Cloning And Expression Of Anti-tumor Polypeptide From Marine Bacillus And The Research Of Its Biological Activity

The incidence rate of cancer shows a rising trend nowadays,the control and treatment of tumors have become the focus of global attention.Drugs have important values in the treatment of cancer,so the development of anti-tumor drugs that have good efficacy,high specificity and small side effects are the focus of medical treatment.Anti-tumor peptides have the advantages of good curative effect,accurate treatment site,low side effects,low molecular weight,easy to transform and synthesize have been applied to the therapy and clinical treatment of cancer.This is of great significance to the treatment and control of cancer.In the early research,we isolated a polypeptide PBN11-8 that had anti-tumor activities from the metabolite of antarctic Bacillus sp.N11-8.PBN11-8 had cytotoxicities on multiple tumor cell lines,and it could inhibit the migration and invasion of human hepatoma cell line BEL-7402.PBN11-8 had the potential to develop to a new marine anti-tumor drug.In this study,we used the anti-tumor polypeptide PBN11-8 as the object of research.The gene that encoding PBN11-8 was obtained by PCR cloning technique,and determined the gene sequence of PBN11-8.The amino acid sequence analysis showed that the exact molecular weight of PBN11-8 was 19.04 kDa and the isoelectric point was 5.37.The contrastive results of the sequence showed PBN11-8 was a kind of metalloproteinase,and the protease activity of PBN11-8 was 1172U/mL(0.85mg/mL)measured using forint-phenol method.EDTA could affect it,s protease activity,it had highest enzyme activity when the temperature was 50? and the pH was 10.0.PBN11-8 was stable when the temperature was below 40? and had high enzyme activity in alkaline conditions.It was an alkaline protease with stable property when in pH7.0-8.0 conditions and at this condition it was easy to store.Mn2+ could improve the activity of PBN11-8,on the contrary,Hg2+ could inhibit the protease activity.Our research group obtained the recombinant PBN11-8 by Escherichia coli prokaryotic expression system using recombinant expression technique.MTT results showed that the recombinant polypeptide had strong anti-tumor activities and could inhibit the growth of diverse tumor cells,including human hepatoma cell line BEL-7402,human ovarian carcinoma cell line NIH: OVCAR-3,human large cell carcinoma NCI-H460,human glioma cell line U251 and human renal clear cell adenocarcinoma cell line 786-0 with the IC50 values of 2.56,2.74,2.97,3.88 and 4.31?g/m L,respectively.Recombinant PBN11-8 could significantly change the morphology of BEL-7402 cell lines,the dosing group of cells gradually became spherical and gathered together,the gap between cells was obvious.The effect of recombinant PBN11-8 on BEL-7402 cells was time-dependent and the the crystal violet adhesion assay results showed that recombinant polypeptide could decrease the adhesive growth capacity of cells and then affect the survival rate.The activity of recombinant polypeptide was 847U/m L(0.73mg/mL),and the enzyme activity was greatly affected by EDTA.The residual protease activity was only 48.5%,13.2% and 9.7% of the original activity when the concentration of EDTA was 0.5mM,1.0mM and 2.0mM.The optimum reaction temperature of the recombinant PBN11-8 was 40? and the optimum reaction pH was 10.0.Recombinant polypeptide were stable when the temperature was below 40? and under neutral conditions.Similar with the native polypeptide,Mn2+ could improve the protease activity of recombinant polypeptide,and Hg2+ had the strongest inhibitory effect on it.Study the related properties of PBN11-8 has important value for its future research and application.The establishment of recombinant expression technology of PBN11-8 can solve the problem such as the low production of natural PBN11-8.This study laid foundation sand provided supports for further study of PBN11-8.