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目的分析我国15群15型问号钩端螺旋体(简称钩体)参考标准株外膜脂蛋白lipL21基因序列,构建该基因原核表达系统并鉴定表达产物的免疫原性,了解lipL21基因自然表达状况。方法高保真PCR扩增上述问号钩体株及双曲钩体Patoc型PatocⅠ株基因组DNA中全长lipL21基因片段,T-A克隆后测序并构建其原核表达系统。分别用钩体TR/PatocⅠ和rLipL21兔抗血清为一抗的Western blot鉴定目的重组蛋白rLipL21的免疫原性,显微镜凝集试验(MAT)检测rLipL21兔抗血清的交叉凝集效价。以盐变-去垢剂处理法提取钩体外膜蛋白,用SDS-PAGE和免疫印迹法检测上述钩体株lipL21基因自然表达情况。结果上述钩体株均存在序列高度保守的lipL21基因,其核苷酸和氨基酸序列相似性分别为98．75％～99．82％和99．46％～100％。rLipL21能与TR/PatocⅠ及rLipl21兔抗血清发生结合反应。rLipL21免疫家兔能产生抗体,该抗体对上述15株问号钩体MAT效价为1：16～1：128。问号钩体外膜标本中均可检出LipL21,双曲钩体则否。结论我国钩体群参考标准株均含有序列保守的lipL21基因并自然表达于外膜,双曲钩体PatocⅠ株虽含有lipL21基因但未表达。rLipL21具有良好的抗原性和免疫反应性,有可能作为新型钩体疫苗或检测试剂盒的候选属特异性表面抗原之一。
OBJECTIVE: To analyze the lipL21 gene sequence of 15 reference strains of Leptospira interrogans (type) Leptospira interrogans in China, and to construct the prokaryotic expression system of the gene and to identify the immunogenicity of the expressed product to understand the natural expression of lipL21 gene. Methods Full-length lipL21 gene fragment was amplified from GenBank accession No. Patoc Patoc Ⅰ by high-fidelity PCR. The T-A cDNA fragment was cloned and sequenced. The prokaryotic expression system was constructed. The immunogenicity of the recombinant protein rLipL21 was identified by Western blot using the rabbit antisera of TR / PatocI and rLipL21 as the primary antibody. The cross-agglutination titer of rLipL21 rabbit antiserum was detected by microscopic agglutination test (MAT). The outer membrane protein of hook body was extracted by salt-detergent treatment and the natural expression of lipL21 gene was detected by SDS-PAGE and Western blotting. Results The sequences of the lipL21 gene were highly conserved among the above-mentioned leptospira strains. The nucleotide and amino acid sequences of the lipL21 gene were 98.75% -99.82% and 99.46% -100%, respectively. rLipL21 can bind to TR / Patoc I and rLipl21 rabbit antisera. Rabbits immunized with rLipL21 could produce antibodies with MAT titers of 1:16 to 1:12 for the above 15 ticks. Question mark hook body outer membrane specimens can be detected LipL21, double hook body is not. CONCLUSION: The reference strain of Chinese hook population contains the conserved lipL21 gene, which is naturally expressed on the outer membrane. Although the homologue Patoc Ⅰ strain contains lipL21 gene, it does not express lipL21 gene. rLipL21 has good antigenicity and immunoreactivity, and may be used as one of the candidate genus-specific surface antigens for the novel hook-like vaccine or detection kit.