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以λgt11作载体构建赖型钩体基因文库及其重组质粒pDL121的…
引用本文:刘洪斌,刘家福.以λgt11作载体构建赖型钩体基因文库及其重组质粒pDL121的…[J].华西医科大学学报,1997,28(1):18-22.
作者姓名:刘洪斌  刘家福
作者单位:[1]川北医学院 [2]华西医科大学
摘    要: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Hha I和Alu I限制性内切核酸酶部分消化,行EcoR I限制酶切位点甲基化,加上EcoRI接头与去磷酸化的λgt11DNA-EcoR1臂连接,体外包装后转染E.coli Y1090,建成含2.1×10^4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中阳性克隆,λDL12,亚克隆

关 键 词:钩端螺旋体  基因文库  λgt11  重组质粒  分子克隆

Construction of genomic library of L. interrogans serovar lai using lambda gt11 as the vector and a study of recombiant plasmid pDL121]
H Liu,B Dai,B Jing,W Wu,S Li,Z Fang,H Zhao,D Ye,R Yan,J Liu,S Song,Y Yang,Y Zhang,F Liu,Y Tu,H Yang,Z Huang,L Liang,L Hu,M Zhao.Construction of genomic library of L. interrogans serovar lai using lambda gt11 as the vector and a study of recombiant plasmid pDL121][J].Journal of West China University of Medical Sciences,1997,28(1):18-22.
Authors:H Liu  B Dai  B Jing  W Wu  S Li  Z Fang  H Zhao  D Ye  R Yan  J Liu  S Song  Y Yang  Y Zhang  F Liu  Y Tu  H Yang  Z Huang  L Liang  L Hu  M Zhao
Institution:Research Group of West China University Medical Science, Chengdu.
Abstract:A genomic library of L. interrogans serovar lai strain 017 has been constructed using lambda gt11 as the vector. DNA was partially digested by two blunt-end restriction enzymes, then methylated with EcoR I methylase; after EcoR I linker was added to the DNA, the linker-ended DNA was ligated to the dephosphorylated EcoR I digested lambda gt11 arms. The recombined DNA was packaged in vitro, and used to transduct E. coli Y1090 for amplification. There were 2.1 x 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-Ga1. A positive clone, designated lambda DL12, was screened with a rabbit anti-serum against L. interrogans serovar lai from the genomic library. The DNA from lambda DL12 was subcloned into plasmid pUC18. A recombinant (designated as pDL121) was obtained. SDS-PAGE analysis indicated that a 23 kd was expressed in E. coli JM 103 harboring pDL121. Western blotting analysis showed that a specific protein band molecular weight of 23 kd could be recognized by the rabbit antiserum against L. interrogans serovar lai strain 017.
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