Expression Of DNase Ⅱ Fusion Protein(PLTD) And Its Inhibition To Tumor Cells

DNaseâ…¡is an acid,cation independent endonuclease,which cleaves DNA at an optimal acid pH(4.5-5.5) producing double strand breaks(DSB) and single strand breaks(SSB) with 3'-phosphate ends.It can involve in apoptosis,cataractous and disease of immune system.Luteinizing hormone-releasing hormone(LHRH) act as antitumor agent s for hormone dependent tumor.LHRH analogue have been developed furthest are in clinical.There many receptors on the surface of cancer cells than noarmal tissue.So it can play an important role in receptor binding with tumor as targeting vector.TAT PTD is amino acid residues from the human immunodeficiency virus type 1 (HIV-1) TAT,which is an trans-activating transcriptional activator.It can cross the membrane of mammalian cells and cause the intracellular delivery of polypeptide, protein,DNA,plasmid and nanometer-sized magnetic bead particles.No toxicity and no side effects to cells.Cancers have been threatening the health of human beings.The chemicals used in clinical now lack selectivity.Side effects are common seen in clinical. Tumor-targeting therapy will be satisfactory for enhancing therapeutic effects of anti-tumor medicines.One strategy for targeted cancer biotherapy is to utilize recombinant protein.In order to develop an agent to treat the malignancies which express LHRH receptors,we prepared the recombinant protein PLTD.The result is following:1) Cloning and expression of mouse DNaseâ…¡geneAfter RT-PCR of total RNA from mouse spleen,the DNaseâ…¡gene was amplified by PCR.Then the product of PCR were cloned into a clone vector pMD18-T. Digested by Ndeâ… and Xhoâ… ,the DNaseâ…¡gene was cloned into an expression vector pET-28a.The gene was sequenced and analyzed by computer software,which confirmed that the gene sequence was consistent with what we had predicted.The recombinant plasmid was transfered into host strain BL21(DE3) and induced by IPTG.The inclusion body expressed was extracted and purified.2) Analysis of enzyme activty of DNaseâ…¡DNaseâ…¡activity was measured byλDNA at different pH.The activity was much higher than other conditions at pH 5.0.Meanwhile,divalent cations had some effects on the DNaseâ…¡activity.The enzyme activity wasn't impacted by Mg²⁺,Ca²⁺,EDTA; a inhibited by Zn²⁺,Mn²⁺ and ATA.3)Construction of recombinant plasmids pET28a-LHRH-TAT-DNaseâ…¡(PLTD)The positive recombinant plasmids PLTD were constructed.Every part of PLTD fusion protein in secondary structure has no significant influence analysis by the bioinformation software,which was important to keep biological activity,a4) Expression and enzyme activity analysis of recobinant proteion PLTDPLTD was transfered into host strain BL21(Rosseta) and induced by IPTG when OD₆₀₀ of the culture was about 0.6.The specific protein PLTD expressed(about 40KDa) was detected by SDS-PAGE.The fusion protein was expressed at high level to 60%of the total bacterial protein as confirmed by thin-layer scanning.The inclusion body expressed was extracted and purified.PLTD activity was measured by digestingλDNA and PLTD had enzyme activiy of DNaseâ…¡.5)Analysis of Antitumor activity of PLTD in vitroThe antitumor activity of PLTD protein in vitro on different cells such as HeLa cells,SP2/0 cells,hepatocarcinoma cells(Bel7402) was determinates by light microscopy,MTT assay,DNA ladder and lacer copolymerization focusing and immunohistochemistry.The results showed that PLTD fusion gene could successfully enter these tumor cells and suppress cell growth.Apoptosis of tumor cells was determinated by light microscopy after HE stains.The results showed that remarkable apoptotic characteristics such as nuclear shrinkage and strong cytoplasm concentration in tumor cells than control,which suggested that the PLTD fusion protein could induce apoptosis in different tumor cells effectively.We also observed PLTD protein distributed by cytoplasm and nuclear membrane. 6)Antitumor activity of PLTD in vivoSP2/0 cell was implanted abdominally in BALB/c mice.BALB/cwere treated with PLTD everyday abdominally for 21 days.The PLTD can effctively inhibit Sp2/0 growth and the inhibition is dose limited.SP2/0 cell was also implanted subcutaneously in back of BALB/c mice.After emerged tumor,BALB/c were treated with PLTD everyday intravenously for15 days. The growth inhibition rate in the group of dose 0.5mg/kg is over 50%,and the inhibition rate is dose limited.In this paper,we have succesfully constructed and expressed the PLTD protein and the PLTD protein can effectively enter cancer cells and inhibit cancer cell growth in vitro and in vivo.The results are significant for further study on the mechanism of PLTD fusion protein to induce apoptosis of cancer cells and application in cancer therapy.