| Antibacterial peptides are a group of small molecule bioactive peptide. Substantial evidence accumulated in recent years indicates that AMPs not only possess direct antimicrobial activities but also have multiple immunomodulatory activities.They play an important role in both the innate and adaptive immunity of the host and antibacterial peptides have the chance to be a new kind of antibacterial drug for clinical use.There are large amounts of substances with special molecular structure, multiplex function contained in mucus which protecting the skin of the amphibians and the the main substance is antimicrobial peptides. The small molecular peptide excretes to the body surface per glandula integumentaria which encodeing by gene and synthesis by ribosome. It has the function of avoiding disoperation of bacteria, protecting the skin, strengthening the immunity. Because of the abuse of antibiotics, the augment of drug-resistant organisms world-wide , especially the harm of MRSA. The antimicrobial peptide had high effective and the broad spectrum of antibacterial activities and hardly inducing drug-resistance and no pharmacal residue so that it can be the effective candidates of the antibiotics.the procedure is tedious and the quantity is too low from directly isolation - purification and the cost is too high from chemosynthesis so we are favor to produce antimicrobial peptide from genetic engineering. As a eukaryotic host strain, the Pichia pastoris has been used for expressing many proteins successfully and its stability and amount of expressing is well, so we chose GS115 as the host strain for expressing antimicrobial peptide gene.The purpose of this research is to obtain AMPs to utilize genetic engineering. The genes of catesbeiania-1and palustrin-Ca were amplified from the library by PCR methods. The yest engineering strains GS115/pPICZαA-Catesbeiania-1 and GS115/pPICZαA-Palustrin-Ca were constructed and induced to express antimicrobial peptide. This study laid the foundations for the application research on antimicrobial peptide from the skin of the Rana catesbeiana and large scale production.Two pairs of primer contain enzyme sites was designed to produce the antibacterial peptide genes from the cDNA Lib. And then the antibacterial peptide gene catesbeiania-1 and palustrin-Ca was cloned, sequenced and analysised by using the software DNA star ,the reading frame of them is totally right, the The molecular of antibacterial peptide was betwen2～8 Dalton. The prediction of second structure was mainlyα-spiral, seldom random coil with some amphipathic properties and they have not so more homology with other AMPs showed they are new cationic antibacterial peptide genes. The gene of catesbeiania-1 and palustrin-Ca was successed to clone into the eukaryotic expression vector pPICZαA, so that the antibacterial peptide gene expression plasmid pPICZαA-Catesbeiania–1 and pPICZαA-Palustrin-Ca were constructed. The linear recombinant plasmids shear by the restricted enzyme Transformed correctly into the GS115 by shock. After screened by grad of Zeocine, homo-expressed recombinant strains were obtained and from PCR verification it was concerned that the objective genes had inserted to the chromosome of GS115.Positive recombinant strains were induced at the conditions, in which PH was 6.0,BMGY : BMMY was 1:1, methanol concentration was 0.5%, and the temperature was 28℃. Then the expression supernatants were show to the protain by the Tris-Tricine-SDS-PAGE and it was justified that the AMPs was secreted exression in Pichia pastoris. Antibacterial activity of the recombinant peptide was tested from bacteriostasis essay in vitro.The expression supernatants was initial purification by Sephadex G-50 gel filtration.As for their in vitro antibacterial activity, catesbeiania-1showed a certain antibacterial activity and antimicrobial spectrum more than palustrin-Ca.Moreover catesbeiania-1 had stronger elimination ability of DPPH but no hemolysis activity and bleeding activity and Erythrocyte agglutination activity to red blood cell of rabbit.The effect of antibacterial peptide Catesbeiania-1 was observed that the antibacterial peptides had effect on microstructure of Bacillus subtilis and APP and E coli O138 using transmission electron microscope. The result shows that the peptides could cause the plasmolysis of the three bacteria. The integrity of the cell wall was clearaged .there were still various kinds of different mechanism of antibacterial peptide should be researched.Catesbeiania-1 with high bacteriostasis activity were studied in inhabiting the grows of bacteria in mice which was infected with E coli O138,the minimal lethal dose(MLD),the minimal protected dose, as well as the comparison between prevention and therapeutic efficacy were determined. The results showed that the group of prevention was better than that of therapy.At the same time,the immune index of CD changes from different groups was determined to prove that the antimicrobial peptide can increase the immune function in mice,defend the invasion of E.coli and keep the gut health.
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