结核分枝杆菌多表位诊断抗原的制备

Preparation of multi-epitope recombinant diagnostic antigen of Mycobacterium tuberculosis

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Mtb PstS1、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Ni2+-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3.124 mg/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。

Abstract:

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, Ag85B, Ag85A and PPE54) was expressed in Escherichia coli BL21 (DE3) and purified by Ni2+-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.