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作 者:侯彬[1] 张卫柱 张侃[1] 张琛[1] 杨章民[1]
机构地区:[1]陕西师范大学生命科学学院,陕西西安710062 [2]西安回天血液制品有限责任公司,陕西西安710075
出 处:《陕西师范大学学报(自然科学版)》2008年第4期76-80,共5页Journal of Shaanxi Normal University:Natural Science Edition
基 金:国家自然科学基金资助项目(38970121)
摘 要:以来自中介蝮(G.intermedius)毒腺cDNA文库的3个新Asn49-PLA2基因(GI4-PLA2,GI5-PLA2及其突变体GI5′-PLA2)为模板,用PCR及DNA重组技术,将三个PLA2基因克隆到原核表达载体pQE-30,并在宿主菌E.coli Rosseta(DE3)中进行诱导表达.Bam HⅠ/HindⅢ双酶切及测序结果提示,成功构建了3个原核表达载体(pQE-30/GI4-PLA2,pQE-30/GI5-PLA2,pQE-30/GI5′-PLA2).SDS-PAGE结果表明,中介蝮的3个Asn49-PLA2基因在大肠杆菌中实现表达,而且在37℃、1mMIPTG的诱导条件下,GI4-PLA2和GI5-PLA2的最佳表达时间为25h,而GI5′-PLA2在15h有较高水平的表达.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. interrneclius were cloned into the prokaryotic expression vector pQE-30, and the expression in E. coli Rosseta (DE3) was induced. Double digestion with Barn H Ⅰ/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 lmM IPTG, the optimal inducing times for the expression of GL-PLA2 and GI5-PLA2 were 25 h, while the optimal time for GI5'-PLA2G was 15 h.
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