Role Of Recombinant Chimeric Protein In Anti-bacterial And Anti-tumor Immunity

Our environment is contaminated by anumerous mumber and variety of pathogenic microorganisms. In resent years, the increasing frequency of bacteria infections in patients together with the emergence of strains resistant to currently used antibiotics. It is an urgent need to discover a new class of antimicrobial drug.Most of endogenous antimicrobial peptides are small cationic peptides, widely distributed all over the body and play a major role in innate immunity. Antimicrobial peptides exhibit remarkable antibacterial, antifungal, antiparasite and antiviral activity against a variety of microorganisms and parasites. However, the antimicrobial activity and the chemical structure of individual antimicrobial peptide can be altered by ions, serum factors and proteases. So, development of a new kind of antibacterial polypeptides through combinations of two or more peptides may be a new way to find new drugs for treatment of microorganism, especially antibiotic-resistant strain infection.In addition to microbicidial activity, Defensins, one of an inportant antimicrobial peptide classes in humans, may also take part in adaptive immune response as a regulators and enhancers. Using p-defensin as immune adjuvant may develop a new DNA vaccine that enhances anti-cancer immune response.In this study, a novel chimeric antimicrobial polypeptide His5-a is constructed to enhance its antimicrobial activity and its chemical stability via the combination of Histatin 5, an antimicrobial peptide derived from human saliva, and HMG N2 α-helical domain that was recently identified to be an antimicrobial peptide from human cervical mucus in our laboratory. And, a chimeric DNAvaccine pcDNA 3.1/hBD2-PSMA is also dveloped to enhance anti-cancer immunity agaist prostate cancer through fusing PSMA, a prostate specific membrane antigen, and human P-denfensin-2.This study includes three parts.Part I is to perfome the N-terminal sequencing of a newly identified antimicrobial polypeptide in our laboratory from human cervical mucus and isolate its full length of cDNA for the use in construction of the chimeric antimicrobial polypeptide. The obtained N-terminal sequence of the newly isolated antibacterial peptide is PKRKADGEAK. Then, degenerate primer was designed and the full length of cDNA was amplified using 3' RACE PCR method. This peptide was identified as High Mobility Group Chromosal protein N2 (HMG N2) by BLAST software analysis. OMIGA protein structure softwar analysis indicated a a-helix region, located from 17th to 47th amino acid reside, was contained in HMG N2. The antibacterial assay showed that only the a-helic structure (17 to 47) had antibacterial activity among three synthetic short peptides (1 to 16, 17 to 47, 48 to 90). The a-helical structure may be antibacterial domain ofHMGN2.Part His to construct chmeric protein prokaryotic expressing plasmid by combination of Histatin 5 and high mobility group chromosal protein N2 a helix through PCR SOEing method. E.coli BL21 strain carrying the recombinant plasmid was cultured in LB medium for 6 hours in the presence of IPTG to induce protein expression. The bacteria were lysed by freezing /thawing in the presence of lysozyme, The fusion protein was separated by affinity chromatography, and tag protein was cleaved by thrombin digestion, at last the recombinant His5-a chimeric peptide was purified using reverse-phase high performance liquid chromatography. In comparison with Histatin 5 alone, the chimeric polypeptide had a higher antibacterial and antifungal activity, and was more stable to tempratuer and trypsin than histatin 5. Using Scan electron microscope, we found that the chimeric peptide was not membrane active for its antimicrobial activity. DNA bingding assay showed that the chimeric peptide binded to bacterial DNA. Fleurecence labling and laser confacal microscopicobservation showed that the chimeric peptide distributed in the cytoplasm and nuclear structure of microorgansms. All these results suggested that targeting and attacking microbial DNA and organelle would be the antimicrobial mechanisms of the chimeric His5-a peptide.The aim of Part mis to construct DNA vaccine containing prostate-specific membrane antigen and hBD-2. PSMA expression could be detected in the DNA vaccine-ingected tissues in two weeks after the last immunization. PSMA specific antibody was detected in the serum of the immunized mice. T cells of the immunized mice were amplificated, especially the percentage of CD4+ T cells was markedly increased. Cytotoxity assay indicated that cytotoxic T lymphocytes to PSMA-expressing cells was also induced. This result provided evendence that P-defensin could be used as a new type of adjuvant in antitumor immunity.