我国4个钩端螺旋体血清群lipL21基因序列分析及其表达产物的鉴定

Sequence analysis of lipL21 genes from the dominant serogroups of Leptospira interrogans prevalent in China and identification of the expression products

目的本文采用高保真PCR从我国主要流行的问号钩体黄疸出血群赖型56601株、波摩那群波摩那型56608株、流感伤寒群临型56609株及腐生性双曲钩体参考标准株三宝垄群patoc型PatocⅠ株基因组DNA中扩增了全长lipL21基因片段。序列分析结果表明,所克隆的4株钩体lipL21基因与已报道的相应序列(GenBankNo.:AY187271)比较,其核苷酸和氨基酸序列相似性分别高达99.64～99.82%和99.46～100%。所构建的问号钩体黄疸出血群赖型56601株lipL21基因原核表达系统在IPTG诱导下,能有效地表达目的重组蛋白rLipL21,其产量约为细菌总蛋白的10%。Westernblot结果证实,兔抗钩体TR/patocⅠ属特异性抗原血清能识别rLipL21并与之结合。上述实验结果提示,lipL21基因序列非常保守,其表达产物有良好免疫原性,可作为研制通用型钩体基因工程疫苗的候选抗原。

In this study, the entire lipL21 gene fragments from L. interrogans serogroup Icterohaemorrhagiae serovar lai strain 56601, serogroup pomona serovar pomona strain 56608, serogroup grippotyphosa serovar lin strain 56609 and saprophytic L. biflexa serogroup Semaranga serovar patoc strain Patoc I were amplified by using high fidelity PCR. The sequencing results indicated that in comparison with the reported corresponding sequences （GenBank No. ：AY187271） the similarities of nucleotide and putative amino acid sequences of the cloned lipL21 genes from these four leptospiral strains were as high as 99.64% - 99.82% and 99.46% - 100 %, respectively. The constructed prokaryotic expression system of the lipL21 gene of L. interrogans serogroup Icterohaemorrhagiae serovar lai strain 56601 could efficiently express the target recombinant protein rLipL21 under inducment of IPTG, and the rLipL21 output was approximate 10 % of the total bacterial proteins. The Western blot assay demonstrated that the rabbit antiserum against leptospiral genus-specific TR/patoc I antigen could recognize rLipL21 as well as combine with the lipoprotein. All these results reach a conclusion that lipL21 gene is highly sequence-conserved and its expression products have fine immunogenicity, indicating the potential use of the lipoprotein as an antigen candidate for developing universal genetic engineering vaccine of Leptospira.