产肠毒素大肠杆菌四价基因工程灭活疫苗研制

Development of a tetravalent genetically engineered inactivated vaccine against enterotoxigenic Escherichia coli

目的利用PCR和基因定点突变技术,分别扩增出K88ac、K99、LTB和突变的ST1基因,构建了重组菌株BL21(DE3)(pXKKSL4),酶切鉴定和序列分析表明构建的pXKKSL4表达载体包含有K88ac-K99-3ST1-LTB融合基因且阅读框架正确。方法SDS-PAGE分析表明融合蛋白占菌体总蛋白含量的35.72%。ELISA检测证实融合蛋白能与ST1单抗、LTB、K88ac和K99抗体相结合。结果乳鼠灌胃实验表明K88ac-K99-3ST1-LTB融合蛋白已失去ST1天然毒性,免疫保护试验表明菌株氢氧化铝佐剂苗和包涵体粗提物氢氧化铝佐剂苗均具有良好的免疫效果,其免疫保护率分为98%和96%。结论上述研究表明构建的四价灭活疫苗不但解决了ST1肠毒素毒性问题,而且又赋予其免疫原性,同时增加K99菌毛可使免疫效果进一步增强,这样从肠毒素和菌毛两种途径,达到预防由ETEC引起的腹泻目的,从而有效控制腹泻的发生,为将来制备产肠毒素大肠杆菌基因工程灭活疫苗打下坚实基础。

Objective The K88ac,K99,LTB and ST1 genes were respectively amplified by PCR and site-directed mutagenesis and the recombinant strain BL21(DE3)(pXKKSL4)was constructed.Enzyme digestion identification and sequence analysis showed that the constructed pXKKSL4 expression vector contained K88ac-K99-3ST1-LTB fusion gene and the reading frame was correct.Method The SDS-PAGE analysis showed that the expression level of the fusion proteins were about 35.72%of total cellular protein.ELISA con-firmed that the fusion protein could bind to ST1 monoclonal antibody,LTB,K88ac and K99 antibodies.Result The results of intragas-tric test of suckling rat showed that the fusion protein had lost the toxicity of natural ST1 enterotoxin.The immune protection test showed that the aluminum hydroxide adjuvant vaccine of strain and crude extract of inclusion body had good immune effect and the immune pro-tection rate was 98%and 96%.Conclusion The above studies indicated that the constructed tetravalent inactivated vaccine not only solved the toxicity problem of ST1 enterotoxin but also gave it immunogenicity.At the same time,K99 pili can further enhance the im-mune effect.This could prevent diarrhea caused by ETEC through both enterotoxin and fimbrium pathways so as to effectively control the occurrence of diarrhea.It lays a solid foundation for the preparation of enterotoxigenic Escherichia coli genetically engineered inactiva-ted vaccine in the future.