表达胃癌MG7-Ag模拟表位的减毒鼠伤寒杆菌疫苗的研制和初步鉴定

Development and preliminary identification of the attenuated Salmonella typhimurium vaccine expressing the gastric cancer MG7-Ag mimotope

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。

Aim To develop an attenuated Salmonella typhimurium vaccine expressing mimotcpe of the gastric cancer MG7-Ag. Methods An complementary sequence of NcoI site and a seqence coding for MG7-Ag mimotope was designed at the 5' terminus of forward primer.Using p1.2II-HBcAg plasmid as template , PCR was performed to get fusion gene of the mimotope and HBcAg gene. The fusion gene was then subcloned into the plasmid pYA3341 complementary to S.typhimurium X4550, the recombinant plasmid was then used to transform attenuated S.typhimurium X4550. Results By sequencing, it was confirmed that the PCR product was afusion gene of MG7-Ag mimotope gene and HBcAg gene. The recombinant plasmid pYA3341-MG7/HBcAg included genes coding for MG7-Ag mimotope and HBcAg. Western blot showed that expression product of recombinant plasmid pYA3341-MG7/HBcAg in the X4550 displayed a protein band, Mr being about 22 000, binding specifically to anti-MG7 mAb. Conclusion The attenuated Salmonella typhimurium vaccine expressing the MG7-Ag mimotope is deveeoped successfully, which lay the foundation for further investigation of its potentiality in the immunotherapy of gastric cancer.