Sequence Analysis And Heterologous Expression Of Thrombin Enzyme From Agkistrodon Acutus Venom

Snake venom thrombin-like enzymes degrade fibrinogen but cause no clotting,thus decrease the plasma fibrinogen level and act as an anticoagulant in vivo. TLEsare clinically used for the treatment of stroke, deep-vein thrombosis etc. TLEs arerelatively abundant in Agkistrodon acutus venom, and exist in forms of isoenzymes.TLEs belong to the serine protease superfamily and exhibit structural and functionaldiversity. So far their molecular structures and structure-function relationshipsneeded to be uncovered, analyzed and presumed. So as the mechanism of themolecular diversity.With the development of molecular biology and the method of sequencing,people raise their efficiency in analyzing of sequences. We can use bio-software toanalyze sequences, analyze the relationship and difference among them, or cananalyze the possible reason about the results. The research on the way of how thegene use the codons can help us to understand the mutation pressure which genomehad endured, the affect about elective translational efficiency and estimation aboutexpressional level on some gene.Primers were designed with reference to the known amino acid sequences or thecDNA sequences of TLEs. The RT-PCR product of A. acutus mRNA was amplifiedby PCR. Product of about 700 bp was obtained and cloned into T-vector. Sequencingresults proved to be a series of TLE cDNAs, some of which were submitted to andaccepted by GeneBank. These results enrich the gene resource of snake venom. Themolecular structure of TLEs takes on characteristics both in diversity andconservatism. The TLE's molecular length, the position of amino acids in catalyticsite, the number and position of disulfide bond are extremely conservative, but theother sites have a fairly high possibility of mutation. The codon method ofadministration reveals that thrombin-like enzyme had endured the pressure from bases composition and natural selection through the long-term evolutionary process.We presume that TLEs and trypase are come from one gene, TLEs had evolved tothe adaption of toxic protein in the evolutionary process. The new disulfide bond cansustain the senior conformation of protein, the molecular diversity can better adaptcomplicated food. The way of TLEs codon usage reveals this evolutionalcharacteristic through another way.Presently the TLE preparations being medically used are extracts from snakevenoms and encounter problems in purity and cost-effectiveness. Sometimes theproduction have a adverse reaction, so people need to produce TLE through geneticengineering.The TLE was cloned into pBAD/Thio-TOPO which fused thioredoxin andexpressed as fusion proteins successfully. This method provided not only a practicalway for gene engineering production of TLEs, but also a fast and convenient systemfor studying the structure-function relationships of TLEs. Up to now, there still areno successful reports on direct expression of TLE. We choose a vector pBV220 fordirect expression. Recombinant plasmid was constructed and optimized, and we hada research on thestructure-function relationship. We found the critical factor aboutexpression is the mRNA' secondary structure, the way how it come to effect has tobe tested in the future study.