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目的制备MAGE-1基因疫苗,观察该疫苗诱导小鼠脾脏的抗原特异性细胞毒性T淋巴细胞CTL活性及产生特异性抗体的影响。方法应用基因重组技术,构建基因疫苗pcMAGE-1;转染HEK293细胞;RT-PCR及Western blot检测MAGE-1mRNA及蛋白的表达;pcMAGE-1免疫3次BALB/c小鼠,末次免疫后7日收集血清及脾细胞。流式细胞术检测血清中抗MA-GE-1多抗的产生,MTT法检测小鼠CTL杀伤活性。结果构建的pcMAGE-1转染HEK293细胞,RT-PCR表明在mRNA水平有MAGE-1的表达;Western blot显示表达产物46kD,与预期一致。基因疫苗免疫组小鼠血清MAGE-1抗体的产生增加,与SMMC-7721结合率显著高于对照组(P<0.05)。杀伤实验结果显示,免疫组小鼠脾细胞对SMMC-7721的杀伤率明显高于两个对照组(P<0.05)。结论成功地构建了MAGE-1基因疫苗,该疫苗能够有效地诱导特异性免疫应答。
Objective To prepare MAGE-1 gene vaccine and observe the effect of the vaccine on the CTL activity of antigen-specific cytotoxic T lymphocytes and the production of specific antibodies in the spleen of mice. Methods The recombinant plasmid pcMAGE-1 was transfected into HEK293 cells. The expression of MAGE-1 mRNA and protein was detected by RT-PCR and Western blot. The BALB / c mice were immunized three times with pcMAGE-1. Collect serum and spleen cells. The production of anti-MA-GE-1 polyclonal antibody in serum was detected by flow cytometry. The CTL killing activity of mouse was detected by MTT assay. Results The constructed pcMAGE-1 cells were transfected into HEK293 cells. The expression of MAGE-1 was detected by RT-PCR and Western blot showed that the expression of 46kD was consistent with the expectation. MAGE-1 antibody was increased in mice immunized with gene vaccine and the binding rate with MMC-7721 was significantly higher than that in control group (P <0.05). The results of killing experiments showed that the killing rate of splenocytes of SMMC-7721 in immunized mice was significantly higher than that of the two control groups (P <0.05). Conclusion The MAGE-1 gene vaccine was constructed successfully and the vaccine can effectively induce the specific immune response.