Construction and identification of the genomic expression library from Campylobacter jejuni strain NCTC11168

The genomic DNA extracted from Campylobacter jejuni strain NCTC11168 was digested using Sau3AⅠ,then the fragments ranging from 0.4 kb to 3 kb were purified and ligated into pET30a(b,c)expression vectors,which were digested by BamHⅠ and dephosphorylated by SAP.The ligated products were transformed into Escherichia coli DH5α,and the sizes of fragments inserted into the vectors as well as the insertion efficiency were analyzed by PCR with pET30 vectors specific primers.Then the plasmids were extracted from recombinant E.coli DH5α and further transformed into BL21(DE3) to develop genomic expression library. The genomic expression library was induced with IPTG,and the protein expression was identified using SDS-PAGE. The genomic DNA was extracted by conventional methods,and was digested using 0.5U/μg Sau3AⅠ at 37℃ for 30min,and the DNA fragments ranging from 400 to 3000bp were purified. The pET30a(b,c) vectors were digested using 2.5U/μg BamHⅠ and dephosphorylated using 0.8U/μg SAP. The ligation was performed according to the genome fragments and vectors mole ratio of 10∶1,and the ligated products were transformed into competent DH5α to construct the genomic expression library of C.jejuni strain NCTC11168.The reservoir capacity of constructed library was 52700 clones,which equated to 4 times of NCTC11168 genome,and the insertion efficiency was 83.3% to 91.7%,the range of inserted fragments were 400 to 3000bp,and the fragments uniform distributed,and the protein was expressed efficiently. The results revealed that the genomic expression library of C.jejuni strain NCTC11168 was constructed successfully.