Construction And Screen Of Genome Subtractive Library Of Uropathogenic E.coli 132

Objective:To construct and screen genomic DNA library of uropathogenic E.coli 132(UPEC132) by suppression subtractive hybridization (SSH). To identify the strain specificDNA sequences from DNA library of uropathogenic E.coli 132 and further to obtainthe open reading frame of novel DNA fragment.Methods:1. Using suppression subtractive hybridization to construct DNA library ofuropathogenic E.coli 132 and the genome DNA of UPEC132 and MG1655 wereobtained as tester and driver. Then tester DNA was hybridized with driver DNAtwice and underwent nested PCR twice. The PCR product was ligated withpMD-18T simple vector and transformed into E.coli Topl0. After screenedthrough antibiotics and X-gal/IPTG, the positive recombinants were selected.2. Randomly select positive clones and extract recombinant plasmid throughalkaline lysis. Then the plasmid was identified by PCR and the PCR productswere dotted on nylon membrane after degeneration. The genomic DNA ofUPEC 132 and MG1655 were probes by digoxin random primer labeling. Positiveclones were selected after prehybridization, hybridization and coloration. Thesignificant clones were sequenced and searched homologically in GenBank.3. To obtain 5' end and 3' end of novel fragment by Genome Walker method. Thegenomic DNA of UPEC132 was divided into 4 groups and digested with differentrestriction enzymes that left blunt ends. The restriction enzymes were Draâ… ,EcoRâ…¤,Stuâ… ,PvUâ…¡. Each batch of digested genomic DNA was then ligatedseparately to the Genome Walker adptor which were reffered to for convenienceGenome Walker library. The first and nested PCR used the adaptor primerprovided in the kit and gene specific primer of novel fragment. The identifiedPCR products were ligated with pMD-18T simple vector and transformed intoE.coli Top10. A full-length DNA sequence was obtained and analyzed by Proscansoftware which determined an open reading frame. Results:1. To obtain genomic DNA subtractive library of uropathogenic E.coli 132 whichcontained 1600 positive clones. Among the 350 colonies randomly chosen whichwere identified by PCR and dot blotting, 93 colonies were sequenced andsearched homologically in GenBank. One of UPEC132 SSH fragments was nohomology with registered UPEC sequences in GenBank. 39 of UPEC132 SSHfragments were high homology with registered UPEC sequences in GenBank. Tenof them were virulence-associated fragments. Another 8 fragments encodedproteins that might be metabolic and transporter. 2 fragments encoded outermembrane protein, 3 fragments encoded regulatory protein, 11 fragments encodedputative and 5 other protein. Another 19 SSH fragments were high homology withregistered non-UPEC sequences(E.coli plasmid or other bacteria) in GenBank.There are 10 repeated fragments and 6 pMD-18T vector fragments. All thesefragments had no homology with the DNA of MG1655. The novel DNA fragmentwhich was named R049 (789bp). The R049 (789bp) was submitted to GenBankand aquired the register number EF488001.2. The two-way fragment of novel fragment R049 (789bp) was amplified byGenome Walker method. The 5' end was 1502 bp and the 3' end was 1207 bp. A3498 bp of full-length DNA sequence was obtained through DNAStar software.An open reading frame of 1311bp was obtained by Proscan(Version 1.7) softwarewhich was named R049 (1311bp) temporarily.Conclusion:The genomic DNA subtractive library of uropathogenic E.coli 132 wassuccessfully constructed . And obtained the specific fragments of UPEC132. Toprovide a good foundation for further research for the pathogenesis of UPEC.