Expression Of Human Lysozyme Gene In Insect Cells And Its Biological Characterization

Human Lysozyme (hlyz) is an alkaline globulin,which is composed of 130 amino acid suquences,and molecular weight is 14.700 kDa. It is an effective antimicrobial agent , full known as 1 , 4-β-N Lysozyme , also was named as visco-peptide N-acetyl-muramyl hydrolase. It is a kind of non-residual,non-resistance of new antibacterial drugs,which represents the future direction of the utilization and development of antibacterial drugs.In the current study, human lysozyme gene fragment was amplified using a pair of specific primers,according to the registered sequence of human lysozyme mRNA in Genbank,with XhoI restraction sites being introduced. The object fragments were obtained by gel extraction kit,then ligated to pGEM-Teasy vector and sequenced,the postive plasmids were named as pGEM-hlyz. Hlyz fragments were subsquently introducted into pFastBacI vector and were identified by XhoI and pstI digestion,the positive recombinant plasmids were named as pFastBacI-hlyz and were transformed into DH10Bac competent cells. PCR was carried out for the identification of recombinant genome cotaining hlyz fragments,using M13 and hlyz specific primers. Positive recombinant genomes were subsequently transfected into Sf9 cells to obtain the recombinant baculovirus,named as rBac-hlyz. Hlyz proteins can be detected in the rBac-hlyz infected Sf9 cells by IFA,SDS-PAGE and Western blot. The results showed that rBac-hlyz recombinant baculovirus were successfully constructed.To examin the activity of hlyz expressed by rhlyz infected Sf9 cells,agar plates were prepared with the M. lysodeikticus and agar mixture. After agar become solidificated,and add culture supernatant and cell lysate supernatant of the rhlyz derived from Sf9,Negative control(uninfected rhlyz Sf9 cells) and the hen egg white lysozyme respectively. The results showed that recombinant human lysozyme activity was clearly detected in the culture supernatants derived from Sf9 cells and cleared a zone against M. lysodeikticus that Measured 20 mm in diameter,compared with 12 mm obtained with the standard (hen egg white c-type lysozyme),thereby suggesting strong lytic activity.