结核分枝杆菌多表位诊断抗原的制备
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摘要
将结核分枝杆菌(Mycobacterium tuberculosis,Mtb)多个B细胞预测表位串联表达(命名为B102),并初步评价其作为诊断抗原的血清学诊断价值。将MtbPstS1、ESAT6、CFP10、Ag85B、Ag85A及PPE54等6个蛋白的11个B细胞预测表位串联,加入合适的连接臂后全基因合成;将多表位片段插入带有TRX标签的表达质粒中,在大肠杆菌BL21(DE3)中诱导表达,并利用Ni~(2+)-Chelating亲和层析和DEAE阴离子交换层析纯化目的蛋白;利用Western blotting (WB)技术对目的蛋白抗原性进行鉴定,并建立Mtb抗体检测竞争法ELISA技术,初步评价此方法对阴阳血清样本的鉴别能力。目的蛋白以包涵体形式存在,其表达量约占菌体总蛋白的31.25%,经纯化及复性后蛋白B102可溶性存在,浓度为3.124mg/mL,纯度为96.71%;WB实验表明目的蛋白能与Mtb阳性血清相应抗体发生反应。对60份Mtb阳性血清及60份Mtb阴性血清进行检测得出其灵敏度为90.00%,特异性为93.33%,阳性预测值为93.10%,阴性预测值为90.32%,符合率为91.67%,McNemer检验的结果提示与"金标准"诊断结果无差异,Kappa=0.833,提示两种方法诊断结果一致性优异。原核表达与层析纯化可以获取抗原性优异的Mtb多表位诊断抗原,作为诊断抗原可以应用于Mtb的血清学检测中。
Multi-epitope recombinant diagnostic antigen(designated 'B102') of Mycobacterium tuberculosis(Mtb) was prepared and evaluated as a serological diagnostic antigen. With TRX at the N-terminal and His tag at the C-terminal, the multi-epitope Mtb recombinant diagnostic antigen including 11 predicted B-cell epitopes from 6 Mtb antigens(PstS1, ESAT6,CFP10, Ag85 B, Ag85 A and PPE54) was expressed in Escherichia coli BL21(DE3) and purified by Ni~(2+)-Chelating affinity and DEAE anion exchange chromatography. Based on the antigenicity of B102 confirmed in Western blotting analysis, we constructed and evaluated a double-antigen sandwich ELISA for diagnosis of Mtb infection. The protein B102 exists in the form of inclusion bodies, accounting for 31.25% of the total proteins of the bacteria. After purification and renaturation,protein B102 exists in soluble form with the concentration 3.124 mg/mL and the homogeneity 96.71%. WB analysis demonstrated that protein B102 could react with antibodies in Mtb positive serum. Using the novel antigen in ELISA, we tested 60 Mtb-related positive and negative serum; The results showed the sensitivity, specificity, positive and negative predictive values and coincidence rate of the detection method is 90.00%, 93.33%, 93.10%, 90.32% and 91.67%, respectively.The McNemer analysis suggested there was no statistical difference between the 'Gold standard' and the novel ELISA with kappa 0.833, which suggested the excellent consistency. By prokaryotic expression and chromatography purification, the multi-epitope recombinant antigen B102 was obtained with excellent antigenicity, which could be applied for Mtb-related serological detection.
Multi-epitope recombinant diagnostic antigen(designated 'B102') of Mycobacterium tuberculosis(Mtb) was prepared and evaluated as a serological diagnostic antigen. With TRX at the N-terminal and His tag at the C-terminal, the multi-epitope Mtb recombinant diagnostic antigen including 11 predicted B-cell epitopes from 6 Mtb antigens(PstS1, ESAT6,CFP10, Ag85 B, Ag85 A and PPE54) was expressed in Escherichia coli BL21(DE3) and purified by Ni~(2+)-Chelating affinity and DEAE anion exchange chromatography. Based on the antigenicity of B102 confirmed in Western blotting analysis, we constructed and evaluated a double-antigen sandwich ELISA for diagnosis of Mtb infection. The protein B102 exists in the form of inclusion bodies, accounting for 31.25% of the total proteins of the bacteria. After purification and renaturation,protein B102 exists in soluble form with the concentration 3.124 mg/mL and the homogeneity 96.71%. WB analysis demonstrated that protein B102 could react with antibodies in Mtb positive serum. Using the novel antigen in ELISA, we tested 60 Mtb-related positive and negative serum; The results showed the sensitivity, specificity, positive and negative predictive values and coincidence rate of the detection method is 90.00%, 93.33%, 93.10%, 90.32% and 91.67%, respectively.The McNemer analysis suggested there was no statistical difference between the 'Gold standard' and the novel ELISA with kappa 0.833, which suggested the excellent consistency. By prokaryotic expression and chromatography purification, the multi-epitope recombinant antigen B102 was obtained with excellent antigenicity, which could be applied for Mtb-related serological detection.
引文
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[20]Huang YT,Lu XM,Yang XR,et al.Renaturation technology of recombinant fusion protein Trx-IFN-CSP.Biotechnol Bull,2016,32(6):219-225(in Chinese).黄演婷,卢雪梅,杨小蓉,等.重组融合蛋白Trx-IFN-CSP复性工艺研究.生物技术通报,2016,32(6):219-225.
[21]Li SJ,Zhang ZG,Yu Q,et al.Application value of detecting Mycobacterium tuberculosis specific IgG/IgM antibodies for tuberculosis diagnosis with colloidal gold immunochromatography assay.Chin JZoon,2018,34(2):139-144(in Chinese).李霜君,张治国,余琴,等.胶体金免疫层析法检测结核分枝杆菌特异性IgG/IgM抗体对结核病诊断的应用价值.中国人兽共患病学报,2018,34(2):139-144.
