问号赖型钩端螺旋体重组质粒pDL121及其表达产物的初步研究

目的:探讨问号赖型钩端螺旋体重组质粒pDL121外源基因及其表达产物23 kDa蛋白的特点。方法:用6种内切酶对pDL121行酶谱分析,用Digoxin标记的pDL121外源基因片段作探针对不同种属的钩体进行杂交,用SDS-PAGE制备23 kDa蛋白,Western blot鉴定其免疫原性。结果:pDL121外源基因没有6种内切酶的酶切位点,重组探针与致病性钩体(serovar lai strain 017,serovar hebdomadis strain56610,serovar pomona strain 56608)有杂交信号,与非致病性钩体(serovar patoc strain Patoc I,serovar illini strain 3055)无杂交信号,亦不识别大肠杆菌。23 kDa兔抗血清可识别pDL121体外表达的23 kDa蛋白带和赖型钩体017株超声抗原成份;其抗体滴度为1/12800;用pDL121细菌裂解液主动免疫豚鼠,可使豚鼠抵抗强毒力株钩体攻击。结论:pDL121外源基因可能是赖型钩体的一个新基因;该重组探针能鉴别致病性钩体和非致病性钩体;23 kDa抗原有良好的免疫原性,可能是赖型钩体017株的保护性抗原。

Preliminary study on the recombinant plasmid pDL121 of leptospira in terrogans serovar Iai and its 23 kDa antigen

AIM: To study the recombinant plasmid pDL121 and its expression product in E.coli. METHODS: pDL121 was analysed by using 6 different restriction endonucleases and dot blotting, and the isolated 23 kDa protein band was cut and injected twice into rabbits to raise anti-23 kDa serum. RESULTS: The restriction map of pDL121 was quite different from other leptospiral genes reported and digoxin labeled recombinant DNA probe of pDL121 could detect the pathogenic leptospires, whereas, not the nonpathogenic leptospires. The anti-23 kDa serum could recognize the sonicated antigen of L.interrogans serovar lai strain 017 and 23 kDa protein expressed in pDL121 and the titer of the antiserum were very high, approximately 1/12 800. Injection of the E.coli lysate of pDL121 with Freund's adjuvant into guinea pigs resulted in some protection of the animal against the challenge with strain 017. CONCLUSION: It indicated that 23 kDa protein had good imunogenicity and could serve as a candidate for protective antigen of L.interrogans and the inserted fragment of pDL121 could be a new gene .