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为探讨赖型钩体抗原基因特点及其表达产物作为疫苗的可行性,采用问号赖型017株钩体经HhaⅠ和AluⅠ限制性内切核酸酶部分消化,行EcoRⅠ限制酶切位点甲基化,加上EcoRⅠ接头与去磷酸化的λgt11DNA-EcoRⅠ臂连接,体外包装后转染E.coliY1090,建成含2.1×106重组子的问号赖型017株钩体基因文库。用抗赖型钩体兔血清从上述基因文库中筛选出一个强阳性克隆,λDL12,亚克隆入pUC18质粒,获重组质粒,pDL121。EcoRⅠ酶切证实该重组质粒含有1kb的外源插入片段。pDL121经IPTG诱导后,在SDS-PAGE上出现23kd特异条带。免疫印迹表明该蛋白带可被抗赖型钩体兔血清识别。
In order to investigate the characteristics of the leptospira antigen and the feasibility of its expression product as a vaccine, the 017 leptospira strain was digested with Hha I and Alu I restriction endonucleases and EcoR I restriction enzyme site methylation , Plus EcoRI linker and dephosphorylated λgt11DNA-EcoRI arm connected, packaged in vitro transfection E. coliY1090 to construct a questioner-type 017 leptospiral gene library containing 2.1 × 106 recombinants. A strong positive clone, λDL12, was screened from the gene library using anti-Lai rabbit serum and subcloned into pUC18 plasmid to obtain recombinant plasmid pDL121. EcoRⅠ digestion confirmed that the recombinant plasmid contains 1kb of exogenous insert. After induced by IPTG, pDL121 showed a 23kd specific band on SDS-PAGE. Western blotting indicated that the protein band could be recognized by anti-Lai rabbit serum.