问号钩端螺旋体疫苗候选基因LB061的抗原性和保守性研究

目的 克隆表达和鉴定问号钩端螺旋体黄疸出血群赖型赖株中疫苗候选基因LB061,研究LB061的免疫原性和在不同血清型钩端螺旋体菌中的保守性。方法 生物信息学软件分析预测LB061的特征。构建原核表达质粒pQE31-LB061,经IPTG诱导后用SDS-PAGE及Western印迹法鉴定表达情况。用表达的重组蛋白免疫BALB/c小鼠,Western印迹法检测其抗原性和在不同血清型钩端螺旋体中的保守性。Western印迹法检测钩端螺旋体全菌兔抗血清中的LB061抗体。结果 生物信息学预测结果显示,LB061含有DUF839家族结构域。成功克隆了重组质粒pQE31-LB061,表达的重组蛋白能刺激BALB/c小鼠产生抗体(效价为1∶32000),并能与相应抗体反应,具有良好的抗原性。在16株不同血清型的钩端螺旋体中均可检测到LB061蛋白的表达,并在钩端螺旋体赖株全菌兔抗血清中检测到其抗体。结论 LB061蛋白可以作为外膜蛋白刺激宿主免疫系统产生抗体,具有良好的抗原性和保守性。本研究为其作为疫苗候选基因的研究奠定了基础。

Research on antigenicity and conservation of Leptospira interrogans vaccine candidate gene LB061

Objective To clone and express the Leptospira interrogans(L.interrogans)vaccine candidate gene,LB061 and to analysis the immunogenicity and conservation of the recombinant protein in different serovars of L.interrogans.Methods The characters of LB061 gene were predicted by bioinformatics software.The prokaryotic expression system of the LB061 was constructed by routine molecular biological techniques.Expression of the recombinant LB061 protein was induced by IPTG,and confirmed by Western blot.Balb/C mice were immunized with ercombinant LB061 and immune serum was used to study the immunogenicity of LB061 and the conservation of expressed LB061 in sixteen different serovars.In addition,antibodies against LB061 in rabbit serum infected with L.interrogans strain Lai(56601)were examined by Western blot.Results LB061 is a putative outer membrane protein in L.interrogans as predicted by bioinformatical analysiss.The prokaryotic expression system of LB061 was constructed successfully.Serum titers of anti-recombinant LB061 antibodies in immunized BALB/c mice were 1:32000.LB061 was detected in sixteen different serovars of L.interrogans.LB061-specific antibodies were detected in rabbit serum infected wit L.interrogans strain Lai by Western blot.Conclusion The vaccine candidate gene,LB061,of L.interrogans serogroup,icterohemorrhagic serovar,Lai strain can elicit antibody responses in rabbit during infection with Leptospira interrogans.Our studies provide a basis for further investigation on the role of LB061 as a vaccine candidate gene.