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目的 制备幽门螺杆菌(H.pylori)HpaA蛋白单克隆抗体,并检测其对H.pylori黏附胃腺癌细胞AGS的抑制作用。方法应用PCR法扩增HpaA基因,构建重组表达质粒pQE30-HpaA,表达并纯化重组HpaA蛋白;以其为抗原免疫BALB/c小鼠,将小鼠脾细胞与骨髓瘤细胞SP2/0融合,筛选阳性杂交瘤细胞株,采用间接ELISA和Western blot法检测单抗的特异性,间接ELISA检测细胞株培养上清和腹水抗体效价,并对其进行亚型鉴定和杂交瘤细胞株稳定性分析;扫描电镜观察重组HpaA蛋白单抗对H.pylori黏附AGS细胞的抑制作用。结果重组表达质粒pQE30-HpaA经双酶切、PCR及测序证明构建正确;表达的重组HpaA蛋白相对分子质量约为30 000,可溶性蛋白含量约占65%,纯化后纯度达85%以上;经细胞融合及筛选克隆获得了2株能稳定分泌抗HpaA单抗的杂交瘤细胞株,2株单抗与其他肠道细菌均无交叉反应,能与H.pylori全菌体发生特异性反应;乳胶凝集试验证明,该单抗属IgG3、κ型;2株单抗细胞培养液的抗体效价分别为1∶128～1∶256和1∶64～1∶128,腹水抗体效价分别可达1∶6 400～1∶12 800和1∶1 600～1∶3 200;杂交瘤细胞经反复冻存、复苏及多次传代,仍能稳定分泌高效价抗体;单抗可抑制H.pylori对AGS细胞的黏附作用。结论已成功制备了H.pylori HpaA蛋白单克隆抗体,为建立新的H.pylori现症感染诊疗方法奠定了基础。
Objective To prepare a monoclonal antibody against HpaA protein of Helicobacter pylori (H.pylori) and test its inhibitory effect on the adhesion of gastric adenocarcinoma cell line AGP to H.pylori. Methods The HpaA gene was amplified by PCR, and the recombinant plasmid pQE30-HpaA was constructed. The recombinant HpaA protein was expressed and purified. BALB / c mice were immunized with the antigen and the spleen cells were fused with myeloma cells SP2 / 0. Positive hybridoma cell lines. The specificity of McAb was detected by indirect ELISA and Western blot. The antibody titers of supernatants and ascites were detected by indirect ELISA. The subtype and the stability of hybridoma cell lines were analyzed. The inhibitory effect of recombinant HpaA monoclonal antibody on the adhesion of H. pylori to AGS cells was observed under electron microscope. Results The recombinant plasmid pQE30-HpaA was confirmed by double enzyme digestion, PCR and sequencing. The recombinant HpaA protein expressed about 30 000 and the soluble protein content was about 65%. The purity of purified recombinant protein was over 85% Two hybridoma cell lines that can stably secrete McAb against HpaA were obtained by fusion and screening clones. The two McAbs did not cross-react with other gut bacteria and could specifically react with H.pylori. The latex agglutination The test proved that the monoclonal antibody belongs to IgG3 and κ type. The antibody titers of the two monoclonal antibody cell culture broths were 1:128 to 1:256 and 1:64 to 1:128 respectively, and the antibody titers of ascites were up to 1: 6 400 ~ 1:12 800 and 1: 1 600 ~ 1: 3 200. The hybridoma cells could still stably secrete high titer antibody after repeated cryopreservation, resuscitation and multiple passages. The monoclonal antibody could inhibit the growth of AGS cells The adhesion effect. Conclusion The monoclonal antibody of H.pylori HpaA protein has been successfully prepared, which lays the foundation for the establishment of a new diagnosis and treatment of H.pylori infection.