淋球菌MlaA重组蛋白表达及多克隆抗体的制备与应用

Prokaryotic expression of Neisseria gonorrhoeae MlaA recombinant protein and its polyclonal antibody preparation and application

目的 原核表达、纯化淋球菌MlaA蛋白,制备并鉴定其多克隆抗体。方法 构建原核表达载体pET-28a-MlaA,转化BL21(DE3)大肠埃希菌,经IPTG诱导重组蛋白的表达。通过亲和层析法纯化目的蛋白,并进行Western blot鉴定。以纯化的重组蛋白作为抗原,与佐剂乳化后免疫6周龄雌性BALB/c小鼠,3次免疫后取小鼠静脉血并分离血清,采用酶联免疫吸附法(ELISA)测定抗体效价,用Western blot、间接免疫荧光试验(IFA)和流式细胞术(FCM)检测MlaA多克隆抗体的反应性和特异性。结果 Western blot显示,pET-28a-MlaA转化BL21后表达相对分子质量为35×10^(3)的淋球菌MlaA重组蛋白。ELISA检测MlaA免疫小鼠血清抗体效价为1∶8 000~1∶32 000。Western blot、IFA和流式细胞术检测制备的抗体能识别淋球菌变性和非变性MlaA蛋白。结论 成功表达并纯化淋球菌MlaA重组蛋白,免疫小鼠制备多克隆抗体,将制备的抗体血清作为一抗对淋球菌及其他不同类菌株进行Western Blot检测后发现,制备的多克隆抗体可以特异性识别淋球菌中的MlaA蛋白。经IFA及FCM检测,结果均表明制备的多克隆抗体与淋球菌MlaA有良好的反应性。

Objective To express and purify MlaA protein of Neisseria gonorrhoeae in prokaryotic cells, and to prepare and identify its polyclonal antibody. Methods The prokaryotic expression vector pET-28a-MlaA was constructed and induced by IPTG in Escherichia coli BL21(DE3) cells. The target protein was purified by affinity chromatography, and was analyzed by Western Blot. The purified recombinant protein was used as antigen, emulsified with adjuvant and immunized 6-week-old female BALB/c mice. After three times of immunization, the venous blood of the mice was taken and the serum was separated to determine the titer by enzyme-linked immunosorbent assay. The reactivity and specificity of MlaA polyclonal antibody were determined by Western blot, indirect immunofluorescence assay(IFA) and flow cytometry(FCM). Results Western Blot results showed that the recombinant MlaA protein was successfully expressed in Escherichia coli, and the relative molecular weight of recombinant MlaA protein was 35×10^(3). ELISA results showed that the antibody titer of immunized mice ranged from 1∶8 000 to 1∶32 000. The results of Western Blot, IFA and flow cytometry showed that the prepared antibody could specifically recognize the denatured and non-denatured MlaA proteins of N. gonorrhoeae. Conclusion According to the expression and purification of MlaA protein of N. gonorrhoeae,immunize mice to prepare polyclonal antibodies. After Western Blot detection of N. gonorrhoeaee and other different strains using the prepared antibody serum as a primary antibody, it was found that the prepared polyclonal antibodies can specifically recognize MlaA protein in N. gonorrhoeae. The results of IFA and FCM showed that the polyclonal antibody had good reactivity with N. gonorrhoeae MlaA.