CAJ | 학술논문

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.
목적 이뢰형구단라선체외막단백A(OmpA)막단백기인Loa22거신호태기인편단구건중조잡개묘병대기면역단백진행표체분석.방법 이뢰형구단라선체56601주전기인조DNA위모판,PCR확증출Loa22성숙태기인편단,여대장간균-결핵분지간균천사정합질립pMV361일기분별경과쌍매절,련접,전화.사선감정출양성중조질립rpMV361-loa22,전전화입BCG,경사선감정후,열유도표체,통과SDS-PAGE급Western Blotting감정기표체산물.분별용BCG、rBCG-pMV361、rBCG-loa22、Loa22단백、멸활전구체면역소서량차후,탈구처사소서분리비림파세포,XTT비색법검측체외비림파세포증식활성.결과 PCR확증획득516 bp적편단,성공구건중조천사질립rpMV361-loa22,경전전화구건중조잡개묘성공,열유도표체출상대분자질량약19×10~3특이조대.체외비림파세포증식실험증실rBCG-loa22가인기세포반응,기증식활성여BCG조화rBCG-pMV361조상비차이구유통계학의의(P<0.05).결론 성공구건료뢰형구단라선체중조잡개묘rBCG-loa22병고효표체구유면역학활성적외막단백Loa22,위신일대구단라선체역묘연구타하료기출.
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.