Partial ESTs Analysis Of Chinese Gloydius Shedaoensis Shedaoensis Venom Gland And The Clone, Expression Of Its Plasminogen Activator

As a complex mixture of proteins, enzymes and polypeptide, snake venomcomprises a wide range of biological activities. Because of the low yield and low purity,it’s not efficient to purify snake venom using traditional techniques, which makes itnecessary to produce high-purity proteins of snake venom by gene recombinationtechnique. The plasminogen activator (PA) in snake venom is a serine protease whichcan specially activate plasminogen to plasmin, dissolve fibrin and thrombus. Theadvantage of its long half-time period and specificity confers it a valuable protein.Objective:1. To obtain and analysis the ESTs of Gloydius shedaoensisshedaoensis venom gland cDNA(GSSGS-cDNA);2. To clone the gene and expressGSSG-PA by genetic engineering technique.Methods and results:1.216GSSG-cDNAs were randomly picked up andanalyzed by single-pass sequencing from the5，end. A total of211high-quality ESTswere generated and sequenced. Bioinformatics blasting results indicated that84ESTscould be annotated as the genes with known function,29ESTs as similar genes withunknown function, and the rest of98ESTs were identified as novel genes2. Thesequence of GSSG-PA was obtained by PCR through GSSG-cDNA library plasmid astemplate, the M13primes in the pDNR-LIB vector as upstream and downstream primesand two sequences as middle primes designed according to the peptides sequenceTLCAGTQQGG of GSSG-PA.3.11Codons of the GSSG-PA were mutated to E.coli’soptimal codons, called GSSG-PA’, and the expression vector pGEX-6P-1-GSSG-PA’was constructed. The protein GSSG-PA appeared as inclusion body by SDS-PAGEanalysis after being induced in E.coli BL21.4. The expression vectorpPIC9K-GSSG-PA was constructed and electro-transformed to yeast strain GS115. It was confirmed that the target gene was integrated onto the host chromosomesuccessfully by colony PCR.5. The expression vector pCold I-GSSG-PA’wasconstructed and the bacteriostasis of GSSG-PA protein was detected when it wasinduced in E.coli BL21.Conclusions:1. The partial ESTs of GSSG were obtained in current work, whichprovides certain useful information for cloning and expressing target protein genes andstudying the biological functions of target proteins from GSSG.2. The gene sequence ofGSSG-PA with independent intellectual property rights was obtained.3. The expressionvectors pPIC9K-GSSG-PA, pGEX-6P-1-GSSG-PA’ and pCold I-GSSG-PA’ wereconstructed successfully.4. GSSG-PA protein in inclusion body was got from E.coliBL21.5. The bacteriostasis of GSSG-PA protein was detected when it was induced togenerate.