Multiplex PCR For Detection Of The Three Enterotoxigenic Genes In Enterotoxigenic Escherichia Coli And The Cloning And Expression Of Recombinant LTB,LT,LTK63,LTR72

Enterotoxigenic Escherichia coli (ETEC) is the main pathogen of colibacillosis of neonatal and post weaned piglets. The pathogenesis of ETEC is strongly related to adhesive fimbriae and enterotoxin. A multiplex polymerase chain reaction (PCR) was developed to detect the three enterotoxigenic genes LT, ST I, ST II in enterotoxigenic Escherichia coli from porcine. Three different sets of oligonucleotide primer were simultaneously used to amplify the enterotoxin genes of heat-labile (LT) and heat-stable (ST I and ST II) enterotoxins of ETEC. These primers amplified the 110, 237 or 368 base pair DNA fragments .With the methods of the restriction endonuclease, the expected target DNA fragments were obtained. There was no cross-reaction with the non-ETEC strains. The results indicate that the PCR is a rapid, sensitive and specific method for detecting ETEC.Based on the published nucleotide sequence of LT, four pairs of primers were designed and synthesized. With the technique of PCR and site-directed mutation PCR, four DNA fragments were amplified from K88 of ETEC and cloned to pMD18-T vector respectively. They are identified to be LT and mutated with the methods of restriction endonuclease digestion, PCR and sequencing. They are identical to the nucleotide sequences reported in Genebank. Comparing with the sequences of LT reported in Genbank, there are mutation nucleotides of five to twenty, and shared 98.9-99.1% homology with each other. Amino acid analysis indicated that there are changes of five to twenty amino acid comparing with LT reported in Genbank, and shared 94.5%-96.6% homology with each other. The second structure and antigenic analysis of LT indicated that LTs have highly conserved feature.By the technique of DNA recombination, the LTB fragment was cloned to plasmid pBV220 vector, and transformed to E. coli. DH5a The recombinant protein was expressed by inducing with the changes of temperature. The result of SDS-PAGE indicated that the production of recombinant LTB reached its peak about 4-5 hours after inducing at 42℃, and the expression protein can take about 20% of the total protein. Most of the recombinant protein exists in sentiment, and a little in supernatant. After ultrasonicated, the sentimentwas isolated and purified primarily, then renatured. rLTB is not only used to vaccine, but also mucosal immune adjuvant.