Research On Diverse Gene Expression Of Active Polypeptides From Rana Chensinensis' Skin

Biologically active peptides are peptides separated from microorganism and those natural products such as animals and plants. These peptides can be either dipeptide composed of only 2 aminoacids, or complex long chain and ring-shaped polypeptide. Biologically active peptides can be divided as physiology bioactive peptide and other biologically active peptides. Among these, physiology bioactive peptides contain antibacterial peptides, antiviral polypeptide, neuroactive peptide, hormone and hormone regulatory peptide, immunoactive peptides and so on. Deep research on principle aspects such as gene structure and expression style as well as physical and chemical properties of active polypeptide is significant because of high cost for producing biologically active polypeptide by artificial chemical composition or separation from natural products, great price and many difficulties in practical application.Rana chensinensis is subspecies of Rana temporaria in China, whose skin contains a great deal of affluent biologically active polypeptide. Degenerate primer was designed in this research according to conserved sequence of antibacterial peptide's signal peptide end within Rana category of Batrachia. Totally 196 pieces of cDNA sequences from signal Rana temporaria chensinensis are obtained by RT-PCR technique and clone, among which, 173 pieces are plus sense cDNA sequences.Thirty five pieces of different antibacterial peptide precursors, thirty three pieces of different mature peptide, 1 piece of bradykinin precursor and some special active polypeptide sequences were encoded. Diversity of active polypeptide gene expression for Rana temporaria chensinensis is substantially demonstrated from this viewpoint. Certain disciplines are summarised for amino acid sequences of polypeptide precursor: conservative propertied are observed for sequences of signal peptide and acid propeptide front zone while greater variations are found for sequences of mature peptide. Eight different antibacterial peptide families and 2 bradykinins can be defined in accordance with sequence diversity of mature peptide which falls into one category of biologically active polypeptide. For antibacterial peptide families, there are temporin-1,brevinin-1,brevinin-2,chensinin-1, chensinin-2, chensinin-3, chensinin-4 and chensinin-5 family. For bradykinin,there are Bradykini and Thr⁶-BK。Based on anylysis of bioinformatics software, cationic charatic properties are observed for all the antibacterial peptides of eight families with theory isoelectric points ranges within 5.76 to 10.07, molecular weight within 1460.8 to 3702.4 and they are generally made up of 17 to 37 amino acid residues. By comparation with antibacterial peptide web, it is concluded the similarity between most of antibacterial peptides of Rana temporaria chensinensis and those in database is higher than 70%. Only the similarity between antibacterial peptide brevinin-1CEf₁ and Nanorana parkeri in Tibet is 58.82%. The similarity between antibacterial peptide brevinin-1CEf₂ and Nanorana parkeri is 50%. The above two (brevinin-1CEf₁ and brevinin-1CEf₂) can be treated as new type antibacterial peptides of Rana category initially. For secondary structure analysis, most of the antibacterial peptides haveα-spiral area, except brevinin-1CEc,brevinin-1CEb and chensinin-2 being secondary structure mainly including extended links.According to category analysis of active polypeptides, Rana temporaria chensinensis'skin contains many biologically active peptides worthy of study including antibacterial peptide and bradykinin and so on. According to analysis of active polypeptide gene and amino acid sequences, sequences of antibacterial peptide are from same source to a great extent, but big variation exists for mature peptide's sequences as well as different secondary structures, which determines diversity of categories, structures and functions of active polypeptides.