中国莱姆病螺旋体FP1外膜蛋白A的克隆表达及其抗原性的初步研究

目的　克隆并表达中国莱姆病螺旋体阿弗西尼疏螺旋体基因种参照菌株FP1的外膜蛋白A(OspA) ,并对其抗原性进行初步研究和基因序列分析 ,为研制中国莱姆病疫苗提供资料。方法设计引物 ,用聚合酶链反应 (PCR)从莱姆病螺旋体FP1全基因组DNA中扩增出OspA编码基因 ,经酶切、连接 ,插入原核表达载体pET 11d ,构成重组质粒pET 11d ospA ,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。结果　rOspA在宿主菌内获得高效、稳定表达 ;相对分子质量 (Mr)约为 31× 10 3,Westernblot显示其与抗OspA的多抗有良好的免疫反应性 ;经测序显示该rOspA的基因片段长度为 783bp ,编码 2 6 1个氨基酸 ,与欧洲标准菌株Pko和瑞士标准菌株VS4 6 1的OspA的DNA碱基序列比较 ,同源性均为 94 %。结论　在国内首次成功地对中国莱姆病螺旋体阿弗西尼疏旋体基因种的代表菌株FP1的OspA基因进行了克隆和表达。并且证实rOspA有良好的抗原性 ,可进一步对其免疫保护性进行研究 ,以便为研制有效的莱姆病疫苗提供数据。

Cloning and expression of OspA gene from a Chinese strain of Borrelia burgdorferi FP1 and preliminary research on the antigenicity of rOspA

ObjectiveCloning and expressing OspA gene from a Chinese Borrelia burgdorferi strain FP1 which is the standard strain of the gene species Borrelia afzelii,and researching on the antigenicity of the rOspA to support Lyme disease vaccine in China. MethodsPCR and gene recombination technique were used to clone the OspA gene from a strain PD91. The ospA gene without its signal peptide sequence was inserted into an expression vector PET-11d and expressed in E.coli. The recombinant plasmid (PET-11d-ospA) was identified with enzyme cuting off and gene sequence comparison. The expressed protein (rOspA) was identified by SDS-PAGE,Western blot. ResultsThe rOspA was highly expressed in E.coli with molecular relative mass (M_r) as 31 000. The rOspA has strong immunological response to the antibodies against the OspA of FP1. The piece of genes coding OspA of FP1 is 783bp without its signal peptide sequence. ConclusionOspA gene from FP1 strains was successfully cloned and expressed. The rOspA has the same antigenicity as the OspA of FP1 strain.