Construction of recombinant prokaryotic vector containing Asn49-phospholipases A2 genes from Gloudius intermedius and the preliminary expression
HOU Bin1, ZHANG Wei-zhu2, ZHANG Kan1, ZHANG Chen1, YANG Zhang-min1*
(College of Life Science, Shaanxi Normal University, Xi′an 710062, Shaanxi, China；2 Xi′an Huitian Blood Products Company Limited, Xi′an 710075, Shaanxi, China)
Abstract:
To obtain pure phospholipases A2 for the further characterization in their evenomation and the underlying mechanism, based on PCR and DNA recombination technology, three novel Asn49-PLA2 genes (GI4-PLA2, GI5-PLA2 and its variant GI5′-PLA2) from the venom gland cDNA library of G. intermedius were cloned into the prokaryotic expression vector pQE-30, and the expression in E. coli Rosseta (DE3) was induced. Double digestion with Bam HⅠ/Hind Ⅲ and sequencing analysis indicated that the recombinant vectors pQE-30/GI4-PLA2, pQE-30/GI5-PLA2 and pQE-30/GI5′-PLA2 had been successfully constructed. Subsequent SDS-PAGE revealed that three target Asn49-PLA2 genes had expressed in the host cells. Under the condition of 37℃ and 1mM IPTG, the optimal inducing times for the expression of GI4-PLA2 and GI5-PLA2 were 25 h, while the optimal time for GI5′-PLA2G was 15 h.
KeyWords:
Gloydius intermedius; phospholipases A2; gene； prokaryotic expression
