中国莱姆病螺旋体PD_(91)外膜蛋白A的克隆表达及免疫保护性的初步研究

Molecular cloning and expression of OspA of a Chinese Borrelia burgdorferi PD_(91) and preliminary research on the protectivity of rOspA

目的 克隆并表达中国莱姆病螺旋体Borreliagarinii基因型代表菌株PD91 的外膜蛋白A(OspA) ,并对其免疫保护性进行初步研究 ,为进一步研制莱姆病疫苗提供基础。方法 用聚合酶链反应 (PCR)从莱姆病螺旋体PD91 全基因组DNA中将OspA基因调出 ,插入原核表达载体P4 2 ,在大肠杆菌BL2 1(DE3)中表达 ,表达产物用SDS PAGE、Westernblot分析 ,并进行基因序列测定。用重组OspA(rOspA)免疫新西兰家兔 ,用间接免疫荧光 (IFA)检测其血清特异性抗体 (IgG) ,并进行体外中和试验 ,从而对其免疫保护性有初步的了解。结果 rOspA在宿主菌内表达高效、稳定 ;Westernblot显示其与抗OspA的多抗有较好的免疫反应性 ;用rOspA免疫新西兰家兔后 ,其血清抗体 (IgG)效价显著升高 (32倍 ) ,体外中和试验表明 ,每毫升兔抗rOspA血清可杀灭 10 5个莱姆病螺旋体。结论 在国内首次成功地对中国莱姆病螺旋体Borreliagarinii基因型的OspA基因进行了克隆和表达。rOspA有较好的免疫保护性 。

Objective Cloning and expressing OspA gene from a Chinese Borrelia burgdorferi strain PD 91 which is the standard strain of the gene species Borrelia garinii , and to identify the protectivity of the rOspA for vaccine development in China. Methods PCR and gene recombination technique were used to clone the OspA gene from a strain PD 91 . The OspA gene was then inserted into an expression vector P42 without its signal peptide sequence and expressed in E.coli . The expressed protein was identified by SDS PAGE, Western blot and gene sequence. The rOspA was used to immunized Newzerland rabbits. IFA was used to test the antibody (IgG), and the in vitro neutralization experiment was used to test the protectivity of the rOspA. Results The rOspA was highly expressed in E.coli . Western blot analysis showed that the rOspA has induced a strong immunological response with antibodies against the OspA of PD 91 . After rabbits was immunized by the rOspA, the antibody titer in serum was at least as 32 times as it was before. The in vitro neutralization experiment showed that the antibodies against rOspA could kill 10 5 Lyme disease spirochetes. Conclusion OspA gene from one of the Chinese Lyme disease spirochetes was successfully cloned and expressed. The rOspA has strong protectivity against Borrelia burgdorferi , and can be function as a component of Lyme disease vaccine. [