摘要
目的建立多房棘球蚴表面抗原EMY162原核表达系统及制备兔多克隆抗体。方法构建表达载体pCzn1-EMY162并转入Arctic Express菌株,用IPTG诱导、Ni柱纯化获得目的蛋白后免疫新西兰兔,以ELISA、SDS-PAGE考察纯化后抗体的效价、浓度及纯度。结果酶切鉴定和测序结果表明重组质粒pCzn1-EMY162构建成功,且EMY162在包涵体部位中表达、可溶部位微量表达。通过Ni-NTA柱亲和层析纯化获得高纯度的EMY162蛋白。免疫兔后收集血清,其ELISA结果表明,抗血清可以特异性结合目标蛋白,抗体效价大于512 K,SDS-PAGE结果表明,纯化后获得了较高纯度的多克隆抗体,可用于后续实验研究。结论成功建立EMY162原核表达系统并制备了高纯度、高效价的多克隆抗体。
Objective To establish the prokaryotic expression system for Echinococcus multilocularis surface antigen EMY162 and prepare its polyclonal antibodies.Method PAS（PCR-based Accurate Synthesis）was used to get the coding sequence of EMY162 and cloned in pCznl.The recombinants wereidentified with Nde I and Xba I double GA endonuclease digestion and sequencing.Recombinant expression plasmids pCznl-EMY162 was transfeet- ed into Arctic Expressbacterial strain.IPTG and Ni-sepharose were utilized to induce and purify EMY162.Antibod- ies of EMY162 were obtained by immunizing New Zealand rabbits with purified EMY162 protein and ELISA and SDS-PAGE were used to identify the purity and antibody titer of polyclonal antibodies.Result Recombiant plasmids pCznl-EMY162 was constructed successfully andidentified by endonuclease digestion and sequencing.SDS PAGEresults showed that EMY162 was expressed in inclusion bodies part of Arctic Express bacterial strain but not solu- ble.High-purity EMY162 protein were obtained by Ni-sepharose purification and ion exchange chromatography. Rabbit polyclonal antibody was successfully obtained, and the antibody titer was more than 512 000. Conclusion This study successfully established the prokaryotic expression system of emy162 and get high-purity, high-efficient polyclonal antibody.
作者
冯琳
李润乐
刘川川
廖瑜
杨全余
汤锋
FENG Lin;LI Run-le;LIU Chuan-chuanI;LIAO Yu;YANG Quan-yu;TANG Feng(Research Center for High Altitude Medicine, Qinghai University, Xining 810001;Clinical medicine of Medical college, Qinghai University, Xining 810016;High Altitude Zoonosis Research Center, Qinghai University, Xining 810001)
出处
《中国高原医学与生物学杂志》
CAS
2017年第4期235-241,共7页
Journal of Chinese High Altitude Medicine & Biology
基金
青海省科技厅项目(2015-ZJ-745
2014-ZJ-716)
青海省重大科技专项(2016-SF-A5)
关键词
EMY162
多房棘球蚴
多克隆抗体
原核表达
EMY162 Echinococcus muhilocularis Polyclonal Antibody Prokaryotic Expression
