幽门螺杆菌HpaA-CtxB融合蛋白的研制及免疫原性研究

【作者】 吴利先；

【导师】 杨致邦；

【作者基本信息】 重庆医科大学 ， 病原生物学， 2004， 硕士

【摘要】 目的：用基因工程技术克隆和表达H.pylori 的hpaA基因，以纯化的重组蛋白为抗原，建立检测血清HpaA抗体的间接ELISA法，探讨以重组蛋白作为抗原在诊断H.pylori感染中的价值。方法：用PCR方法从H.pylori DNA中扩增hpaA基因片段。插入原核表达载体pQE30，转化受体菌DH5a，克隆及序列分析后在大肠杆菌中进行高效表达，表达产物经纯化和Western blot鉴定后，作为抗原,建立检测血清H.pylori HpaA抗体的间接ELISA法，与科研诊断标准比较评价其应用的可行性。结果： 1.重组表达质粒pQE30-hpaA构建成功,序列分析所得序列完整，插入的基因片段全长783bp，与基因文库中的hpaA基因同源性达97.3%；2. SDS-PAGE显示表达产物相对分子量为30 000，表达量占全菌总量的31.67%；3. Western blot显示该蛋白可被病人血清所识别；4.SDS-PAGE分析表达形式，蛋白主要存在于细菌裂解液上清中，部分存在于包涵体中,经Ni2+-NTA树脂对上清进行纯化后，可得到纯度90%以上的蛋白质；5.重组蛋白免疫兔后的免疫血清双扩<WP=8>效价为1:16；6. 通过对临床标本的检测结果显示HpaA抗体检测的敏感性和特异性分别为100.0%和90.1%。结论：H.pylori hpaA基因片段克隆表达成功，获得高纯度重组蛋白并具有良好的免疫原性和抗原性，为研制疫苗和制备诊断H.pylori感染试剂盒提供了可靠材料。以重组蛋白为抗原初步建立检测血清HpaA抗体的间接ELISA法，敏感性、特异性高，为制备商品化的试剂盒奠定了基础。更多还原

【Abstract】 Objective: To obtain the recombinant adhesion gene hpaA in H.pylori by gene-engineering, which would lay a foundation for the development of diagnostic reagent and vaccine for H.pylori infection. To establish an indirect ELISA with the recombinant protein，which would provide a solid basis for obtaining diagnostic reagent kit detecting H.pylori infection.Methods: The high homogeneity gene fragment was found in comparing adhesin gene hpaA of domestic typical H.pylori strains. By using a pair of primers designed according to the hpaA sequence from Gene Bank, the hpaA gene was amplified by PCR and cloned into prokaryotic expression vectors pQE30 to construct pQE30-hpaA. The pQE30-hpaA was transformed into DH5<WP=10>α E.coli and expressed in the presence of IPTG. The expression product was analyzed by SDS-PAGE and its antigenicity was confirmed by Western blot. The recombinant protein was purified by Ni2+-NTA agarose. The feasibility of its clinical applications with comparative evaluation was done by H.pylori diagnostic standard of reseach. Results: 1.The prokaryotic expression vectors pQE30-hpaA was constructed successfully. The total length of the gene cloned was 783 bp with a 97.3% sequence homology with adhesion genes in Gene Bank; 2.SDS-PAGE showed that the recombinant protein that Mr was about 30 000 represented 31.67% of total cell protein; 3. Western blot showed that the recombinant protein could be recognized by H.pylori antiserum of patients; 4.SDS-PAGE showed that the recombinant protein existed in the supernatant and precipitation of the lysed bacteria broken by ultrasonic wave, and its purity was over 90% after purified with Ni2+-NTA agarose of supernatant; 5. The specific antibody titer was 1:16 in rabbit immunized with HpaA protein detected by double immunodiffusion; 6. The indirect ELISA based on HpaA protein was successfully established to assay the anti-HpaA in serum, and its sensitivity and specificity was 100.0% and 90.1% <WP=11>respectively.Conclusion: The successful expression and purification of the HpaA of H.pylori , provided antigen basis for the development of the vaccine and the diagnostic reagent for H.pylori infection. The indirect ELISA was created to assay anti-HpaA in serum provided a basis for the preparation of diagnostic reagent kit detecting H.pylori infection.更多还原