目的克隆表达和鉴定问号钩端螺旋体(L.interrogans)黄疸出血群赖型赖株中一个新的外膜蛋白(Omp)A家族基因LA0301,研究LA0301编码蛋白的抗原性和在15个钩端螺旋体(简称钩体)血清群代表株中的保守性,探讨其在疫苗研究中的意义。方法生物信息学软件分析预测LA0301的特征。构建原核表达重组体pQE31-LA0301,经IPTG诱导后用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)及蛋白质印迹法(Western blot)鉴定表达情况。用表达的重组蛋白免疫BALB/c小鼠,Western印迹检测其免疫反应性和在不同血清型钩体中的保守性。酶联免疫吸附试验(ELISA)和Western印迹检测兔抗钩体全菌血清中的LA0301编码蛋白的抗体。结果生物信息学预测结果显示,LA0301具有OmpA家族的结构域。克隆表达了重组质粒pQE31-LA0301,重组蛋白能刺激BALB/c小鼠产生特异性抗体,效价为1:32000。在兔抗钩体全菌血清中检测到特异的LA0301蛋白抗体,并在15个血清群的代表株钩体中均可检测到LA0301蛋白。结论LA0301蛋白是问号钩体中一个新的OmpA家族蛋白,具有良好的抗原性和保守性,并且能在钩体感染的过程中刺激机体产生相应的抗体。为进一步研究钩体新型疫苗候选基因奠定了基础。

Objective To clone and express a novel OmpA gene (LAO301) from Leptospira interrogans , serogroup ictemhaemorrhageae serovar from the Lai strain and to analyze the antigenicity and conservation of this protein from fifteen different strains belonging to 15 prevalent semgmups of L. interrogans from China. Methods The characteristics of LA0301 were predicted by bioinformatics software.The prokaryotic expression system of LA0301 was constructed by routine molecular biological techniques and expression of the recombinant protein was identified by SDS-PAGE and Western blot after induction with IP1'G. Western blot was used to identify the antigenicity and the conservation of the expressed LA030I in fifteen strains from 15 prevalent serogroups in China.The LA0301 antibody from rabbit sera infected with whole leptospires from the Lai strain was identified by Western blot and F.IISA. Results LA0301 is a novel gene belonging to the OmpA family of L. interrogans . The prokaryotic expression system of LAO301 was constructed successfully. The anti-recombinant protein serum titer in BALB/c mouse was 1: 32, 000. The LA0301 protein was detected in all tested strains of L. interrogans . Rabbit serum infected with L. interrogans from the Lai strain was positive for recombinant protein antibody as detected by Western blot and Fl ISA. Conclusion LAO301, a novel gene belonging to the OmpA family, not only displays good antigenicity and conservation but also can elicit antibody responses in rabbits that are infected with L. interrogans from the Lai strain. Our studies on LA0301 provide a basis for developing novel vaccine candidate of leptospira.