Studies On Expression Of Antibacterial Peptide Gene Max From Skin Of Rana Catesbiana And Its Biological Activity

Antibacterial peptides are a group of small molecule bioactive peptide, which encode by genome and synthesis by ribosome, and produced by plants, insects, amphibians and mammals including human beings. These peptides have some characteristic including small molecular weight, poor antigenicity, stabilization, particular action mechanism, hardly inducing drug-resistance and so on. Apart from activity of antibacterial, these peptides also have many biological activities including antifungal, antivirus, antitumor and antiparasite. Because a lot of drug-resistance strains are emerging prominently at present, antibacterial peptides have the chance to be a new kind of antibacterial drug for clinical use. The antibacterial peptides are the most important nature defense material of the amphibian because of the special living environment. So far there are many antibacterial peptides which possess broad-spectrum antibacterial activities have been segregated one after another, and some of them possess special antifungal activities. So the antibacterial peptides of amphibian will be wildly used in the future. Extracting nature amphibian antibacterial peptides have the limits of little raw material, high cost, low yield and complex process, the purpose of this research is to overcome these defects and to produce the peptide by the method of gene engineering in order to be research a new path for prepare abundance antibacterial peptide.Cloning and sequencing of the antibacterial peptide gene from the skin of Rana catesbiana. On the basis of the frog batrachian antibacterial peptide genes (AY849019,AY849010,AY848986)reported in GenBank, one pair of primer was designed and the antibacterial peptide gene was amplified by RT-PCR from total RNA of skin. And then the antibacterial peptide gene max was cloned, sequenced and analysised by using the software DNAstar. The result showed that, the length is 109bp, the largest reading frame is 54bp and 17 amino acid residues were encoded. The molecular of antibacterial peptide was 1820.13 Dalton and the isoelectric point was 8.852. At the environment of pH 7.0, antibacterial peptide carried positive charge. The prediction of second structure was mainlyα-spiral andβ-folding, seldom random coil with some amphipathic properties. The apparentely variation between the cloned gene and the reported gene in Genbank showed that this is a new antibacterial peptide gene.Expression of the recombinant antibacterial peptide in E.coli. The gene was amplified from the recombinant plasmid pMD18-T-Max which contained the antibacterial peptide gene max by PCR, and then cloned into the prokaryotic expression vector pGEX-3X, so that the antibacterial peptide gene expression plasmid pGEX-3X-Max was constructed. Transformed correctly positive plasmid into the BL21(DE3), and induced with IPTG. The antibacterial peptide gene was expressed from the recombinant cell and the protein was purified. At the same time, the conditions of induction were optimized. The result show that time 5 hours, the temperature 37℃, the pH 7.2 and the concentration of IPTG 0.8mmol/L was the best conditions of induction. Under these conditions, the expressed recombinant protein existed in form of inclusion bodies. In these conditions the expression level of recombinant peptide is 20.285% contrasted the total protein of E.coli.. The recombinant peptide was purified and its antibacterial activity was tested. The result showed that it could inhibit Streptococcus (ATCC55121) and Bacillus paratyphosus(S.C500).Expression of the recombinant antibacterial peptide using Pichia pastoris secreting type expression system. We considered Pichia pastoris GS115 as the host strain and pPIC9K as the secreting type expression vector.The recombinant antibacterial peptide was leaded to secreting type expression byα-factor. The expression system was then consisted of complete antibacterial peptide gene, the secreting type expression vector pPIC9K and Pichia pastoris GS115. After screened by grad of G418, auxotype and PCR, twenty-five recombinant strains were obtained and then one strain showed higher activity was screened by assay of the antibacterial activity in vitro. The recombinant strain was induced at the conditions, in which BMGY : BMMY was 1:1, methanol concentration was 1%, and the temperature was 28℃. Then the expression supernatant was concentrate and antibacterial activity of the recombinant peptide was tested. The result was that recombinant antibacterial peptide could inhibit Streptococcus (ATCC55121), Bacillus paratyphosu(sS.C500), Bacillus cereus (CMCC(B)63301) and Candida albicans (ATCC10231) .