[2]Steingart KR,Ramsay A,Pai M.Optimizing sputum smear microscopy for the diagnosis of pulmonary tuberculosis.Expert Rev Anti-Infect Therapy,20075(3):327-331.
[3]Diel R.Long-term effect of bacille calmette-Guérin vaccination in tuberculin skin testing:a new reality for TB prevention.Chest,2017,152(2):235-236.
[4]Casela M,Cerqueira SMA,de Oliveira Casela T,et al.Rapid molecular test for tuberculosis:impact of its routine use at a referral hospital.J Bras Pneumol2018,44(2):112-117.
[5]Bekmurzayeva A,Sypabekova M,Kanayeva DTuberculosis diagnosis using immunodominant secreted antigens of Mycobacterium tuberculosis Tuberculosis,2013,93(4):381-388.
[6]Steingart KR,Henry M,Laal S,et al.Commercial serological antibody detection tests for the diagnosis of pulmonary tuberculosis:a systematic review.PLoSMed,2007,4(6):e202.
[7]Nguyen TLT,Sarmiento ME,Calero R,et al.Immunoinformatics study on highly expressed Mycobacterium tuberculosis genes during infection Tuberculosis,2014,94(5):475-481.
[8]Zhu ZY,Zhang D,Wang HB,et al.Expression and serological diagnosis of Mycobacterium tuberculosis CFP-10 and Rv2626c proteins.Genet Mol Res,2014,13(3):7398-7406.
[9]Luo Q,Li SJ,Xiao TY,et al.Cloning expression and serological evaluation on Mycobacterium tuberculosis four new antigens.Chin J Epidemiol2018,39(4):514-518(in Chinese).罗巧,李霜君,肖彤洋,等.结核分枝杆菌4种新抗原的克隆表达及血清学评价.中华流行病学杂志,2018,39(4):514-518.
[10]Burbelo PD,Keller J,Wagner J,et al.Serological diagnosis of pulmonary Mycobacterium tuberculosis infection by LIPS using a multiple antigen mixture.BMC Microbiol,2015,15:205.
[11]Infantes-Lorenzo JA,Whitehead CE,Moreno I,et al.Development and evaluation of a serological assay for the diagnosis of tuberculosis in alpacas and llamas.Front Vet Sci,2018,5:189.
[12]Wang S,Wu J,Chen JZ,et al.Evaluation of Mycobacterium tuberculosis-specific antibody responses for the discrimination of active and latent tuberculosis infection.Int J Infect Dis,2018,70:1-9.
[13]Yan ZH,Yi L,Wei PJ,et al.Evaluation of panels of Mycobacterium tuberculosis antigens for serodiagnosis of tuberculosis.Int J Tubercul Lung Dis,2018,22(8):959-965.
[14]Pukazhvanthen P,Anbarasu D,Basirudeen SA,et al.Assessing humoral immune response of 4recombinant antigens for serodiagnosis of tuberculosis.Tuberculosis,2014,94(6):622-633.
[15]Andersen P.The T cell response to secreted antigens of Mycobacterium tuberculosis.Immunobiology,1994,191(4/5):537-547.
[16]Sun L,Min CY,Qian Y,et al.Progress on PE/PPEproteins of mycobacterium tuberculosis.Chin J Zoon,2013,29(5):499-503(in Chinese).孙林,闵晨雨,钱源,等.结核分枝杆菌PE/PPE蛋白研究进展.中国人兽共患病学报,2013,29(5):499-503.
[17]He XY,Zhuang YH,Zhang XG.The immunogenic characteristics of the recombinant 38 000 antigen from Mycobacterium tuberculosis.Chin J Tubercul Respirat Dis,2000,23(8):485-488(in Chinese).何秀云,庄玉辉,张晓刚.结核分支杆菌重组38000蛋白的免疫原性特征.中华结核和呼吸杂志,2000,23(8):485-488.
[18]Dillon DC,Alderson MR,Day CH,et al.Molecular and immunological characterization of Mycobacterium tuberculosis CFP-10,an immunodiagnostic antigen missing in Mycobacterium bovis BCG.J Clin Microbiol,2000,38(9):3285-3290.
[19]Chai XM,Li YL.The research progression of tuberculosis vaccine on antigen85 complex.Chin JAntitubercul,2013,35(4):282-285(in Chinese).柴雪敏,黎友伦.结核分枝杆菌Ag85复合物相关疫苗研究进展.中国防痨杂志,2013,35(4):282-285.
[20]Huang YT,Lu XM,Yang XR,et al.Renaturation technology of recombinant fusion protein Trx-IFN-CSP.Biotechnol Bull,2016,32(6):219-225(in Chinese).黄演婷,卢雪梅,杨小蓉,等.重组融合蛋白Trx-IFN-CSP复性工艺研究.生物技术通报,2016,32(6):219-225.
[21]Li SJ,Zhang ZG,Yu Q,et al.Application value of detecting Mycobacterium tuberculosis specific IgG/IgM antibodies for tuberculosis diagnosis with colloidal gold immunochromatography assay.Chin JZoon,2018,34(2):139-144(in Chinese).李霜君,张治国,余琴,等.胶体金免疫层析法检测结核分枝杆菌特异性IgG/IgM抗体对结核病诊断的应用价值.中国人兽共患病学报,2018,34(2):139-144.