以λgt11作载体构建赖型钩体基因文库及其重组质粒pDL121的克隆和在E.coli表达的研究

Construction of Genomic Library of L. interrogans Serovar lai Using λgt11 as the Vector and A Study of Recombiant Plasmid pDL121

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。

A genomic library of L.interrogans serovar lai strain 017 has been constructed using λgt11 as the vector. DNA was partially digested by two blunt end restriction enzymes, then methylated with EcoRⅠ methylase; after EcoRⅠ linker was added to the DNA, the linker ended DNA was ligated to the dephosphorylated EcoRⅠ digested λgt11 arms. The recombined DNA was packaged in vitro, and used to transduct E.coli Y1090 for amplification. There were 2.1×10 6 recombinant bacteriophages as recognized by their ability to form white plaques plated on Lac host in the presence of both IPTG and X Gal. A positive clone, designated λDL12, was screened with a rabbit anti serum against L. interrogans serovar lai from the genomic library. The DNA from λpDL12 was subcloned into plasmid pUC18. A recombinant (designated as pDL121) was obtained. SDS PAGE analysis indicated that a 23kd was expressed in E.coli JM 103 harboring pDL121. Western blotting analysis showed that a specific protein band molecular weight of 23kd could be recognized by the rabbit antiserum against L.interrogans serovar lai strain 017.