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目的表达幽门螺杆菌(Helicobacter pylori,H.pylori)脂蛋白Lpp20基因的重组蛋白,并检测其免疫活性。方法应用PCR技术从H.pylori标准株26695染色体DNA中扩增出Lpp20的编码基因片段,将其克隆至表达载体pGEX-6P-2上,在大肠杆菌(Escherichia coli,E.coli)BL21中表达并进行GST亲和层析纯化,用Western blot方法检测纯化产物重组Lpp20(recombinantLpp20,rLpp20)的免疫反应性。免疫C57BL/6小鼠后,以rLpp20为包被抗原建立间接ELISA法对免疫小鼠血清进行特异性抗体检测;ELISA双抗体夹心法检测小鼠脾淋巴细胞培养上清中IFN-γ、IL-2、IL-4水平;MTT比色法检测小鼠脾淋巴细胞增殖反应等指标以分析rLpp20的免疫活性。结果扩增的lpp20全长528 bp,与GenBank上公布的H.pylori 26695株lpp20序列作BLAST比较,结果完全一致;成功构建了pGEX-6P-2/Lpp20重组质粒,表达的rLpp20 M_r 43 000,纯化产物可被鼠抗H.pylori抗体识别;以rLpp20为包被抗原建立的间接ELISA法检测免疫小鼠血清,与对照组相比rLpp20免疫组可见阳性反应;rLpp20免疫组小鼠脾淋巴细胞经特异性抗原刺激后,培养上清中IFN-γ、IL-2和IL-4含量均明显升高;脾淋巴细胞增殖反应测定,rLpp20免疫组小鼠脾淋巴细胞经特异性抗原刺激后,刺激指数也明显升高,与对照组有明显差异。结论rLpp20具有良好的免疫活性,在小鼠体内可诱导较强的特异性体液免疫和细胞免疫应答,为筛选高效的H.pylori疫苗抗原奠定了基础。
Objective To express the recombinant protein of Lpp20 gene of Helicobacter pylori (Hpylori) lipoprotein and test its immunogenicity. Methods The gene encoding Lpp20 was amplified from the chromosomal DNA of 26695 strain of H.pylori by PCR and cloned into the expression vector pGEX-6P-2 and expressed in Escherichia coli (E.coli) BL21 And purified by GST affinity chromatography. The immunoreactivity of purified recombinant Lpp20 (recombinant Lpp20, rLpp20) was detected by Western blot. After immunizing C57BL / 6 mice, the specific antibody was detected by indirect ELISA with rLpp20 as coating antigen. The levels of IFN-γ and IL-10 in the spleen lymphocyte culture supernatants were detected by ELISA. 2, IL-4 levels; MTT colorimetric assay mouse splenic lymphocyte proliferation response and other indicators to analyze the immunological activity of rLpp20. Results The amplified lpp20 was 528 bp in length and was identical to the lpp20 sequence of H. pylori 26695 strain published in GenBank. The result was identical. The recombinant plasmid pGEX-6P-2 / Lpp20 was successfully constructed and expressed rLpp20 M_r 43 000, The purified product could be recognized by mouse anti-H.pylori antibody. The serum of the immunized mice was detected by indirect ELISA with rLpp20 as coating antigen. The rLpp20 immunized group showed positive reaction compared with the control group. The splenic lymphocyte After stimulated with specific antigen, the contents of IFN-γ, IL-2 and IL-4 in culture supernatants were significantly increased. The proliferation of splenic lymphocytes was measured. The splenic lymphocytes of rLpp20 immunized mice were stimulated by specific antigen Index also increased significantly, with the control group were significantly different. Conclusion rLpp20 has good immunocompetence and can induce strong specific humoral and cellular immune responses in mice, which lays the foundation for the screening of highly effective H.pylori vaccine antigens.