hAPOB的克隆、原核表达及抗体效价分析  

Cloning, Prokaryotic Expression and Antibody Titer Analysis of hAPOB

作　　者：徐志伟[1] 王期[2] 徐一然[3] 陈永霞 徐小波[5] 于建宁[5] XU Zhi-wei;WANG Qi;XU Yi-ran;CHEN Yong-xia;XU Xiao-bo;YU Jian-ning(Zhenjiang Agricultural Scientific Research Institute of Jiangsu Hilly Area,Jiangsu Jurong 212400,China;School of Life Sciences,Nanjing Normal University,Jiangsu Nanjing 210023,China;Institute of Civil Engineering and Mechanics,Jiangsu University,Jiangsu Zhenjiang 212013,China;Jurong Animal Disease Prevention and Control Center of Jiangsu Province,Jiangsu Jurong 212400,China;Institute of Animal Science,Jiangsu Academy of Agricultural Sciences,Jiangsu Nanjing 210014,China)

机构地区：[1]江苏丘陵地区镇江农业科学研究所,江苏句容212400 [2]南京师范大学生命科学学院,江苏南京210023 [3]江苏大学土木工程与力学学院,江苏镇江212013 [4]江苏省句容市动物疫病预防控制中心,江苏句容212400 [5]江苏省农业科学院畜牧研究所,江苏南京210014

出　　处：《西南农业学报》2019年第2期446-450,共5页Southwest China Journal of Agricultural Sciences

基　　金：江苏省农业科技自主创新资金项目[CX(16)1054];镇江市科技局社会发展项目(SH2018003);句容市科技局民生计划项目(SF2018022693)

摘　　要：【目的】人载脂蛋白B(hAPOB)是保持人体血脂恒定的重要蛋白,检测hAPOB具有重要的临床价值。【方法】本研究将hAPOB(97-526AA)序列连接到原核表达载体pGEX-4T-1中,构建pGEX-4T-1-hAPOB(97-526AA)原核表达质粒,然后将其转入宿主菌BL21,宿主菌表达出与预期分子量大小相符的80 kDa的融合蛋白;将纯化后的融合蛋白免疫湖羊,制备抗hAPOB血清并检测抗体效价。【结果】每次注射0.4 mg的原核表达抗原,湖羊抗血清的产生效果最佳;经间接酶联免疫分析(ID-ELISA)方法检测,原核表达蛋白免疫湖羊得到的hAPOB抗体效价为l︰40000。【结论】本试验获的抗血清能够满足商用标准,为后续拓展其应用奠定了基础。【Objective】Human apolipoprotein B(hAPOB)is an important protein that keeps the blood lipid constant.Detecting hAPOB has a important clinical value.【Method】In this study,the hAPOB(97-526AA)sequence was ligated into the prokaryotic expression vector pGEX-4T-1 to construct the pGEX-4T-1-hAPOB(97-526AA)prokaryotic expression plasmid which was then transferred into the host strain BL21,and an 80 kDa fusion protein was expressed with an expected molecular weight;The purified fusion protein was immunized against Hu sheep to prepare anti-hAPOB serum,and antibody titers were detected.【Result】The antiserum produced by Huyang was the best when 0.4 mg prokaryotic expression antigen was injected each time.Indirect enzyme-linked immunosorbent assay(ID-ELISA)showed that the titer of the hAPOB antibody obtained by immunizing Hu sheep with the prokaryotic expression protein was 1∶40000.【Conclusion】The antiserum obtained from this experiment can meet the commercial standards and lay the foundation for the subsequent application.

关 键 词：hAPOB PGEX-4T-1 抗血清 抗体效价 湖羊 

分 类 号：S826.99[农业科学—畜牧学]