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

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

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真核和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HLA-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HLA-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。

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 ob- tained by RT-PCR from human melanoma A375, and then cloned into the pcDNA3. 1/VS-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 ex- pression 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 a- nalysis 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.