中国B.afzelii基因型莱姆病螺旋体GDsh1表面蛋白VlsE保守区段的克隆表达及抗原性分析

Cloning and expressing a conserved region of surface protein VlsE gene from a Chinese Borrelia afzelii strain GDsh1and analyzing antigenicity of rVlsE protein

目的克隆表达中国莱姆病螺旋体B.afzelii基因型菌株GDsh1的表面蛋白VlsE保守区段,并对其抗原性进行分析,为制备中国莱姆病重组抗原ELISA检测试剂盒提供依据。方法结合文献,下载并比对PubMed上所有莱姆病螺旋体B.a型菌株的VlsE基因序列,确定保守区段,设计引物,扩增GDsh1的VlsE基因片段。将扩增产物与载体PET-32a连接,转入大肠杆菌BL21(DE3)中表达。对重组载体进行序列测定,表达产物用SDS-PAGE和Western blot分析。利用重组VlsE蛋白制备ELISA试剂盒,检测83份莱姆病阳性血清,90份阴性血清以及90份梅毒血清,计算重组试剂盒的灵敏度和特异性。并与科室已有的全菌蛋白ELISA试剂盒检测结果进行比较。结果成功克隆表达了B.afzelii型VlsE保守区蛋白,Western blot结果显示VlsE保守区蛋白与免疫兔血清有较强的抗原抗体反应。ELISA结果表明:重组VlsE蛋白的灵敏度60.2%低于全菌蛋白的灵敏度92.8%(P<0.001);特异性分别为73.3%、68.9%,差异无统计学意义(P=0.511)。在检测梅毒血清上特异性分别为83.3%、18.9%,重组蛋白的特异性远远高于全菌蛋白(P<0.001)。结论重组VlsE基因保守区段蛋白在莱姆病的检测中具有一定的灵敏度和特异性,且在区分梅毒血清与莱姆病血清上,其特异性远远高于全菌蛋白,在莱姆病血清学检测中具有不容忽视的重要意义。

We cloned and expressed a conserved region of surface protein VIsE gene from a Chinese Borrelia afzelii strain GDshl, and analyzed the antigenicity of the rVlsE protein to support the development of Chinese ELISA recombinant antigen detection kit for Lyme disease. According to literature, we downloaded and aligned VIsE sequences of all B. afzelii strains from PuhMed to identify conserved segment and design primers. The target fragment was amplified and inserted into an expres- sion vector PET-32a and expressed in E. coli B21. The expressed protein was identified by SDS-PAGE, western blot and gene sequence. ELISA was performed with the rVlsE protein to test 83 Lyme disease sera, 90 negative serum samples and 90 sam- ples of syphilis serum, thereby to determine the sensitivity and specificity of the rVlsE protein. SDS-PAGE analysis showed the conserved region was successful expressed in E. coli. Western blot results confirmed that recombinant protein was able to re- sponse with immune rabbit serum. ELISA results showed that the sensitivity of rVlsE protein and the whole borrelia burgdor- feri protein were 60.2% and 92.8%（P〈0. 001）, the specificity were 73.3% and 68.9%（P=0. 511）. The specificity of this method in the detection of syphilis serum was 83.3%, much higher than that using the whole bacterial protein（18.9% specificity） （P〈0. 001）. The method using conserved fragments of VIsE protein has certain sensitivity and specificity in the distinguishing of syphilis and Lyme serum samples, its specificity is much higher than the whole cell protein. Therefore, the conserved fragments of VIsE in Lyme disease serological detection should not be ignored.