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目的 :探讨问号赖型钩端螺旋体重组质粒pDL12 1外源基因及其表达产物 2 3kDa蛋白的特点。方法 :用 6种内切酶对pDL12 1行酶谱分析 ,用Digoxin标记的pDL12 1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 2 3kDa蛋白 ,Westernblot鉴定其免疫原性。结果 :pDL12 1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 (serovarlaistrain 0 17,serovarhebdomadisstrain 5 6 6 10 ,serovarpomonastrain 5 6 6 0 8)有杂交信号 ,与非致病性钩体 (serovarpatocstrainPatocI,serovarillinistrain 30 5 5 )无杂交信号 ,亦不识别大肠杆菌。2 3kDa兔抗血清可识别pDL12 1体外表达的 2 3kDa蛋白带和赖型钩体 0 17株超声抗原成份 ;其抗体滴度为 1/12 80 0 ;用pDL12 1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 :pDL12 1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 ;2 3kDa抗原有良好的免疫原性 ,可能是赖型钩体 0 17株的保护性抗原
Objective: To investigate the characteristics of exogenous gene of Leptospira interrogans leptospiral plasmid pDL12 1 and its expression product 2 3 kDa protein. Methods: Six kinds of endonucleases were used to analyze pDL121. Digoxin-labeled pDL12 1 gene fragment was used as a probe to probe hybrids of different species. The 23 kDa protein was prepared by SDS-PAGE and identified by Western blot Its immunogenicity. Results: There was no restriction endonuclease digestion site of 6 endonucleases in the exogenous gene of pDL12 1. The recombinant probe hybridized with the serovar laistrain 0 17 (serovarhebdomadisstrain 5 6 6 10, serovarpomonastrain 5 6 6 0 8) And non-pathogenic hook (serovarpatocatrainPatocI, serovarillinistrain 30 5 5) no hybridization signal, also does not recognize E. coli. 2 3kDa rabbit antiserum can recognize the 21kDa protein band of pDL12 1 expressed in vitro and 0 17 strains of the antigen of the clade. The antibody titers are 1/12 80 0; the guinea pigs can be immunized immunized with pDL12 1 bacterial lysate Guinea pigs to resist virulent strains hook body attack. CONCLUSION: The exogenous gene of pDL12 1 may be a new gene of Leptospira interrogans; this recombinant probe can identify pathogenic leptospira and non-pathogenic leptospira; 2 3 kDa antigen has good immunogenicity may be due to Leptospira 0 17 strains of protective antigen