问号钩端螺旋体脂蛋白LipL32膜定位及其免疫应答

Membrane location of Leptospira interrogans lipoprotein LipL32 and LipL32-associated immune responses

目的确定问号钩端螺旋体（简称钩体）属特异性脂蛋白抗原LipL32膜定位及其自然抗体应答情况和抗体类型。方法IPTG诱导目的重组蛋白rLipL32-1和rLipL32-2表达，Ni-NTA亲和层析法提纯rLipL32。采用显微镜凝集试验（MAT）检测四川地区钩体患者血清标本及rLipL32兔抗血清与我国问号钩体参考标准株的交叉凝集情况。采用胶体金免疫电镜技术对LipL32进行膜定位。建立基于rLipL32的ELISA，检测钩体患者血清中特异性抗体类型及其水平。结果黄疸出血群是四川地区最主要的优势钩体血清群。rLipL32兔抗血清均能与我国问号钩体参考标准株发生MAT效价为1：80～1：320的交叉凝集反应。LipL32是位于钩体外膜表面的蛋白质分子。156例MAT阳性钩体患者血清标本中，rLipL32-1和rLipL32-2特异性IgM阳性率分别为91．0％～92．9％和90．4％～92．3％，特异性IgG阳性率分别为99．4％和97．4％～98．1％。结论LipL32是问号钩体属特异性表面蛋白抗原。自然感染钩体时，LipL32-1和LipL32-2可诱导机体产生IgM和IgG两类血清抗体。rLipL32-1和rLipL32-2可作为研制检测试剂盒的候选抗原。

Objective To determine the location on the outer envelope and natural antibody response and its types of genus-speciflc lipoprotein antigen LipL32 of Leptospira interrogans. Methods The target recombinant proteins rLipL32-1 and rLipL32-2 were expressed under inducement of IPTG. Ni-NTA affinity chromatography was performed to extract rLipL32. Microscope agglutination test （MAT） was used to examine the leptospirosis patients＇ serum samples from Sichuan area, China, and the cross immunoagglutination of rabbit anti -rLipL32 sera and L. interrogans reference strains in China. Immune aurosol electron microscopy was selected to locate the position of LipL32 on the leptospiral envelope. ELISA based on rLipL32 was established to confirm the level and types of specific antibody. Results L. interrogans serogroup Icterohaemorrhagiae remains to be the most dominant leptospiral serogroup in Sichuan area. Both the rabbit anti-rLipL32 sera could produce the cross immuno-agglutinations to all the L. interrogans reference strains with MAT titers of 1 ： 80-1 ： 320. LipL32 was the protein molecules that located on the external surface of leptospiral envelope. In the 156 cases of MAT positive leptospirosis patients＇ serum samples, the positive rates for rLipL32-1 or rLipL32-2 specific IgM were 91.0%- 92.9% and 90.4%-92.3%, respectively, while for rLipL32-1 or rLipL32-2 specific IgG were 99.4% and 97.4% -98.1%, respectively. Conlcusion LipL32 are the genus-specific superficial protein antigen of L interrogans. Both the LipL32-1 and LipL32-2 can induce serum antibodies IgM and IgG with extensive antigeniccross reaction during naturally infection with L. interrogans in populations. Therefore, rLipL32-1 or rLipL32-2 can be used as the antigen candidate for detection kit.