Construction Of Low Temperature-induced Expression Vector And The Study On The Regulation Mechanism Of The CspA Promoter Region

Both prokaryotes and eukaryotes exhibit a cold-shock response upon an abrupttemperature downshift. About 26-27 cold-shock proteins are synthesized to overcome thedeleterious effects of low temperature stress. CspA, the major cold-shock protein ofEscherichia coli, has recently been studied with respect to its structure, function andregulation at the level of transcription, translation and mRNA stability, especially the functionof 5'-UTR of its mRNA. Overexpression of proteins in E. coli at low temperature improves their solubility andstability. We apply the unique features of the cspA gene to develop a series of low temperatureinduced expression vectors, termed pMBC vectors. To detect the level of expression,cecropinB was choosed as the report gene. CecropinB was used in this case is a precusor ofcecropinB, a kind of Drosophila melanogaster cecropins, which were tested againstpathogens including E.coli from the normal enviroment of Drosophila as part of the innateimmune system. Nine sequences with cspA promoter and different regulatory motifs have been amplifiedfrom the E.coli genome by utilizing PCR. The products have been ligated into the pET28a(+)vector plasmids instead of the T7 promoter. The total RNA has been isolated from theDrosophila, and the cDNA sequence of cecropinB has been amplified from thesingle-stranded cDNA reverse transcripted from the total RNA. We obtained 4 lowtemperature induced expression vectors successfully (pMBC2,pMBC3,pMBC4 and pMBC5respectively), as well as a IPTG-induced expression plasmid pCEC that cecropinB reportergene was inserted in the multiple clone sites of the pET28a(+) vector. The low temperature-induced expression vectors and pCEC vector were induced in thiscase. The growth curve of the host cells showed the expressed fusion protein was deleteriousto the host cells; SDS-PAGE assay and RT-PCR result of cecropinB showed pMBC2epression efficiency was identical to pCEC, compared to pMBC2, pMBC3,pMBC4 andpMBC5 expression level of the reporter gene cecropinB was lower. Additionally, deletion experiment proved that the 3' terminal of 5'-UTR was no effectto the expression, as well as the deletion of the up regulate element was adiaphorous.