关键词: 毒腺, cDNA文库, 安克洛酶, 序列分析, 蛋白质

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 2.3×10⁶. 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, His⁴¹,Asp⁸⁶, Ser¹⁸⁰; and six disulfide bridges Cys⁷ -Cys¹³⁹, Cys¹²⁶ -Cys⁴², Cys⁷⁴ -Cys²³², Cys¹¹⁸ -Cys¹⁸⁶, Cys¹⁵⁰-Cys¹⁶⁵, Cys¹⁷⁶-Cys²⁰¹. Conclusion The capacity of cDNA library of venom gland is above 2.3×10⁶, overtop the level of 10⁵ 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.

Key words: venom gland, cDNA library, ancrod, sequence analysis, protein