MAGE-1基因疫苗的构建及其免疫学活性的研究

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C几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-1 mRNA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MAGE-1多抗的产生，MTT法检测小鼠CIL杀伤活性。结果：构建的pcMAGE-1转染瑚：K293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SMMG7721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。

Construction and immunologic competence of MAGE-1 gene vaccine

Objective:To construct MAGE-1 DNA vaccine and to assay the cytotoxic activities of the spleen cells specific to MAGE-1 expressing cells and serum antibody of the immunized mice.Methods:Eukaryotic expression plasmid pcMAGE-1 was constructed by gene recombination technique, then transfected into HEK293 cells through cation lipid transfection. The expression of MAGE-1 at the level of mRNA and protein was detected through RT-PCR and Western blot. Serum antibody against MAGE-1 were detected by FACS, and CTLs were assayed through MTT.Results:The eukaryotic expression plasmid of pcMAGE-1 was constructed correctly and transfected into HEK293 cells. The result of RT-PCR showed that MAGE-1 mRNA was expressed. Western blot showed that the expressed protein of MAGE-1 was about 46 kD, absent in cells transfected with empty plasmid. Production of antibody increased in the immunised group with pcMAGE-1 showed by higher percentage of combination with SMMC-7721 cells than the control(P<0.05), and much more cytotoxic activity of the spleen cells in the immunised group was detected, compared to the control group(P<0.05).Conclusion:The gene vaccine of MAGE-1 is constructed successfully and it can induce specific immunological responses effectively.