Molecular Cloning And Expression Of The Gene Encoding Lysozyme From Screened Strains

N-acetylmuramidase can hydrolyze theβ-1,4 glycosidic bond between the N-acetylglucosamine and N-acetylmuramic acid of the peptidoglycan of bacterial cell wall.The lysozymes origining from microorganisms have bothβ-1,4-N-acetylmuramidase andβ-1,4-N,6-O-diacetylmuramidase activities.They have wider lysis spectrums and potential application values.Actinomycetales possessing high lytic activities were isolated from soils by the double layer plate containing intact cells of Staphylococcus aureus.The strains were identified by 16S ribosome DNA.Three of them were Streptomyce, with the identity of Streptomyces exfoliates,Streptomyces graminearus and Streptomyces koyangensis,respectively.The other was Kribbella sp.Because different characters and different applications can be obtained from lysozymes with different origin,the genes of N,O-diacetylmuramidase were cloned and expressed in E.coli in order to obtain lysozymes with good application characters.The homologous sequences of lysozymes were analyzed using Clustalw software.Their CODEHOP primers were designed online.The conserved partial N,O-diacetylmuramidase sequences were amplified by different gradient temperature PCR.The amplification products were ligated to pMD18-T vector and transformed into E.coli DH5α.The positive clone was slected,identified and sequenced.The nest primers were designed based on the acquired conserved lysozyme sequences to amplify the 3′and 5′sequences adjoin to the partial lysozyme sequence.The sequence and characters of the gene products,such as pI and Mw, were analyzed.One of the genes was amplified to construct a recombinant plasmid pET-26b(+)-lysozyme and transformed into E.coli BL21(DE3)pLysS.After being analyzed by colony PCR and sequencing,it has been confirmed that heterogeneous gene was inserted into the vector,as a result,the recombinant protein was expressed after IPTG induction.