[Construction of genomic library of L. interrogans serovar lai using lambda gt11 as the vector and a study of recombiant plasmid pDL121]

Hua Xi Yi Ke Da Xue Xue Bao. 1997 Mar;28(1):18-22.
[Article in Chinese]

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cloning, Molecular
  • DNA, Bacterial / genetics*
  • DNA, Recombinant / biosynthesis*
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Genetic Vectors
  • Genomic Library
  • Leptospira interrogans / genetics*
  • Plasmids
  • Recombinant Fusion Proteins / biosynthesis*
  • Transfection

Substances

  • DNA, Bacterial
  • DNA, Recombinant
  • Recombinant Fusion Proteins