鼠疫菌PsaA抗原的表达及抗体制备和应用

PsaA Antigen Expression of Y. pestis and Its Antibody Development and Application

目的:制备鼠疫菌PsaA抗原及抗体,建立针对PsaA抗体的快速检测方法,并检测鼠疫感染猴血清标本中的PsaA抗体的阳性率。方法:利用PCR方法扩增出PsaA蛋白基因片段,在大肠杆菌原核表达系统中表达出重组PsaA抗原,以镍柱亲和层析纯化包涵体形式的表达蛋白,以尿素梯度透析复性成可溶蛋白。再以表达蛋白为免疫原,常规免疫家兔,收集兔血清制备多抗,并以正辛酸-硫酸铵法提纯获得PsaA抗体IgG。利用得到的PsaA抗原和抗体为材料,建立两种检测PsaA抗体的快速检测方法,即间接ELISA法和上转换发光(Up-converting Phospher Technology,UPT)免疫层析试纸条法。最后利用这两种方法检测18份猴血清标本中的PsaA抗体。结果:鼠疫菌感染猴血清标本中PsaA抗体的阳性率为62%(8/13)。结论:成功建立了针对鼠疫菌PsaA抗体的快速检测方法,并检测到13份鼠疫感染猴血清标本中的PsaA抗体的阳性率为62%。

Objective：To develop the PsaA antigen of Y.Pestis and its antibody,and to develop the fast detection assays,and to detect the positive ratio of PsaA antibody in 18 plague monkey serum samples.Methods：The PsaA gene was amplificated by PCR method.The production was cloned and expressed using an E.coli expression system,purified with the Ni-NTA affinity chromatography,and renatured the inclusion body expressed proteins using a urea gradient dialysis method.The corresponding polyclonal antibody was prepared by immunizing rabbits using the conventional method.Bloods were obtained from rabbits for collection of sera and purification of specific immunoglobulin G（IgG） with a caprylic acid-saturated ammonium sulfate precipitation method.Then both indirect ELISA method and Up-converting Phospher Technology（UPT） lateral flow assay were developed to detect PsaA antibodies of samples.Lastly,18 monkey serum samples were tested.Results：The data showed that the positive ratio of the PsaA antibody in plague monkey serum samples was 62%（8/13）.Conclusion：The fast detection assays were successfully developed for the PsaA antibody test.And the positive ratio of the antibody in plague monkey serum samples was 62%.