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目的 构建LTB- LipL32/ 1和CTB- LipL32 / 1融合基因原核表达系统并鉴定其表达产物的免疫和佐剂活性及保护作用,了解问号钩端螺旋体(简称钩体)野生株LipL32基因携带、表达及钩体病人血清LipL32基因产物抗体产生频率。方法 采用常规分子生物学技术构建LTB- LipL32 / 1和CTB- LipL32 /1融合基因及其原核表达系统。采用SDS PAGE检测目的重组蛋白rLTB -LipL32 / 1及rCTB -LipL32 / 1表达情况。分别采用Westernblot和GM1 -ELISA检测rLTB -LipL32 / 1及rCTB -LipL32 /1的免疫反应性和佐剂活性。采用PCR和MAT分别检测97株问号钩体野生株LipL32基因及其表达情况。采用ELISA检测2 2 8例钩体病人血清LipL32基因产物的抗体。采用豚鼠保护试验检测重组蛋白的免疫保护作用。结果 与报道的相关序列比较,LTB -LipL32 /1和CTB- LipL32 / 1融合基因核苷酸序列相似性分别为99.12 %～99.71%和98.5 4 %～99.4 2 % ,氨基酸序列相似性分别为97.5 8%～99.6 3%和96 .77%～99.6 3%。rLTB- LipL32 1和rCTB- LipL32 /1表达产量均约为细菌总蛋白的10 % ,并主要以包涵体形式存在。rLTB -LipL32 / 1和rCTB- LipL32 /1均分别能与LipL32 /1兔抗血清和牛GM1结合。97.9%(95 97)问号钩体野生株含有LipL32基因,95 .9% (93 97)问?
OBJECTIVE: To construct the prokaryotic expression system of LTB-LipL32 / 1 and CTB-LipL32 / 1 fusion gene and to identify the immune and adjuvant activity and the protective effect of the expressed product, and to understand the expression of LipL32 gene in the wild strain of Leptospira interrogans Lysate patient serum LipL32 gene product antibody production frequency. Methods The fusion gene LTB-LipL32 / 1 and CTB-LipL32 / 1 and their prokaryotic expression system were constructed by conventional molecular biology techniques. SDS PAGE was used to detect the expression of the target recombinant proteins rLTB-LipL32 / 1 and rCTB-LipL32 / 1. The immunoreactivity and adjuvant activity of rLTB-LipL32 / 1 and rCTB-LipL32 / 1 were detected by Western blot and GM1-ELISA, respectively. PCR and MAT were used to detect the 97 strains of Leptospira wild-type strain LipL32 gene and its expression. Antibody of serum LipL32 gene product was detected by ELISA in 238 cases of leptospira patients. Guinea pig protection assay was used to test the protective effect of recombinant protein. Results The nucleotide sequences of LTB-LipL32 / 1 and CTB-LipL32 / 1 fusion genes were 99.12% -99.71% and 98.5% -99.42%, respectively, with similar amino acid sequence identities of 97.5 8% ~ 99.6 3% and 96.77% ~ 99.6 3%. The yield of both rLTB-LipL32 1 and rCTB-LipL32 / 1 was about 10% of the total bacterial proteins, and mainly existed as inclusion bodies. Both rLTB-LipL32 / 1 and rCTB-LipL32 / 1 were able to bind to LipL32 / 1 rabbit antiserum and bovine GM1, respectively. 97.9% (95 97) Questionnaire hook wild-type plants contain LipL32 gene, 95.9% (93 97) ask?