Purification, Gene Cloning and Expression of an Acidic Phospholipase A2 from Agkistrodon s

Purification, Gene Cloning and Expression of an Acidic Phospholipase A2 from Agkistrodon

shedaoensis Zhao

Qian JIN; Li-Xia YANG; Hao-Mang JIAO; Bin LU; Yu-Qun WU; Yuan-Cong ZHOU

【期刊名称】《《生物化学与生物物理学报：英文版》》 【年(卷),期】2004(036)001

【摘要】A protein with the activity of phospholipase A2 named asAPLA2 was purified to homogeneity from the venom of Agkistrodon shedaoensis Zhao through DEAE-Sepharose CL-6B anion exchange column,Source S and Mono Q FPLC. Its molecular weight was estimated as 19 kD by SDS-PAGE and its pI was about 3.5 by IEF analysis. It inhibits the platelet aggregation that was induced by 1 μmol/L ADP, and the IC50 was determined to be 6 μmol/L. Degenerate primer was designed and synthesized according to the Nterminal amino acid sequence of asAPLA2. Its full-length cDNA was cloned by RT-PCR from the total RNA extracted from the snake venom gland. According to the deduced amino acid sequence, its molecular weight and pI are determined to be 13,649 and 4.39 respectively as calculated by DNAclub and DNAstar softwares.The gene was then cloned into the expression plasmid pET-40b(+) and expressed in E. Coli BL21(DE3).Western blot analysis indicated that the expressed protein cross-reacted with the antibody against the nativeenzyme.