Study On Function Of Usp45Gene And Its Application In Expression Vector

In the study of expression of Lactococcus lactis1.2472’s, a secretion protein in50kDa wasalways detected with amount of expression in the culture supernatant.The protein was Usp45confirmed by LC-MS mass spectrometric identification and encoded from usp45gene(ID:M60178.1). The function of this protein is unknown by now. The secretion single peptides of it,however, was widely used in construction of LAB gene expression vector.Our study analyse thefunction of the usp45gene after it was expressed in E.coli and constructed Lactococcus lactisgene expression vector with it,which bring a solid basis for further study of mucosal targeting oftherapeutic molecules using modified lactic acid bacteria.Fristly, Usp45gene was amplified by PCR from genome which is1371bp and cloned intopMD-18T Simple Vector to build pT-usp45, which was confirmed by BamH I/EcoR I analysisand DNA sequencing. A usp45prokaryotic expressing plasmid, pET-28a-usp45was constructedby subclone and transformed into E.coli BL21(DE3) induced by IPTG. A50kDa protein wasdetected by SDS-PAGE analysis which was Usp45protein confirmed by Westen blot further.After optimizing the induction conditions including inducing teprature, time and IPTGconcentration, the best expression condition were confirmed which is induced5hours at37℃while IPTG concentration is1.0mM.Secondly, His-tag was added into usp45gene which was expressed in recombinant bacteria.The Usp45protein was purified by Ni-NTA affinity chromatography, and confirmed bySDS-PAGE. The concentration of the protein is13.488mg/mL determined by BCA Kit.Thirdly, usp45gene and Usp45protein were analysed by biosoft to confirm its sequence,struction and function. The immunological character of Usp45protein was analysied throughtreating Caco-2with purified Usp45protein and examining the expression of cytokine byRT-PCR.Finally, construced a LAB expression vector using side sequence of usp45. Emr gene wasamplified by PCR from plasmid pMG36e and cloned into pMD-18T Simple Vector to buildpT-Emr. The fragment of cloned Emr was obtained by Sal I/EcoR I from pT-Emr and insertedinto pEx-EGFP to got pEX-EM which is a new expression vector.The new plasmid was electric transformed into Lactococcus lactis1.1936competent cells and the target gene EGFP wasdetected in15generation of positive clones by PCR.In summary, usp45gene of Lactococcus lactis1.2472was expressed in E.coli BL21(DE3)and the protein was purified by affinity chromatography. We analyzed the structure and functionof the gene, discussed the character of Usp45protein and constructed a new expression vectorusing the sequence of usp45gene.