1. The Screening Of Na~+ Channel Protein Binding Proteins From The Spider Toxins 2. Expression And Function Assessment Of Two Venom Peptides From The Chinese Bird Spider Orinithoctonus Huwena

Na~v1.8 is expressed in the pathic neron,and plays a key role in the course of pain.It is meaningful for the further research of Na~+ channel proteins and designing the effective and safe pain-killer medicine in the clinical of screening the special inhibitior to the Na,,1.8 channel protein. Natural peptide venom provides a rich source for the research of ion channels.The venom of Orinithoctonus huwena has at least 400 kinds of peptides which provide a rich source for the screening of inhibitior of new type Na~+ channel proteins.We devoted to constructing a platform for screening the genes which ecoding the protein binding to Na~+ channel proteins from the cDNA library of the Orinithoctonus huwena venom gland.We chose the MatchMaker two-hybrid systemⅢpurchased from Clontech.We chose the S5-S6 segment of Na_v1.8 domainⅣas bait.The S5-S6 transmembrane segment of Na_v1.8 domainⅣwere cloned into the pGBKT7 vector which were transformed to the yeast strain AH109.We chose the sequencial transformation protocol.By spreading the transformants to the SD/-Trp/-Leu/-His agar plate,after several days,it grew out 22 colonies.Striking the colonies on the SD/-Trp/-Leu/-His/-Ade agar plates with X-α-gal,19 blue color colonies were found.By extracting the plasmid of the 19 colonies and sequencing, we got six cDNA sequences with no repeat.Unfortunately,four AD plasmids got self activation and two didn't take on blue colour in the retest screening.The 19 blue colonies turned out to be false positives. We compared the performace of X-α-gal with that of X-β-gal by agar plat analysis and filter paper analysis in screening blue colonies.We also summarized the high efficient method of preparing electrotransformation competent cells and extracting plasmid from yeast cells.All we did established a solid foundation for the further screening work. We found two cDNAs encoding toxin-like peptides,named HWTX-713 and HWTX-671,respectively,from the venom gland cDNA library of the bird spider Orinithoctonus huwena.The molecular weight of the HWTX-713 is 3733.52 Da by theoretical prediction.Only one clone was found in the 468 ESTs from the cDNA library and also never been separated from the crude venom.In order to study the special molecule,we decided to get the peptide by the molecular cloning and expression.We chose the eukaryotic secretion expression system.The HWTX-713 was expressed and purified using the vector of pVT102U/αand its yield was up to 10mg/L.It can form the disulfide bonds.The expression peptide showed no toxicity to the mice and cockroach.The patch clamp showed that the HWTX-713 had no effect to the Na~+,K~+ and Ca~+ ion channels of the mice DRG cells and cockroach DUM cells.It also didn't inhibit the growth of Escherichia coli,Bacillus subtilis, Staphyloccocus aureus Rosenbach,Salmonella typhi and Pseudemonas aeruginosa.The HWTX-671 shares 77%sequence similarity with SHL-I which contain six cysteines cross-linked by three disulfide bonds.The amidation signal GRR is located in the C-terminal of the HWTX-671 which was separated from the O.huwena crude venom.Unfortunately the yield is too low to further research.So we decided to increase the yield using the eukaryotic secretion expression system.But the yield is also so low, furthermore,unevenly cleavage and incorrectly amidation modification were found at the C-terminal.So we cut short the C-terminal to -G or-~* by adding the stop code ahead of schedule.The yield was the 590μg/L and 600μg/L,respectively.We found that the HWTX-671 has the hemagglutination at the minimal effective concerntation of 1.5mg/mL, 5.9mg/mL separately.