人黑色素瘤抗原MAGE-3 肿瘤疫苗的构建及其诱导HLA-A *0201 转基因小鼠CTL活性的观察

 目的 通过基因工程的方法获得人黑色素瘤抗原MAGE-3的DNA和蛋白疫苗，并观察其对HLA-A＊0201转基因小鼠的CTL活性的影响。方法 用RT-PCR的方法，从人黑色素瘤细胞株A375中扩增MAGE-3的cDNA片段，分别将其插入到pcDNA3．1／v5-His真核扣pET32a原核表达载体。将pcDNA-MAGE-3转染B16肿瘤细胞观察其是否能在真核细胞中表达；将pET32a-MAGE-3转化大肠杆菌BL21，经IPTG诱导，SDS-PAGE观察融合蛋白表达情况。并用镍柱亲和层析的方法纯化融合蛋白。然后，采用DNA疫苗初次免疫和蛋白疫苗加强的策略免疫HL-A＊0201转基因小鼠，LDH方法检测CTL活性。结果 酶切结果证实，成功地构建了pcDNA-MAGE-3真核表达载体和pET32a-MAGE-3原核表达载体，并在真核细胞B16和原核细胞大肠杆菌中获稳定表达，原核表达产物为融合蛋白且分布于包涵体。用DNA疫苗进行初次免疫，镍柱纯化的融合蛋白加强免疫后，可成功地诱导HL-A＊0201转基因小鼠CTL活性。结论 成功地获得了MAGE-3肿瘤抗原，为进一步完善MAGE-3肿瘤疫苗打下了基础。

Construction of MAGE-3 Tumor Vaccine and Its Effect on CTL Activity in HLA-A * 0201 Mice

Objective To obtain the DNA and protein vaccine of human MAGE-3 gene and to observe the effect on CTL activity induction in HLA-A*0201 mice.Methods The MAGE-3 gene fragment was obtained by RT-PCR from human melanoma A375,and then cloned into the pcDNA3.1/V5-His and pET-32a vectors,respectively.The expression vectors were confirmed by restriction enzyme digestion analysis.The mRNA expression of MAGE-3 gene was detected after transfection of the recombinant karyotic expression vector into B16 tumor cells and the expression of pET-32a-MAGE-3 fusion protein was analyzed in E.coli by SDS-PAGE.The fusion protein was purified by Ni2 affinity chromatography.According to the DNA-priming and protein-boosting immune protocol,HLA-A*0201 transgenic mice were vaccinated with DNA and followed by the protein of MAGE-3.Then,the activity of CTL against specific peptide pulsed by SW480 tumor cells was tested by the method of LDH.Results Restriction enzyme digestion analysis showed that the recombinant expression vectors pcDNA3.1-MAGE-3 and pET32a-MAGE-3 were successfully constructed and expressed in B16 or E.coli as a fusion protein.The DNA and purified protein vaccine were obtained respectively and the CTL activity in HLA-A*0201 transgenic mice was obviously observed.Conclusion MAGE-3 tumor vaccine was obtained successfully,which is helpful for the further study of the MAGE-3 vaccine for tumor therapy.