Research On The CDNA Cloning And The Fused Expression In Pichi Pastoris Of The Antibacterial Peptide From The Rana Dybowskii

The antibacterial peptides secreted by the skin of amphibians are small molecule polypeptides with the functions of antibiosis, anticancer and so on. Such peptides are attracting increasing attention due to the growing problem of pathogenic microorganisms resistant to conventional antibiotics. Research in antibacterial peptide has lasted for years and some peptide antibiotics are being the phase of clinical trial in abroad. It’s initial stage of research in antibacterial peptide of Rana dybowskii on molecular level with several sequences reported only. Accordingly, it’s very important for practical application of antibacterial peptide with the study of gene structure, physico-chemical property, function and the construction of engineering bacteria and so on.In this study, degenerate primer is designed based on 5’conservative sequence of variety of Rana antibacterial peptide gene and used to amplify the cDNA library which reverse-transcripted by the total RNA extracted from the skin of Rana dybowskii. The gene library of the antibacterial peptide from Rana dybowskii was got and sequenced randomly. The result alignmented by CLUSTALW, the physico-chemical property estimated and analyzed by the softwares of EXPASY .There are 11 kinds of antimicrobial peptides of Rana dybowskii. The dybowskin-5, 6, 7, 8, 9 belonging to the Temporin family and dybowskin-1, 2, 3 belonging to Brevinin-1 family. The dybowskin-4 is similar to the Japonicin-1 of Rana japonica with 1 residue difference only. Both dybowskin-10 and 11 belong to a new family series which are small similarity to other peptides reported previously with different significant characters: more positive charge; no amidation modification of C terminal; higher pI, strong alkaline and hydrophilic.The hEGF sequence and mature peptide gene sequence of Dybowskin-2CDYa were connected with the thrombin cutting site, and connected to the expression vector pPIC9K. The plasmid was transformed into Pichia pastoris GS115, and the recombination peptide was expressed by the methanol induction. The expression production was precipitated in TCA and analyzed with the 15 % Tricine-SDS-PAGE, the band of 9 kDa was got. The results of RT-PCR show that there is a specific fragment at the location of a 250 bp, which suggests that the target gene has been inserted into the genome of the host and can be transcribed. RP-HPLC results show that the expression product is 0.30 g/L. Anti-bacterial test results show that both the broth and the peptides purificated by RP-HPLC have broad spectrum anti-bacterial activity.The study of the Rana dybowskii antimicrobial peptide settled a theoretical basis for the development and application of it.