幽门螺杆菌Lpp20重组蛋白的表达、纯化及其免疫活性初步研究

Expression and purification of the Lpp20 recombinant protein of Helicobacter pylori and primary study on its immunocompetence

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。

We aimed to express recombinant Lpp20 protein of Helicobacter pylori and study its immunocompetence.We amplified Lpp20 gene by polymerase chain reaction（PCR） and obtained Lpp20 gene fragments（528 bp）.Then subcloned the fragment into the expression vector pGEX-6P-2 to generate recombinant plasmid pGEX-6P-2/Lpp20.Recombinant Lpp20（rLpp20） was expressed in Escherichia coli（E.coli） BL21 and purified by GST affinity chromatograph,thus we obtained a soluble fusion protein with molecular weight about 43 000.Then we analyzed the immunoreactivity of rLpp20 by Western blotting and found that rLpp20 could be recognized by the polyclonal mouse anti-H.pylori antibody.Six weeks old C57BL/6 mice were immunized with purified rLpp20,GST,or Elution buffer intramuscularly at 2-week interval for four times.ELISA showed significant specific antibody titers,and demonstrated that the cytokines IFN-γ,IL-2,and IL-4 in mice spleen lymphocyte culture medium after stimulating by rLpp20 were increased significantly in rLpp20 groups.By using MTT assay,we found that the proliferation response of spleen cells of rLpp20 group was significantly higher than those of mice in control group.All these results suggest that rLpp20 protein has good immunocompetence and can induce strong humoral and cellular immunity in C57BL/6 mice,which lay a foundation for further development of effective vaccine against H.pylori infection.