【摘要】 目的选取钩端螺旋体黄疸出血群(56601株)外膜脂蛋白LipL32进行克隆、表达和纯化,以获得能应用于致病性钩体检测的纯化重组蛋白。方法用PCR法扩增黄疸出血群(56601株)外膜脂蛋白LipL32基因,与表达载体pET-30a连接并在大肠杆菌BL21(DE3)中表达重组蛋白,免疫印迹(WB)鉴定活性后用电洗脱法进行纯化。将纯化的重组蛋白用间接和夹心ELISA法应用于22株兔免疫血清的检测,检测结果与钩体显微镜凝集试验(MAT)进行比较。结果重组表达质粒在大肠杆菌中表达高产量的重组蛋白,WB结果显示重组蛋白具有免疫活性。纯化的重组蛋白对15株钩体标准致病株兔免疫血清全部检出,对7株腐生性钩体株兔免疫血清全部未检出,与MAT检测结果相符。结论黄疸出血群(56601株)外膜脂蛋白LipL32基因在大肠杆菌中成功表达,重组蛋白可应用于致病性钩体血清抗体的检测,为研制致病性钩体抗体检测试剂奠定了基础。更多还原

【Abstract】 The outer membrane lipoprotein LipL32 in pathogenic Leptospira icterohaemorrhagiae strain 56601 was cloned,expressed and purified in order to obtain the purified recombinant protein used for the detection of pathogenic Leptospira,in which the gene encoding the lipoprotein LipL32 was amplified by PCR and linked to expression vector pET-30a,and then expressed in E.coli BL21(DE3)to obtain the recombinant protein.The recombinant protein was identified by Western blotting and purified by electroelution.This purified protein was used in the indirect and sandwich ELISA assays to test the antibodies in sera of rabbits infected with leptospira,and the results were compared with those obtained by microscopy agglutination test(MAT).Results of this study showed that this recombinant protein was expressed with high yields in E.coli,meanwhile,the results demonstrated by Western blotting revealed that it possessed good antigenic activity.The antibodies from rabbits infected with pathogenic leptospira showed positive reactions with immune sera of 15 standard strains of leptospira,but negative reactions were found in immune sera of 7 strains of saprophytic leprospira.These results were in consistence with those obtained by MAT.These results suggest that this recombinant protein can be used as an antigen for the detection of antibodies against pathogenic leptospira.更多还原