8 or CD4 positive T lymphocytes level,and the change of CD8 was not stable.CTL killing results showed the recombinant bacteria with this reconstructed plasmid could up-regulated high level cell killing ability of mice.Conclusion Lactobacillus vector pMG36e-NS3-CD40L was constructed successfully,it could induce high level of cell killing activity after transferred into the lactobacillus and immunized the mice.