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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 prepare PsaA antigen and antibody against PsaA and to establish a rapid detection method for PsaA antibody. The positive rate of PsaA antibody in plague infected monkey serum was also tested. Methods: The PsaA gene fragment was amplified by PCR. Recombinant PsaA antigen was expressed in prokaryotic expression system of Escherichia coli. The expressed protein in inclusion body was purified by nickel column affinity chromatography and recoverable by urea gradient dialysis protein. Then the expressed protein was used as immunogen to routinely immunize rabbits. The rabbit serum was collected to prepare polyclonal antibody and purified with n-octanoic acid-ammonium sulfate to obtain PsaA antibody IgG. Using the obtained PsaA antigen and antibody as materials, two rapid detection methods for detecting PsaA antibody were established, namely indirect ELISA method and Up-converting Phospher Technology (UPT) immunochromatography strip method. Finally, the two methods were used to detect PsaA antibody in 18 monkey serum samples. Results: The positive rate of PsaA antibody in monkeys infected with Y. pestis was 62% (8/13). Conclusion: The rapid detection of PsaA antibodies against Yersinia pestis was successfully established and the positive rate of PsaA antibodies in 13 serum samples from plague-infected monkeys was 62%.