摘要
本试验旨在制备牦牛早孕因子(early pregnancy factor,EPF)多克隆抗体,为牦牛妊娠早期高效快速诊断技术研究奠定基础。采用RT-PCR方法,从牦牛胎盘和卵巢组织中提取RNA,反转录为模板后进行EPF基因扩增,并将其克隆到改造后的载体pET28-His10-Sumo上,构建pET28-His10-Sumo-EPF重组质粒,经PCR和测序鉴定后将阳性重组质粒转化至感受态大肠杆菌BL21(DE3)中。用IPTG诱导表达并优化表达条件,使用His标签镍柱纯化重组EPF蛋白并免疫小鼠制备抗体。结果显示,经PCR扩增的牦牛EPF基因在300 bp左右,符合预期结果,并通过测序验证获得重组阳性质粒。pET28-His10-Sumo-EPF重组质粒转化后经IPTG的诱导,表达分子质量大小为29 ku的蛋白(其中包括早孕因子目标蛋白和SUMO标签蛋白),且在37℃、IPTG浓度为300 nmol·L^(-1)、诱导6 h时获得最高表达量。Western blot结果显示,蛋白在上清和包涵体中均有表达,免疫后的小鼠抗血清能够与纯化后的重组蛋白发生免疫反应。本试验通过原核表达系统成功表达了重组牦牛EPF蛋白,制备了免疫原性较好的鼠抗EPF多克隆抗体。
The purpose of this study was to prepare polyclonal antibody against early pregnancy factor(EPF)of yak and lay the foundation for developing an efficient and rapid diagnosis of early pregnancy for yak in clinical field.EPF gene was amplified by RT-PCR using RNA extracted from yak placenta and ovarian tissue and reverse transcribed as template.Then the EPF gene was subcloned into the modified vector pET28-His10-Sumo to construct the pET28-His10-Sumo-EPF recombinant plasmid.After PCR and sequencing identification,the positive recombinant plasmid was transformed into BL21(DE3)for expression.IPTG was used to induce and optimize the reaction conditions.The recombinant EPF protein was purified and immunized mice to produce polycolonal antibodies.The results showed that,after PCR amplification,the target was about 300 bp,which was consistent with the expected results,sequencing identification confirmed the recombinant positive plasmid was successfully constructed.After EPF protein was expressed in E.coli,the protein molecular weight was 29 ku(includes target proteins and SUMO tagged proteins)using IPTG inducer.The protein expression level reached the highest at 37℃than other controls and induced concentration of IPTG was 300 nmol·L^(-1)for 6 h.Western blot results showed that the protein was expressed in both supernatant and inclusion body,and the mouse immunized antiserum could react with the purified recombinant EPF protein.The recombinant EPF protein was successfully expressed by prokaryotic expression system,and the mouse anti-EPF polyclonal antibodies with good immunogenicity was prepared.
作者
曹艳桃
樊江峰
余四九
周应聪
杜培岩
李一娟
马进彪
赵生贤
CAO Yantao;FAN Jiangfeng;YU Sijiu;ZHOU Yingcong;DU Peiyan;LI Yijuan;MA Jinbiao;ZHAO Shengxian(Research Center of Bovine and Sheep Embryo Engineering Technology of Gansu Province,College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China)
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2022年第2期470-480,共11页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
“十三五”国家重点研发计划子课题(2018YFD0502303)
甘肃省科技重点研发计划(农业类)项目(20YF8NA131)
陇原青年创新创业人才项目(2021LQGR47)
甘肃农业大学科技创新基金(青年导师基金)项目(GAU-QDFC-2018-10)
国家自然科学基金(31660732)
甘肃省高校基本科研业务费。
关键词
牦牛
早孕因子(EPF)
原核表达
多克隆抗体
yak
early pregnancy factor(EPF)
prokaryotic expression
polyclonal antibody
