摘要
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.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2002年第2期138-140,143,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金资助
No.398707424 39800057