Location:Home > Engineering science > Chemical Engineering > Chemical Processes > Chemical Synthsis, Activity Evaluation and Expression of the Novel Conotoxins from South China Sea

Chemical Synthsis, Activity Evaluation and Expression of the Novel Conotoxins from South China Sea

Downloads: []
Tutor: CuiZhiFang;DaiQiuYun
School: Shandong University of Science and Technology
Course: Chemical processes
Keywords: conus,conotoxins,synthesis,expression,disulfide-bridge,analgesic activity
CLC: TQ464
Type: Master's thesis
Year:  2011
Facebook Google+ Email Gmail Evernote LinkedIn Twitter Addthis

not access Image Error Other errors

Conotoxins (CTx) secreted from venom of cone snails are a class of small bioactivity-peptides and rich in disulfide bonds. Conotoxins could specifically bind to channels (Na+, K+, Ca2+, etc) and membrane receptors, and are useful for neuropharmacological researches. Some conotoxins with potent analgesic activity, such as MVIIA, have been utilized as new analgesic agents. It is estimated that there are about 50,000 conotoxins all of the world, but only a few of which have been identified functionally. Conus species are rich resources in South China Sea, but few of them have been studied.This subject aims to obtain novel conopeptide products by both chemical synthesis and genetic engineering methods, then determine the disulfide-bridge connectivity, and evaluate preliminarily analgesic activities of some peptides so as to lay the foundation for the further study on the identification of functions and structure-activity relationships. Main works are as follows:(1) 25 linear peptides including 4 conantokins,15 a-conotoxins and 6 M-superfamily conotoxins were synthesized by Fmoc solid-phase method. Then correct folded conotoxins were obtained using air oxidative folding, most of which could fold efficiently except for the M-conotoxins due to their special structures. Furthermore, Mass Spectral analysis showed that these folded products have correct molecular weights. The co-elution assay of the oxidized products with two-step folding products suggested that they have the disulfide connectivity arrangement of (Cys1-Cys3, Cys2-Cys4), which is identical to that of the native conotoxins reported.(2) The analgesic activity of 7 folded products from 6 a-conotoxins (liu-5, liu-6, liu-7, liu-16, liu-21 and T4) was assessed using the partial sciatic nerve ligation model (PSL). The results indicated that all peptides possess significant analgesic activities except for the liu-6 and liu-7, and the effect lasted longer than that of positive control Vcl.1. At the dose of 3 nmol/rat and 1.5h after administration, liu-5 could increase the pain threshold value of the rats by about 97%, and the analgesic activity of a4/7-liu-16, as well as a4/8-T4, was slightly lower than that of Vcl.1.(3) Conotoxins were expressed by fusion protein expression system, and the conditions were explored. The target gene was inserted into vector pGEX-6p-1 and expressed with GST in the form of fusion protein.12-15 mg GST fusion protein was obtained from 1 L culture under the optimized inducing conditions (0.5 mM IPTG,23¡æfor 8 h). Affinity chromatography was employed to give fusion protein GST-conotoxin with high purity, whose molecular weight is consistent with the calculated value. Several small fragments formed after cleavage by enterokinase. More researches on qualitative analysis and purification are progressing.
Related Dissertations
Last updated
Sponsored Links
Home |About Us| Contact Us| Feedback| Privacy | copyright | Back to top