Genetic Engineering Of The Changbai Mountain The Mangabeys Viper Phospholipase A <sub> 2 </ Sub>

Based on N-terminal sequencing and peptide prints analysis of the purified phospholipase A2 protein from Agkistrodon blomhoffii ussurensis, its cDNA was obtained by means of reverse transcription polymerase chain reaction (RT-PCR) and Rapid Amplification of cDNA Ends (RACE). The open reading frame (ORF) of the mature gene of Gln49-PLA2 consists of 366 bases encoding 122 amino acids.Amino acids sequence analysis and molecular model of the Gln49-PLA2 show the phospholipase A2, named Gln49-PLA2, is a new phospholipase A2 with a Gin at position 49. Gln49-PLA2 is devoid of phospholipase A2 activity, which is probably because of failing to bind the co-factor Ca2+.Comparison with other PLA2S, the amino acid sequence of the Gln49-PLA2 shows higher similarity to Asp49-PLA2 variants (93% to 61%) than that of Lys49-PLA2 variants (60% to 56%). The phylogenetic tree indicates that the Asp49-PLA2 variants are more closely related to the Gln49-PLA2 than the Lys49-PLA2 variants.GIn49-PLA2 was cloned into the vector pET-32a(+), then transformed into E.coli strain BL21(DE3). After induced by isopropylb-D-thiogalactoside (IPTG), the gene was expressed in the form of soluble protein. Western blot confirmed that the protein specifically reacts with yolk IgY against the native Gln49-PLA2 from Agkistrodon blomhqffii ussurensis. Its expression level was studied and the optimal culture conditions is obtained as follows: Harvesting cells after 6 hr of IPTG induction at 30C. By immobilized metal affinity chromatography, ion-exchange chromatography and hydrophobic interaction chromatography, the recombinant Gln49-PLA2 was purified with 91% purity. The recombinant Gln49-PLA2 has apparent anticoagulant activity in vitro and the growth of Hela cell can been inhibited by the recombinant Gln49-PLA2.