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目的构建问号钩端螺旋体(简称钩体)lipL32/1-lipL41/2融合基因及其原核表达系统并鉴定其表达产物的免疫性。方法采用连接引物PCR构建lipL32/1-lipL41/2融合基因,常规方法构建其原核表达系统。采用SDS-PAGE和BioRad凝胶图象分析系统检测目的重组蛋白rLipL32/1-LipL41/2表达情况。采用Western blot鉴定rLipL32/1-LipL41/2的免疫反应性。采用ELISA检测228例钩体病人血清中lipL32/1、lipL41/2基因和lipL32/1-lipL41/2融合基因表达产物的抗体。结果 lipL32/1-lipL41/2融合基因核苷酸和氨基酸序列与报道的相关序列相似性分别为99 .9 %和99 .8 %。rLi-pL32/1-LipL41/2表达量约为细菌总蛋白的10 %。rLipL32/1和rLipL41/2兔抗血清均能识别并与rLipL32/1-LipL41/2结合。97 .4 %、78 .5 %和99 .1 %病人血清rLipL32/1、rLipL41/2和rLipL32/1-LipL41/2抗体阳性。结论本研究成功地构建了问号钩体lipL32/1-lipL41/2融合基因及其原核表达系统,rLipL32/1-LipL41/2不仅同时具有两个单一重组抗原的免疫反应性,且能提高钩体病人血清中抗体检测阳性率,可作为研制钩体属特异性基因工程疫苗或检测试剂盒的候选抗原。
Objective To construct the lipL32 / 1-lipL41 / 2 fusion gene of Leptospira interrogans (Leptospira interrogans) and its prokaryotic expression system and identify the immunogenicity of its expressed product. Methods The lipL32 / 1-lipL41 / 2 fusion gene was constructed by PCR and the prokaryotic expression system was constructed by conventional methods. The expression of the target recombinant protein rLipL32 / 1-LipL41 / 2 was detected by SDS-PAGE and BioRad gel image analysis system. The immunoreactivity of rLipL32 / 1-LipL41 / 2 was identified by Western blot. The antibodies of lipL32 / 1, lipL41 / 2 gene and lipL32 / 1-lipL41 / 2 fusion gene expression in serum of 228 leptospira patients were detected by ELISA. Results The nucleotide and amino acid sequences of the lipL32 / 1-lipL41 / 2 fusion gene were 99.9% and 99.8% similar to the reported sequences, respectively. The expression level of rLi-pL32 / 1-LipL41 / 2 is about 10% of the total bacterial protein. Both rLipL32 / 1 and rLipL41 / 2 rabbit antisera were recognized and bound to rLipL32 / 1-LipL41 / 2. 97.4%, 78.5%, and 99.1% of patients were positive for rLipL32 / 1, rLipL41 / 2 and rLipL32 / 1-LipL41 / 2 antibodies. Conclusions This study successfully constructed the fusion gene of lipL32 / 1-lipL41 / 2 and its prokaryotic expression system. The rLipL32 / 1-LipL41 / 2 not only has the immunoreactivity of two single recombinant antigens simultaneously, The positive rate of antibody detection in the patient serum can be used as a candidate antigen for the development of a genetically-engineered vaccine or detection kit for Leptospira.