中国莱姆病螺旋体鞭毛蛋白中央区的基因克隆和表达

目的　对中国莱姆病螺旋体Borreliagarinii基因种参照菌株PD91的鞭毛蛋白中央区的编码基因进行克隆表达 ,对重组鞭毛蛋白作为莱姆病血清学诊断抗原进行初步的研究 ,并进行基因序列和氨基酸序列分析。方法　设计引物 ,用PCR技术获得PD91的鞭毛蛋白中央区的编码基因片段 ,经酶切、连接 ,插入质粒pET 30a中 ,构成重组质粒pET30a mfla,转化到大肠杆菌BL2 1,提取质粒进行酶切、DNA测序和氨基酸序列分析鉴定 ,诱导表达 ,筛选高效表达株 ,应用SDS PAGE和Westernblot鉴定重组蛋白及其抗原性。结果　成功地获得了鞭毛蛋白中央区的编码基因片段和基因重组 ,重组蛋白在宿主菌BL2 1中高效表达。Westernblot结果显示重组鞭毛蛋白的中央区与PD91的鞭毛蛋白具有相同的抗原性。经测序显示该中央区基因片段为鞭毛蛋白基因的 4 0 9～ 786bp ,与北美莱姆病螺旋体标准株B31的DNA碱基序列比较分析 ,同源性 92 %。结论　成功地对中国莱姆病螺旋体Borreliagarinii基因种的鞭毛蛋白中央区进行了克隆表达 ,并证实具有抗原性 ,为我国莱姆病血清学诊断研究提供资料。

Cloning and expression of the central fragment of flagellin gene from the Chinese strain PD91 of Borrelia garinii

ObjectiveCloning and expressing the specific fragment of flagellin from Borrelia burgdorferi (PD91 strain),evaluating the feasibility of the recombinant protein as a diagnosis antigen,and comparing the gene sequence and amino acid sequence with flagellin gene from North American Borrelia burgdorferi B31. MethodsGenes coding Borrelia burgdorferi (PD91strain) from 409bp to 786bp was made by PCR method,and then,transformed them into E.coli BL21 strain. The recombinant plasmid was identified by enzyme cutoff and gene sequence comparison. The expression product was analyzed by Western blot method. ResultsThe recombinant protein expressed in host bacteria showed biologic function. By means of Western blot the immunological response of r-flagellin specific fragment was the same as PD91 flagellin. The piece of genes coding the specific fragment of flagellin is from 409-786bp of flagellin,and showed 92% homology with the reference strain. ConclusionThe specific fragment of flagellin gene of Chinese Borrelia burgdorferi was successfully cloned and expressed. And the product is a potential diagnostic antigen.