问号赖型钩体与七日热型钩体内鞭毛蛋白基因的克隆及序列分析

Cloning and Sequence Analysis of the Flagellin Gene from Leptospira Interrogans Serovar Lai and Serovar Hebdomadis

目的 筛选钩体核酸疫苗的候选株 ,并从基因水平解释钩体血清型的特异性。方法 通过 PCR方法扩增赖型 0 17株及七日热型 5 6 6 10株钩体的内鞭毛蛋白 (fla B)基因 ;经 T/A快速克隆法将 PCR产物连接于PGEM- T Easy Vector载体。用 α-互补法、PCR法及酶切分析鉴定重组质粒 ,获得了重组质粒 p DHTF0 17和p DHTF6 10 ;双脱氧终止法对两个重组质粒中的克隆化 fla B基因进行测序。结果 两型 fla B基因长 85 2 bp,编码2 83个氨基酸。两个基因的限制性内切酶位点相似 ,含多个常见酶切位点。基因编码的蛋白质预测相对分子质量为31.3× 10 3。结论 几个血清型的 fla B基因同源性达 90 %～ 99%。

Objective To screen the candidate strain of Leptospiral nucleic acid vaccine and explain serovar differentiation. Methods We amplified the endoflagellin flaB gene from Leptospira interrogans serovar Lai strain 017 and serovar hebdomadis strain 56610 with PCR and cloned the flaB gene into the PGEM T Easy Vector with the method of T/A directly cloning. Two recombinant plasmids (pDHTF017 and pDHTF610) were established by identification using PCR and restriction enzymes digestion. The two cloned flaB genes were sequenced with Sanger's DNA sequencing method. Results The DNA sequencing result indicated that the flaB genes both contain 852bp and encode 283 amino acids. By analysis of the two flaB genes with software, it was found that their restriction sites are similar and include many general restriction enzymes. Conclusion Homologous analysis of DNA sequence of the flaB gene from different serovars reveals that the homology between different serovars is very high (90% 99%).