摘要
目的 :探讨幽门螺杆菌融合蛋白粘附素 霍乱毒素B亚单位 (HpaA CtxB ,HCTB)经口服免疫小鼠后 ,机体产生的免疫应答。方法 :用PCR方法扩增HpaA和CtxB两个目的基因片段 ,将它们分别克隆至pET 32a( + )和pQE 30质粒上 ,然后同时插入pQE 30表达载体中 ,构建含双基因的的表达质粒pQE HCTB ,转化E .coliDH5α,经IPTG诱导表达融合蛋白HCTB ;West ernblot分析其免疫反应性。融合蛋白经镍离子柱纯化后 ,HCTB和HpaA分别给小鼠灌胃进行免疫 ,ELISPOT和ELISA分别检测小鼠胃粘膜、派伊尔小结 (Peyer′spatches ,PP)抗原特异性抗体分泌细胞 (ASC) ,和血清IgG、IgA、小肠粘液sIgA和粪便sIgA。结果 :经测序HCTB融合基因片段由 116 1bp组成 ,为编码 387个氨基酸残基的多肽。经SDS PAGE分析相对分子量 (Mr)约为4 0 0 0 0。可溶性表达占全菌的 2 5 %以上 ,经亲和层析后可获得纯度为 92 %以上的重组蛋白。Westernblot分析其能分别与HpaA抗血清和CT抗血清特异性反应。灌胃免疫小鼠后ELISPOT和ELISA检测结果 ,胃和PPsIgA ASC、IgG ASC数量明显增加 ,尤以sIgA ASC为甚 ,同时血清IgA、IgG ,粪sIgA ,肠粘液sIgA也明显高于HpaA组和对照组 (P <0 0 5和P <0 0 1)。结论 :融合蛋白HCTB经口服免疫可有效诱导粘膜免疫应答 。
Objective:To explore the immune response in mice fed orally with the conjugated antigen of HpaA-CtxB(HCTB).Methods:A recombinant strain which could express bivalent antigen of HpaA and CtxB subunit was constructed. HpaA and CtxB gene was amplified by PCR. The DNA products of HpaA and CtxB were inserted into a prokaryotic expression vector pQE-30 respectively, and then translated into E.coli strain DH5α to express HCTB fusion protein. Its immunogenicity was analyzed by Western blot. After purification, to fed mice by oral immunization. The change of antigen-specific ASC antibody(IgG and IgA) in the mucosa of the animals were detected by ELISPOT and ELISA assay. Results:HCTB fusion gene was sequenced as 1 161 bp, the fusion protein encoded polypeptides of 387 amino acid residues.The molecular weight was 40 kD analysed by SDS-PAGE. The level of soluble expression product was about 41.67% of total cell protein. After affinity chromatography, the purity of fusion protein was above 92%.Western blot analysis confirmed that fusion protein could be specifically recognized by the serum of anti-HpaA and anti-CT. Antigen-specific ASC and antibody response in animals immunized with HCTB or HpaA was determined by ELISPOT and ELISA. The results showed that the number of sIgA and IgG-ASC increased significantly in PP and gastric mucosa, especially those of the sIgA-ASC by orally immunization with HCTB. The levels of specific antibody were also higher than those of controls. Conclusion:The results indicated that the oral immunization with HCTB induces effective mucosal immune respones and produced higher levels sIgA. The recombinant fusion protein HCTB can be used as an effective oral vaccine for prevention and treatment of infection of Hp.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2004年第5期306-310,共5页
Chinese Journal of Immunology
