摘要
目的 克隆并表达我国莱姆病螺旋体分离株 Bm p A即 P39基因 ,为制备莱姆病螺旋体基因工程重组抗原以用于我国莱姆病的基础与临床诊断研究奠定基础。方法 应用 PCR方法及基因重组技术对我国莱姆病螺旋体分离株 P39蛋白进行全基因克隆 ,并在此基础上去掉信号肽序列 ,将 P39基因插入原核表达载体 L KB2 ,在大肠杆菌 BL 2 1(DE3)中进行诱导表达。表达产物以 SDS— PAGE电泳、Western blot、薄层扫描分析。结果 成功地克隆了 P39基因 ,序列分析显示 ,P39基因全长 10 2 0 bp,其核苷酸序列及氨基酸序列同源性与国外分离株均较高 ,与 Sh- 2 - 82株皆为 99% ,P39基因在大肠杆菌中以天然蛋白形式得到高效表达 ,扫描分析其分子量为 37k Da,单株表达量占菌体总蛋白的 5 8% ,Western blot实验表明其可与莱姆病患者血清发生特异性反应。结论 成功克隆了莱姆病螺旋体国内分离株 P39蛋白基因并且在原核系统中得到高效表达。重组 P39蛋白具有较好的免疫活性 。
Aim Molecular cloning and expressing BmpA(P39) gene from a Borrelia burgdorferi strain isolated from China for preparing recombinant antigen to the basic and chinical diagnostic study on Lyme disease in China.Method PCR and gene recombination technique were used to clone the whole P39 gene from a strain BT01. The P39 gene was then inserted into a expression vector LKB2 without its signal peptide and expressed in E.coli. The expressed protein was identified by SDS-PAGE,Western blot, and scanning analysis.Results Sequence analysis showed the P39 gene was 1020 base pairs with high homology to foreign isolates.Especially it had 99% nucleotide and amino acid identity with the Sh 2 82 strain. The recombinant plasmid BT-p39 expressed the protein with high level and a native form in the host cell BL21(DE3),the protein was about 37KDa,and it accounted for 58% of total bacterial protein by scanning analysis. Westernblot assay further proved it had good antigenicty to the specific antibody from patients with Lyme disease. Conclusion The P39 gene of the Chinese Borrelia burgdorferi isolate was successfully cloned and highly expressed in prokaryotic expression system. The recombinant P39 protein may provide a promising candidate antigen for diagnostic usage in Lyme disease.
出处
《中国人兽共患病杂志》
CSCD
北大核心
2000年第4期9-11,共3页
Chinese Journal of Zoonoses
