抗呋喃它酮代谢物衍生物单链抗体-碱性磷酸酶融合蛋白表达条件的优化  

Expression Condition Optimization for Single Chain Variable Fragment Antibody-alkaline Phosphatase Fusion Protein Against Furaltadone Metabolite Derivative

作　　者：陈薪竹 杜华英 洪艳平 王丹 王玉波 杨武英 熊建华 CHEN Xinzhu;DU Huaying;HONG Yanping;WANG Dan;WANG Yubo;YANG Wuying;XIONG Jianhua(School of Food Science and Engineering,Key Laboratory of Agricultural Products Processing and Quality Control of Nanchang City,Jiangxi Agricultural University,Nanchang 330045,China)

机构地区：[1]江西农业大学食品科学与工程学院/南昌市农产品加工与质量控制重点实验室,江西南昌330045

出　　处：《江西农业大学学报》2021年第2期426-434,共9页Acta Agriculturae Universitatis Jiangxiensis

基　　金：国家自然科学基金项目(31660486);江西省自然科学基金面上项目(20202BABL205021);江西省教育厅科学技术研究项目(GJJ190218);江西省大宗淡水鱼产业技术体系项目(JXARS-3)。

摘　　要：【目的】优化基因工程菌的诱导表达条件,以提高抗呋喃它酮代谢物衍生物单链抗体-碱性磷酸酶融合蛋白在大肠杆菌中的可溶性表达量。【方法】采用液体发酵法培养抗AMOZ衍生物单链抗体-碱性磷酸酶融合蛋白的基因工程菌,分别研究不同宿主菌、培养基、初始pH、诱导时期、诱导时间和蛋白诱导表达剂异丙基硫代半乳糖苷(isopropylβ-D-thiogalactopyranoside,IPTG)对融合蛋白表达量的影响,并通过蛋白质印迹法和直接竞争酶联免疫分析法对其活性进行鉴定。【结果】以E.coli BL21(DE3)作为宿主菌,在以初始pH为7.0的SB液体培养基作为发酵培养基,将含有目的蛋白的基因工程菌培养至OD600为0.6时,用浓度为0.6 mmol/L的诱导表达剂IPTG诱导表达9 h的条件下,抗AMOZ衍生物单链抗体-碱性磷酸酶融合蛋白的表达量由最初的7.94%增加到了11.45%,总体增加了约3.51%,且融合蛋白具有良好的生物活性。【结论】成功提高了抗AMOZ衍生物单链抗体在大肠杆菌中的可溶性表达量,为后期大规模生产抗AMOZ衍生物单链抗体奠定了一定的基础。[Objective]In this experiment,the induced expression condition for fusion protein AMOZscFv-AP were studied and optimized in order to improve the expression level effectively[Methods]First,the recombinant plasmid plip6/GN-AMOZscFv was transformed into four strains of Escherichia col(i E.coli)to determine the optimal host bacteria.Then the effects of different media and their initial PH values,induction periods,induction time and protein inducer IPTG concentrations on the expression of fusion protein were studied after determining the optimal host bacteria.Finally,their activity was identified by Western-blotting and dcELISA[Results]The results showed that when E.coli BL21(DE3)was used as a host bacterium,the gene engineering bacteria were cultured in SB liquid medium with initial pH of 7.0 to a OD600 value of 0.6,and the expression was induced by IPTG with a concentration of 0.6 mmol/mL for 9 h,fusion protein AMOZscFv-AP expression increased from 7.94%to 11.45%[Conclusion]The soluble expression level of AMOZscFv in E.coli increased efficiently in the study,which laies a foundation for the later mass production and expression of AMOZscFv-AP fusion protein.

关 键 词：呋喃它酮代谢物 单链抗体 碱性磷酸酶 融合蛋白 表达条件 

分 类 号：TS207.3[轻工技术与工程—食品科学]