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- W2384656477 abstract "Objective To construct a cDNA library by using mRNA from Gloydius ussuriensis(G. ussuriensis) venom gland, to clone and analyze serine proteinase gene from the cDNA library. Methods Total RNA was isolated from venom gland of G. ussuriensis, mRNA was purified by using mRNA isolation Kit. The whole length cDNA was synthesized by means of smart cDNA synthesis strategy, and amplified by long distance PCR procedure, lately cDAN was cloned into vector pBluescript-sk. The recombinant cDNA was transformed into E.coli DH5α. The cDNA of serine proteinase gene in the venom gland of G. ussuriensis was detected and amplified using the in situ hybridization. The cDNA fragment was inserted into pGEMT vector, cloned and its nucleotide sequence was determined. Results The capacity of cDNA library of venom gland was above 2.3×10 6. Its open reading frame was composed of 702 nucleotides and coded a protein pre-zymogen of 234 amino acids. It contained 12 cysteine residues. The sequence analysis indicated that the deduced amino acid sequence of the cDNA fragment shared high identity with the thrombin-like enzyme genes of other snakes in the GenBank. The query sequence exhibited strong amino acid sequence homology of 85% to the serine proteas of T. gramineus, thrombin-like serine proteinase Ⅰ of D. acutus and serine protease catroxase Ⅱ of C. atrox respectively. Based on the amino acid sequences of other thrombin-like enzymes, the catalytic residues and disulfide bridges of this thrombin-like enzyme were deduced as follows: catalytic residues, His 41 ,Asp 86 , Ser 180 ; and six disulfide bridges Cys 7 -Cys 139 , Cys 26 -Cys 42 , Cys 74 -Cys 232 , Cys 118 -Cys 186 , Cys 150 -Cys 165 , Cys 176 -Cys 201 . Conclusion The capacity of cDNA library of venom gland is above 2.3×10 6, overtop the level of 10 5 capicity, The constructed cDNA library of G. ussuriensis venom gland would be helpful platform to detect new target genes and further gene manipulate. The cloned serine proteinase gene exhibits strong amino acid sequence homology of 85% to the serine proteas of T. gramineus, thrombin-like serine proteinase Ⅰ of D. acutus and serine protease catroxase Ⅱ of C. atrox respectively." @default.
- W2384656477 created "2016-06-24" @default.
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- W2384656477 date "2005-01-01" @default.
- W2384656477 modified "2023-09-22" @default.
- W2384656477 title "Cloning and sequence analysis of serine proteinase of Gloydius ussuriensis venom gland" @default.
- W2384656477 hasPublicationYear "2005" @default.
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