Study On Expression And Antibacterial Effect Of Mycobacterium Phage Guo1 LysA

Objective Bioinformatics analysis of Mycobacterium phage Guo1 Lysin A,and cloning,expression and purification of Guo1 Lys A,and explore its role in the resistance to mycobacteria.Methods1.Bioinformatics analysis of Mycobacteriophage Guo1 Lysin A Through NCBI-BLAST,the domain of encoding protein and possible3 D structure of mycobacteriophage Lys A was predicted.Then both its gene and amino acid sequences was compared to other been known and studied mycobacteriophage Lysin A by MEGA 7.0 software.2.cloning,expression and purification of Mycobacterium phage Guo1 Lys A The primers of Lysin A were designed for the template of Mycobacterium phage Guo1 genome DNA,then the mycobacteriophage Guo1 Lysin A was cloned by PCR,and ligated into p MD20-T vector,through double enzymes Nde I /Xho I digestion,the target gene Guo1 Lysin A was ligated into prokaryotic expression vector PET-28 a,then the recombinant vector p ET28a-Lysin A was transformed into BL21(DE3).Then the Guo1 Lys A protein was expressed by the inducing of IPTG and purified by his-tag Ni-NTA.Then the expressed and purified protein was visualized on a 12% SDS-polyacrylamide gel.3.Refolding of Mycobacterium phage Guo1 Lys A The inclusion body of purified mycobacteriophage Guo1 Lys A protein was refolded by gradient urea dialysis(Final concentration of urea was 8M,6M,4M,2M,1M,0M,0M respectively),in which containing 0.1%PEG2000,1m M EDTA,0.1m M GSSG,1m M GSH,and 0.6m M L-arginine.4.The antibacterial effect of Guo1 Lys A The antibacterial effects of lyase Guo1 Lys A was explored both on Mycobacterium and non-Mycobacterium,their antibacterial effects were observed by colony counting method,edge azure coloration and scanning electron microscope respectively.Results1.Bioinformatics analysis of Guo1 Lysin A Based on bioinformatics analysis of Mycobacterium phage Guo1 Lyase encoding gene Lysin A,we found that it has the domain of encoding endopeptidase and amidase domain,and their 3D structure were predicted.It is speculated that its encoded protein may have cleavage enzyme function,then both its gene and amino acid sequences were compared with other Mycobacteriophage Lysin A which have been known and published,found that they are distinctly different.It is proved that it has continued research value.2.cloning,expression and purification of Guo1 Lysin A The 1316 bp of Guo1-Lysin A was synthesised by PCR,latter was ligated into PET28 a vector,yield recombinant vector p ET28a-Lysin A.By sequencing verification,the correct p ET28a-Lysin A was transformed into BL21(DE3),and the expected protein about 48.3k D was expressed through IPTG inducing.Then the expressed protein was purified by His-labeled Ni-NTA,both the expressed and purified protein were visualized on 12% SDS-PAGE gel.3.Refolding of Mycobacterium phage Lyase Guo1 Lys A The activity of being refolded mycobacterium phage Guo1 Lys A was judged through detecting its antibacterial activity on mycobacterium smegmatis.4.The antibacterial effects of Guo1 Lys A The antibacterial effects of Guo1 Lys A protein was identified both on Mycobacterium and non-mycobacterium respectively,the significant morphologic changes of Mycobacterium smegmatis treated by 1mg/ml of Guo1 Lys A were observed by scanning electron microscope,compared to the 1X PBS buffer treated group,the Guo1 Lys A treated group became cataclastic,swelling and dissolving,while the control group of 1x PBS buffer processing remains slender,bend and regular rod-shaped;Compared to the 1x PBS group,the significantly inhibited of growth was observed by Guo1 Lys A treatment in Mycobacterium smegmatis,Mycobacterium tuberculosis through plating method;Further,the resazurin chromogenic agent was added into the treatment of cleavage Guo1 Lys A treated groups and 1x PBS buffer treated control groups,found that by 1mg/ml of Guo1 Lys A treated groups maintained a day blue or light blue indicated that there was no growth of viable bacteria or less growth of bacteria,while the culture medium of 1x PBS treated turned pink,indicating there were more viable bacteria.In the same way,we found weak anti non-mycobacterial effects.Conclusion Mycobacteriophage Guo1 Lys A can be obtained by cloning and prokaryotic expression,purified and refolded Guo1 Lys A protein has obvious anti Mycobacterium smegmatis and Mycobacterium tuberculosis,also show its weak anti non-Mycobacterium.Therefore,Mycobacteriophage lyase Guo1 Lys A is expected to become a new anti-tuberculosis drug,which lays the foundation for the researchment of new drugs for the treatment of tuberculosis.