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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。
Objective To construct a recombinant Lactococcus lactis expression vector with the fusion protein of extracellular domain of NS3 and CD40L of hepatitis C virus and investigate the immunogenicity of Lactococcus lactis after electroporation. Methods The recombinant plasmid pMG36e-NS3-CD40L was constructed by using recombinant plasmid pMG36e. The vector electrotransformed Lactococcus lactis MG1363, an oral immunization of male ICR mice. The anti-NS3 antibody in peripheral blood of mice was detected by ELISA, the spleen T lymphocyte subsets were detected by flow cytometry, and the cytotoxic activity of splenic lymphocytes was detected by CTL cytotoxicity assay. Results Lactococcus lactis expression vector pMG36e-NS3-CD40L was constructed and successfully transformed into Lactococcus lactis MG1363. ELISA test results showed that the recombinant lactic acid bacteria oral immunization mice, mice can induce high titers of anti-NS3 antigen antibodies. The results of flow cytometry confirmed that the bacteria could up-regulate the ratio of CD8 + T lymphocytes and CD4 + T lymphocytes in mice, and the increase of CD8 + T lymphocytes was more significant. CTL killing results show that the recombinant lactic acid bacteria can increase the mouse CTL killing capacity. Conclusion The Lactococcus lactis expression vector pMG36e-NS3-CD40L was successfully prepared. The vector was transformed into Lactococcus lactis and was orally immunized to induce high levels of cytotoxic activity.