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目的 研制可表达胃癌ＭＧ７－Ａｇ模拟表位的减毒鼠伤寒杆菌疫苗。 方法将ＮｃｏⅠ位点和ＭＧ７－Ａｇ模拟表位编码序列的互补序列设计到上游引物的５′端。以含有ＨＢｃＡｇ全序列的质粒ｐ１．２Ⅱ为模板，通过ＰＣＲ扩增，获得ＭＧ７－Ａｇ模拟表位与ＨＢｃＡｇ的融合基因。将目的基因插入载体ｐＵＣｍ－Ｔ，经酶切鉴定和序列测定证实后，再亚克隆至与减毒鼠伤寒杆菌Ｘ４５５０互补的质粒ｐＹＡ３３４１中，再以此重组质粒转化减毒鼠伤寒杆菌Ｘ４５５０。结果序列测定证实，ＰＣＲ产物为胃癌ＭＧ７－Ａｇ模拟表位与ＨＢｃＡｇ的融合基因片段；最终得到含有目的基因片段的重组表达载体ｐＹＡ３３４１。重组质粒在Ｘ４５５０中的表达产物，经Ｗｅｓｔｅｒｎ ｂｌｏｔ表明，在相对分子质量（Ｍｒ）约在２２ ０００处有１条可与抗ＭＧ７ ｍＡｂ特异性结合的多肽表位。结论成功地研制可表达胃癌ＭＧ７－Ａｇ模拟表位减毒鼠伤寒杆菌疫苗，为进一步研究其在胃癌免疫治疗中的作用奠定了基础。
Objective To develop an attenuated Salmonella typhimurium vaccine that expresses MG7-Ag mimotopes in gastric cancer. Methods The complementary sequence of the Nco I site and the MG7-Ag mimotope coding sequence was designed to the 5 ’end of the upstream primer. The fusion gene of MG7-Ag mimotope and HBcAg was obtained by PCR amplification using the plasmid p1.2Ⅱ containing the full sequence of HBcAg as a template. After the target gene was inserted into the vector pUCm-T, it was confirmed by restriction enzyme digestion and sequence analysis, then subcloned into the plasmid pYA3341 which is complementary to attenuated Salmonella typhimurium X4550, and transformed into attenuated Salmonella typhimurium X4550 with the recombinant plasmid. Results The sequence analysis confirmed that the PCR product was fusion gene fragment of gastric cancer MG7-Ag mimotope and HBcAg. Finally, a recombinant expression vector pYA3341 containing the target gene fragment was obtained. The expressed product of recombinant plasmid in X4550 showed by Western blot that there was one polypeptide epitope that could specifically bind to anti-MG7 mAb at a molecular weight of about 22,000. Conclusion The attenuated Salmonella typhimurium vaccine expressing gastric cancer MG7-Ag mimotopes is successfully developed, which lays the foundation for further study on its role in gastric cancer immunotherapy.