Construction of cDNA library of Gloydius saxatilis venom gland cloning and sequence analysis of defibrase

Purpose To construct a cDNA library by using mRNA from Gloydius saxatilis venom gland, to clone and sequence analysis defibrase from the cDNA library. Methods Total RNA was isolated from venom gland of G. saxatilis , and mRNA was purified by using mRNA isolation kit. Full length cDNA was synthesized by means of smart cDNA synthesis strategy, and amplified by long distance PCR procedure. Lately cDAN was linked to vector pBluescript-sk. The recombinant cDNA was transformed into E.coli DH5 α. It detected and amplified the cDNA of defibrase gene in the venom gland of G. saxatilis using the hybridization in situ method. 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 the 2.0×106 . Its open reading frame is composed of 777 nucleotides and codes a pre-zymogen of 258 amino acids. It contains 12 cysteine residues. Conclusion The capacity of cDNA library of venom gland was above the general level of cDNA library. The constructed cDNA library of G. saxatilis venom gland would be a helpful platform to detect new target genes and further gene manipulate. The sequence analysis indicates that the deduced amino acid sequence of the defibrase shares more than 86% identity with the Thrombin-like enzyme genes of other snakes in the gene bank.