Purification, Molecular Cloning And Gene Analysis Of Ussurin,a Novel Platelet Agglutination-inhibited Metalloproteinases/ Disintegrin From Gloydius Ussuriensis

Total RNA was isolated from venom gland of Glydius Ussurisis,mRNAwas purified with mRNA isolation kit. Whole length cDNA was obtained bymeans of SMART cDNA synthesis strategy and followed by PCR amplified.The DNA fragment of preprometalloproteinase containing Disintegrin wasobtained through PCR amplification,and later cloned into pGEM-T Easyvector,which contained M13 sequence for following sequence analysis.We confirmed preprometalloproteinase cDNA's sequence by CEQ-2000made by Beckman corporation and it's exactly what we are looking for.Software such as DNAstar,NCBI Blast was used to analyze the homologybetween the amplified fragment and the corresponding sequences of westerndiamondback rattlesnake preprometalloproteinase,bothrops insularis clusterBITM06A metalloproteinase precucor,Deinagkistrodon acutus metalloproteinaseprecucor etc.The results revealed that the coloned fragment was veryconservative,especially in the coding regions of the gene,and the homologycould reach up to 93%. Insertion and deletion happened in the coding region ofpresignal peptide in preprometalloproteinase of Gloydius Ussurisis wereobserved,in which gene homology up to 87%.Amino acid sequence deduced from the encoding genes was also...