重组CFP10-PPE68融合蛋白检测结核分枝杆菌感染的初步应用

目的以结核分枝杆菌(Mycobacterium tuberculosis,MTB)RD1区特异性培养滤液蛋白10(culture filtrate pro-tein10,CFP10)与戊糖-5-磷酸-3-差异构象酶68(pentose-5-phosphate-3-epimerase68,PPE68)的融合蛋白CFP10-PPE68作为包被抗原,建立检测结核病患者血清中MTB抗体的间接ELISA法。方法采用亲和层析法纯化重组CFP10-PPE68、CFP10和PPE68蛋白;分别以3种蛋白作为包被抗原,建立检测结核病患者血清中MTB特异性抗体的间接ELISA法;采用方阵滴定法对建立的间接ELISA法的条件进行优化;并以临床诊断为金标准,对间接ELISA法的特异性、敏感性和准确性进行验证。结果纯化的重组CFP10-PPE68融合蛋白纯度约为70%。分别以重组CFP10-PPE68、CFP10和PPE68蛋白作为包被抗原的最佳包被浓度分别为2、4、4μg/ml,血清最佳稀释度均为1∶1 000,酶标二抗最佳稀释度均为1∶5 000。3种蛋白对肺外结核的诊断效果均优于肺结核,且重组CFP10-PPE68融合蛋白检测肺外结核的特异性、敏感性及准确性均最佳;3种蛋白诊断结核病的特异性较高,而敏感性相对较低。结论以重组CFP10-PPE68融合蛋白作为包被抗原建立的间接ELISA法诊断MTB感染具有较高的敏感性和特异性,特别是对难以诊断的肺外结核病的诊断有一定的参考价值。

Preliminary application of recombinant culture filtrate protein 10-pentose-5-phosphate-3-epimerase 68 fusion protein to detection of Mycobacterium tuberculosis infection

Objective To develop an indirect ELISA method for detection of Mycobacterium tuberculosis(MTB) infection in sera of patients with tuberculosis(TB) using the fusion protein of culture filtrate protein 10(CFP10) and pentose-5-phosphate-3-epimerase 68(PPE68) as diagnostic antigen.Methods Recombinant CFP10-PPE68,CFP10 and PPE68 proteins were purified by affinity chromatography,and used as coating antigens for development of indirect ELISA method for MTB infection in sera of patients with TB.The condition for the developed ELISA method was optimized by block titration.The developed ELISA method was verified for specificity,sensitivity and accuracy using the result of clinical diagnosis as gold standard.Results The purified recombinant CFP10-PPE68 fusion protein reached a purity of 70%.The optimal concentrations of recombinant CFP10-PPE68,CFP10 and PPE68 as coating antigens were 2,4 and 4 μg / ml respectively,while the optimal dilutions of serum and enzyme-labeled second antibody were 1 ∶ 1 000 and 1 ∶ 5 000 respectively.The diagnostic effectiveness of extrapulmonary tuberculosis by the three recombinant proteins was higher than that of pulmonary tuberculosis.All the specificity,sensitivity and accuracy of recombinant CFP10-PPE68 fusion protein for extrapulmonary tuberculosis were higher than those of other two proteins.The specificities of three recombinant proteins for diagnosis of TB were high,while the sensitivities were low.Conclusions The indirect ELISA method for diagnosis of MTB infection developed using recombinant CFP10-PPE68 fusion protein as coating antigen showed high sensitivity and specificity,especially for extrapulmonary tuberculosis which was difficult to be diagnosed.