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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.