摘要
目的 对中国莱姆病螺旋体伽氏疏螺旋体基因种参照菌株PD91的鞭毛蛋白编码基因进行克隆表达与基因序列分析。方法 设计引物,用聚合酶链反应(PCR)技术获得PD91的鞭毛蛋白编码基因片段,经酶切、连接,插入质粒pET-11d中,构成重组质粒pET11d-fla,转化致大肠埃希菌BL21,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,应用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和免疫印迹(WB)方法,鉴定重组蛋白及其抗原性。结果成功地获得了鞭毛蛋白的编码基因片段和基因重组,重组蛋白在宿主菌BL21中高效表达。WB结果显示重组鞭毛蛋白与PD91的鞭毛蛋白具有相同的抗原性。经测序显示该鞭毛蛋白的基因片段长度为1011bp,与北美莱姆病螺旋体标准株B31的DNA碱基序列及氨基酸序列比较分析,同源性分别为94.70％、95.85％。结论 在国内首次成功地对中国莱姆病螺旋体伽氏疏螺旋体基因种的鞭毛蛋白基因进行了克隆表达,并证实具有良好的抗原性,为莱姆病血清学诊断研究提供了较好的基础。
Objective To study the clonging and expression of flagellin gene from Chinese Borrelia burgdorferi ,PD91 strain and to evaluate the feasibility of using recombinant protein as diagnostic antigen when comparing the gene sequence with flagellin gene from North American Borrelia burgdorferi B31. Methods The piece of genes coding flagellin from Chinese Borrelia burgdorferi PD91 by polymerase chain reaction (PCR) method was obtained, and constructed recombinant plasmid, before transformed into E. coli BL21 strain,and induced. The recombinant plasmid was identified with enzyme cutoff and gene sequence comparison. Efficient expression strain was selected and the expression product was analyzed with sodium amplified polymorphic-polyacrylamide gel eloctrophoresis(SDS-PAGE) and Western-blot method. Results The recombinant protein (r-flagellin) expressed in host bacteria was successful. By means of western-blot assay, the immunological response showed the same antigenieity between r-flagellin and PD91 flagellin. The piece of genes coding flagellin of PD91 was 1011 bp, but when comparing with that of North American Borrelia burgdorferi it showed 94.70 % homology. Homology between the sequence of amino acid of the r-flagellin and that of B31 flagellin was 95.85 %. Conclusion Flagellin gene of Borrelia garinii of Chinese Lyme disease spirochete was successfully cloned and expressed for the first time. It was proved that the immunoreactivity of r-flagellin was the same as the natural flagellin.
出处
《中华流行病学杂志》
CAS
CSCD
北大核心
2004年第9期783-786,共4页
Chinese Journal of Epidemiology
基金
国家"十五"科技攻关资助项目(2001BA 705B07)
国家自然科学基金(31070820)
