赖型钩端螺旋体OmpA外膜基因Loa22重组卡介苗的构建及其免疫蛋白表达

目的 以赖型钩端螺旋体外膜蛋白A(OmpA)膜蛋白基因Loa22去信号肽基因片段构建重组卡介苗并对其免疫蛋白进行表达分析.方法 以赖型钩端螺旋体56601株全基因组DNA为模板,PCR扩增出Loa22成熟肽基因片段,与大肠杆菌-结核分枝杆菌穿梭整合质粒pMV361一起分别经过双酶切,连接,转化.筛选鉴定出阳性重组质粒rpMV361-loa22,电转化入BCG,经筛选鉴定后,热诱导表达,通过SDS-PAGE及Western Blotting鉴定其表达产物.分别用BCG、rBCG-pMV361、rBCG-loa22、Loa22蛋白、灭活全钩体免疫小鼠两次后,脱臼处死小鼠分离脾淋巴细胞,XTT比色法检测体外脾淋巴细胞增殖活性.结果 PCR扩增获得516 bp的片段,成功构建重组穿梭质粒rpMV361-loa22,经电转化构建重组卡介苗成功,热诱导表达出相对分子质量约19×10~3特异条带.体外脾淋巴细胞增殖实验证实rBCG-loa22可引起细胞反应,其增殖活性与BCG组和rBCG-pMV361组相比差异具有统计学意义(P<0.05).结论 成功构建了赖型钩端螺旋体重组卡介苗rBCG-loa22并高效表达具有免疫学活性的外膜蛋白Loa22,为新一代钩端螺旋体疫苗研究打下了基础.

Construction and Expression of Recombinant Mycobacterium bovis BCG with the OmpA-like Membrane Protein Gene Loa22 of Leptospira Interrogans Serovar Lai

Objective To study the immunity of Loa22 from Leptospira interrogans serovar Lai strain 56601 by expressing its protein in BCG. Methods Amplified the mature peptide of Loa22 gene from the genome of of Leptospira interrogans serovar Lai strain 56601 and constructed recombinant plasmid rpMV361-loa22 with the E.coli-BCG integrating shuttle plasmid pMV361 and the Loa22 mature peptide gene. The rpMV361-loa22 plasmid was transformed into BCG by electroporation. The rBCG bearing rpMV361-loa22 was induced by high temperature of 45 ℃ and expressed protein was identified by SDS-PAGE and Western Blotting. Fifth 6-week-old BALB/c mice were randomly divided into five groups, which were inoculated intraperitoneally two times at 0-day and 21-day with BCG,rBCG-pMV361,rBCG-loa22,Loa22 and killed whole-leptospires respectively. All animals were dislocated from cervical vertebra on the 14~(th) day after the last immunization. The proliferative reaction of splenic lymphocyte in vitro were tested by XTT. Results The rpMV361-loa22 plasmid was constructed successfully and transformed into BCG. The rBCG expressed a 19×10~3 specifical protein identified by SDS-PAGE and Western Blotting. The splenic lymphocyte proliferate activity (SI) in rBCG-loa22 group in vitro was significantly higher than those in BCG group and rBCG-pMV361 group. Conclusion We explored the expressing feasibility of Loa22 in Mycobacterium bovis BCG, may therefore make further researches on the induction of protective immunity against human and animal leptospirosis.