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目的为克隆表达幽门螺杆菌(Helicobacter pylori,H.pylori)临床株的塑性区hp4565基因,并制备抗体,以探讨其生物学功能。方法以标准株NCTC shfi470的hp4565基因组全长设计引物,以H.pylori临床株为模板,PCR扩增临床株中hp4565基因,经原核表达后,选定最适条件大量诱导表达并经Ni2+-NTA柱纯化的目的蛋白。用MTT法检测重组蛋白对胃上皮细胞GES-1增殖影响,定磷法检测重组蛋白ATP酶活力。用纯化蛋白为抗原免疫家兔后制备多克隆抗体,ELISA检测抗体效价。结果成功克隆表达了hp4565基因,全长942 bp,编码313个氨基酸,原核表达后SDS-PAGE电泳显示有41 k Da的融合蛋白,Western blot鉴定是预期HP4565蛋白。纯化后获得较高纯度的重组蛋白,MTT法结果显示HP4565蛋白在低浓度(≤40 mg/L)时对细胞增殖无明显影响;蛋白浓度在60 mg/L以上时,细胞增殖随着培养时间的延长和浓度的增加呈现逐渐降低的趋势;重组蛋白具有一定的ATP酶活力,免疫家兔制备的多克隆抗体效价为1∶1.6×105。结论成功克隆表达了H.pylori临床株塑性区hp4565基因,得到纯度较高的蛋白和滴度较高的抗体,重组蛋白对胃上皮细胞增殖有一定影响,并具有一定的ATP酶活力。
Objective To clone and express the hp4565 gene in the plastic zone of Helicobacter pylori (H.pylori) clinical strain and to prepare the antibody to explore its biological function. Methods The hp4565 genomic full length cDNA of NCTC shfi470 was designed and the hp4565 gene was amplified by PCR from clinical strains of H.pylori. The recombinant plasmid pGEX - hp4565 was cloned and expressed in E.coli BL21 Purification of the target protein. The effect of recombinant protein on the proliferation of gastric epithelial cells GES-1 was detected by MTT assay. The activity of recombinant protein ATPase was assayed by fixed phosphorus assay. Polyclonal antibody was prepared by immunizing rabbits with the purified protein as antigen and the antibody titers were detected by ELISA. Results The hp4565 gene was successfully cloned and expressed, which was 942 bp in length and encoded 313 amino acids. The fusion protein was 41 kDa after SDS-PAGE electrophoresis, and the expected HP4565 protein was identified by Western blot. After purification, the recombinant protein with higher purity was obtained. MTT assay showed that HP4565 protein had no significant effect on cell proliferation at low concentration (≤40 mg / L). When the protein concentration was above 60 mg / L, And the concentration of polyclonal antibody tended to decrease gradually. The recombinant protein had certain ATPase activity, and the titer of polyclonal antibody prepared by immunized rabbits was 1: 1.6 × 105. Conclusion The hp4565 gene was cloned and expressed in the plasticity region of H.pylori successfully. The higher purity of the protein and the higher titer of the antibody were obtained. The recombinant protein had some effects on the proliferation of gastric epithelial cells and had certain ATPase activity.