摘要
为原核表达抗伏马菌素单链抗体-碱性磷酸酶融合蛋白并分析其活性,本研究根据抗伏马菌素单链抗体H2的基因序列设计引物,PCR扩增获得目的基因,经限制性核酸内切酶SfiⅠ和NotⅠ的酶切位点克隆到pDAP2/S载体中,转化大肠杆菌(Eschrichia coli)菌株XL1-Blue并鉴定阳性转化子。IPTG诱导H2-AP融合蛋白基因的表达,利用Western blot检测其表达情况,AP显色反应和ELISA鉴定其活性。结果显示成功构建了pDAP2/S-H2原核表达载体,融合蛋白在大肠杆菌中实现可溶性表达,并保留单链抗体和碱性磷酸酶的活性。因此,H2-AP融合蛋白通过原核表达后可用于发展伏马菌素的快速免疫检测方法。
To express fusion protein containing alkaline phosphatase(AP) and single-chain variable fragment(scFv)antibody against fumonisin and analyze its activity, primers were designed according to the sequence of the fumonisin-specific scFv(H2 antibody) and the target gene was amplified by PCR technology. Subsequently, the scFv genes were cloned into pDAP2/S vector through SfiⅠ and NotⅠ restriction endonuclease digestion site. The recombinant vector was transformed into the competent cells of Escherichia coli XL1-Blue and the positive transformants were identified. Then, The gene expression of H2-AP fusion protein was induced by isopropyl-β-D-thiogalactoside(IPTG) and detected by Western blot. Simultaneously, the activity of fusion protein was identified by AP color reaction and enzyme-linked immunosorbent assay(ELISA). The results showed that the prokaryotic expression vector of pDAP2/S-H2 was successfully constructed and the scFv-AP fusion was achieved to be expressed in E. coli in soluble form. In addition, the sc Fv-AP fusion retained the activity of AP and scFv antibody. Therefore, the prokaryotic expressed H2-AP fusion protein can be applied to develop rapid immunoassays for fumonisin detection.
作者
刘江丽
赵雪
尚国富
于欢
彭晓燕
王赟
周静
刘丽娜
曾柱
胡祖权
Liu Jiangli;Zhao Xue;Shang Guofu;Yu Huan;Peng Xiaoyan;Wang Yun;Zhou Jing;Liu Lina;Zeng Zhu;Hu Zuquan(Immune Cells and Antibody Engineering Research Center of Guizhou Province,Key Laboratory of Biology and Medical Engineering,Guizhou Medical University,Guiyang,550025;Key Laboratory of Environmental Pollution Monitoring and Disease Control,Ministry of Education,Guizhou Medical University,Guiyang,550025;School of Biology and Engineering,Guizhou Medical University,Guiyang,550025;School of Basic Medical Sciences,Guizhou Medical University,Guiyang,550025)
出处
《基因组学与应用生物学》
CAS
CSCD
北大核心
2021年第1期252-257,共6页
Genomics and Applied Biology
基金
国家自然科学基金(21906036,31771014,11762006,31860262)
贵州省科技计划项目(黔科合基础[2018]1412,黔科合平台人才[2016]5676,黔科合人才团队[2015]4021,黔科合LH字[2016]7357)
贵州省科技支撑项目(黔科合支撑[2019]2787号)
贵州医科大学环境污染与疾病监控省部共建教育部重点实验室开放课题基金(黔教合KY字[2017]380)
2011协同创新中心(黔教合协同创新字[2015]04)
贵州省细胞与基因工程创新群体(黔教合KY字[2016]031)共同资助。
关键词
伏马菌素
单链抗体
碱性磷酸酶
融合表达
Fumonisins
Single chain variable fragment(scFv)
Alkaline phosphatase(AP)
Fusion expression
作者简介
同等贡献:刘江丽;同等贡献:赵雪;通信作者:曾柱,zengzhu100@sina.com;通信作者:胡祖权,huzuquan@gmc.edu.cn。
