Molecular Cloning And Sequence Analysis Of CDNA Encoding Metalloproteinase From The Venom Of Agkistrodon Acutus From Guangxi Province

Objective: To clone and sequence the cDNA encoding metalloproteinase from the venom of Agkistrodon acutus from northen mountain area of Guangxi province. Methods: One step method was used to extract total RNA from the venom of Agkistrodon acutus found in northern mountain area of Guangxi province. Different kinds of cDNA encoding metalloproteinase were amplified by one step method (RT-PCR and PCR reactions occurred in the same tube) using different primers. The PCR products were cloned into the pGEM-T easy vector. Plasmid DNA was obtained from positive clones identified by means of PCR reaction. Their cDNA sequences were determined with positive clones or purified PCR products directly. Results: One of the PCR product is about 600 bp, the other PCR product is about 1.5 Kb,1.3 Kb and 800 bp. The 600 bp and 800 bp PCR products were cloned into the pGEM-T easy vector. Their cDNA sequences were determined with positive clones or purified PCR product. Conclusion: Compared the 600 bp PCR product with the amino acids sequence for the fibrinolysin metalloproteinase from the Venom of Agkistrodon acutus from the southern of Anhui province, their homology is 90.6%. The cDNA sequence with 789 bp encodes an uncompleted open reading frame (ORF) of 263 amino acids, which included a signal peptide of 18 amino acids, a proposed propeptide of 171 amino acids, a partial mature protein of 74 amino acids. The sequences of signal peptide and propeptide are very similar to ones from Wannan Agkistrodon acutus reported by Fan Chunyan, but it only contains partial amino acids of mature protein. The 800 bp is likely to be a part of gene encoding metalloproteinase Agkistrodon acutus from Guangxi province.