Abstract
Streptococcus mutans 6715 (serotype g) chromosomal DNA has been cloned into Escherichia coli K-12 using cosmid pJC74 by λ in vitro packaging system. A 45. 1-kilobase recombinant plasmid pYA721, which coded Streptococcus mutans extracellular protein, has two molecules of pJC74 and two DNA fragments (8.5 and 3.2-kilobase) inserted from Streptoccccus mutans chromosomal DNA. Subclone pYA724, which has one pJC74 and 8.5-kilobase fragment, was cut with restriction enzyme BamHI and self-ligated with T4 DNA ligase. The ligated chimera plasmids were transformed into Escherichia coli K-12, HB101 and selected ampicillin resistance clones. Cloned plasmids obtained were analyzed by digestion with restriction enzymes BamHI and EcoRI. Synthesis of extracellular protein was studied by immunodiffusion analysis using periplasmic space fraction and anti Streptococcus mutans extracellular protein antibody. The new clone plasmids had the same size as pYA724 but different EcoRI digestion patterns from pYA724. The results showed that the new clone constructed had 8.5-kilobase fragment inserted in the opposite orientation from that of pYA724, and produced extracellular protein. These data suggested that 8.5-kilobase fragment had own Streptococcus mutans promotor region and being used as functional gene expression in Esherichia coli.
