Construction Of Pichia Pastoris Vector Co-expressing Ec-cLYZ And MEL Genes And Evaluation Of Its Antibacterial Activity

Lysozyme is a kind of antibacterial proteins with enzyme activity from animals,plants and microorganisms.Fish lysozyme(Ec-cLYZ)plays a key role in resisting the invasion of foreign microorganisms.Antimicrobial peptides are peptide chemicals that exist in organisms,which secreted by the immune system.Grouper type c lysozyme is an important part of fish immune system with strong heat resistance.It is a stable protein with broad-spectrum bacteriostatic effect,antifungal and antiviral activity;Antimicrobial peptides can prevent and inhibit the invasion of various harmful substances into the body.Melittin is the main component of bee venom,which has a wide range of anti-bacterial,antifungal and anti-virus acticity,but its application is limited by the hemolytic activity of natural melittin.Its hemolytic activity can be greatly reduced by modifying the structure of melittin(MEL).Antibacterial activity of lysozyme and antimicrobial peptides makes them widely known in the research and development of new antibacterial drugs.In this study,the target gene Ec-cLYZ and the modified MEL sequence were connected with T2A connecting peptide,and the separate expression groups of Ec-cLYZ and MEL were constructed as control.The target gene was cloned into Pichia pastoris expression vector pPICZ?A to obtain high efficiency and stable expression recombinant vectors expressing pPICZ?A-MEL?pPICZ?A-Ec-cLYZ and pPICZ?A-Ec-cLYZ-MEL.The target gene was detected by PCR,double enzyme digestion and sequencing.The correctly sequenced recombinant plasmid was electroporated into Pichia pastoris expression strain GS115 after digestion by restriction enzyme Sac I.The highly expressed Mut⁺strain was obtained after screening with universal primer 5?AOX1/3?AOX1 and high concentration zeocin resistance,which was named GS115/pPICZ?A-MEL?GS115/pPICZ?A-Ec-cLYZ?GS115/pPICZ?A-Ec-cLYZ-MEL.The recombinant expression vector was induced by 0.5%methanol,supplemented with methanol to 0.5%per 24 hours,cultured at 28?and 250 rpm.The supernatant was taken per 12 h and the total protein concentration was measured.The results showed that The best expression time of GS115/pPICZ?A-MEL?GS115/pPICZ?A-Ec-cLYZ?GS115/pPICZ?A-Ec-cLYZ-MEL was 96 h,72 h and 96 h respectively.After the supernatant was purified by His-tag protein purification kit,the concentrations of MEL,Ec-cLYZ and Co-expression Ec-cLYZ and MEL were 27.31,28.73and 35.49 mg/L respectively.CCK-8 test was used to analyze the toxic effects of recombinant proteins MEL,Ec-cLYZ and Co-expression Ec-cLYZ and MEL with concentrations of 150,75,37.5,18.75,9.38 and 4.7mg/L on HEK293A cells at 12 h,24 h and 36 h.The hemolytic activities of recombinant proteins with concentrations of 150,75,37.5,18.75,9.38,4.69 and 2.35 mg/L on rabbit erythrocytes were detected respectively.The results of CCK-8 test showed that within the measured concentration range,the cell survival rate of co-expression Ec-cLYZ and MEL treatment group had no significant difference between PBS treatment group within 36 hours.It was almost non-toxic.The results of hemolysis test showed that the hemolysis rate of erythrocytes in co-expression Ec-cLYZ and MEL treatment group was 2.3%at the concentration of 150 mg/L,which was much lower than that in MEL treatment group(79%).The antibacterial activity was evaluated by calculating the antibacterial rate and comparing it with that of ampicillin at the same concentration.Oxford cup method was used to determine the inhibition circle diameter of recombinant protein on experimental strains.The results showed that the purified recombinant protein co-expression Ec-cLYZ and MEL had antibacterial activity against E.coli K88,S.aureus CMCC(B)26001,S.epidermidis ATCC12228,S.agalactiae,S.dysagalactiae ATCC9809,K.pneumoniae and P.multocida.Compared with the non antibacterial activity of MEL group against K.pneumoniae and Ec-cLYZ against Pasteurella multocida,the antibacterial range of Co-expression Ec-cLYZ and MEL co-expression group is wider.The inhibitory rates of co-expression Ec-cLYZ and MEL at the same concentration on E.coli K88,S.dysagalactiae ATCC9809 and S.agalactis were higher than those in ampicillin treatment group(p<0.01).The protein expressed by the Pichia pastoris expression vector GS115/pPICZ?A-Ec-cLYZ-MEL constructed in this study showed good antibacterial activity without cytotoxicity or hemolytic activity,which provided a new idea and reference for the prevention and treatment of bacterial diseases.