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机构地区:[1]新余高等专科学校,江西新余338000 [2]浙江工业大学药学院,浙江杭州310032 [3]爱思进生物技术有限公司,浙江杭州310021
出 处:《江西师范大学学报(自然科学版)》2010年第1期93-97,共5页Journal of Jiangxi Normal University(Natural Science Edition)
基 金:江西省教育厅青年科学基金(GJJ09625)资助项目
摘 要:PCR扩增来自黄杆菌ATCC27551的有机磷水解酶基因opd,直接与XcmⅠ酶切去除Ampr基因片段的pZXL-T连接,将opd克隆于锚定单元Lpp-OmpA编码序列下游,转化E.coliBL21(DE3).经抗性筛选、PCR鉴定和测序证实,成功构建了具有全细胞催化效应的大肠杆菌细胞表面展示工程菌.SDS-PAGE结果表明,工程菌能表达产生51 kD的融合蛋白.细胞表面展示的有机磷水解酶具有较高全细胞酶活性,用蛋白酶K消化处理重组菌表面蛋白可使其全细胞酶活降低90%.Opd genes of Flavobacterium sp. ATCC27551 strain coding organophosphate hydrolase is amplified by PCR, then ligated with vector pZXL-T which is digested with Xcm I to remove Ampr, so opd gene is cloned into anchoring motif Lpp-OmpA downstream and the recombinant plasmid is transformed into E. coli BL21(DE3).After drug-resistant selection and identification by PCR and DNA sequencing, the cell-surface display engineering strain,which have a whole cell catalytic activity of organophosporus hydrolase, is successfully constructed. SDS-PAGE analyses eonfirm that the fusion protein can be expressed in engineering strain with a molecular of about 51 kD, and it is found that the engineering strain exhibits high whole cell activity of OPH. Additionally, OPH activity declines by 92 % Of total whole cell activity by treating the surface proteins with proteinase K.
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