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奶山羊EGFR蛋白胞外区的原核表达及其多克隆抗体的制备
马 跃, 王建珏, 黄江涛,等
西北农林科技大学 动物科技学院,陕西省农业分子生物学重点实验室
摘要:
【目的】原核表达奶山羊表皮生长因子受体(epidermal growth factor receptor,EGFR)蛋白胞外区25-644位共620个氨基酸序列(ECD序列),纯化蛋白并免疫家兔,制备针对山羊EGFR蛋白的特异性多克隆抗体,为山羊EGFR蛋白功能研究奠定基础。【方法】以pMD19-T-EGFR为模板,采用PCR方法扩增得到EGFR胞外区(ECD)基因序列。将ECD序列连接pET-32a(+)质粒构建pET-32a(+)-ECD原核表达载体,将该重组载体导入大肠杆菌BL21(DE3)中并诱导表达。通过Ni-Charged MagBeads纯化重组蛋白后免疫2月龄白色獭兔,采用间接ELISA (iELISA)法测定抗体效价,Western blot 检测抗体特异性。【结果】PCR扩增得到1 860 bp的EGFR胞外区基因序列,成功构建pET-32a(+)-ECD载体。加入终浓度为0.8 mmol/L的IPTG,于37 ℃诱导表达6 h成功得到重组蛋白,该蛋白分子质量约94 ku,且为以包涵体形式存在的变性蛋白。iELISA检测抗体效价为1∶16 000;Western blot检测表明,制备的多克隆抗体能特异性识别原核表达的ECD蛋白及在山羊乳腺上皮细胞和HEK293细胞中的EGFR蛋白。【结论】成功制备山羊EGFR多克隆抗体,该抗体可以用于科研试验。
关键词:  表皮生长因子受体(EGFR)  多克隆抗体  原核表达
DOI:
分类号:
基金项目:国家自然科学基金项目(31672398);陕西省科技统筹创新工程计划项目(2016KTZDNY02-05)
Prokaryotic expression,purification and preparation of polyclonal antibody for extracellular domain of epidermal growth factor receptor (EGFR) of dairy goat
MA Yue, WANG Jianjue, HUANG Jiangtao,et al
Abstract:
【Objective】The amino acids from 25 to 644 of epidermal growth factor receptor (EGFR) of dairy goat (ECD sequence) were expressed and purified before immunizing rabbit to provide basis for research of EGFR protein function in dairy goat.【Method】The goat ECD sequence was cloned by PCR using pMD19-T-EGFR plasmid as template and then ligated with pET-32a(+) to construct prokaryotic expression vector pET-32a(+)-ECD.The pET-32a(+)-ECD vector was then transformed into E.coli BL21(DE3) competent cells for induction expression.Recombinant protein was purified by Ni-Charged MagBeads before immuning 2-month-old Rex Rabbit.Titer and specificity of the polyclonal antibody were analyzed through indirect ELISA(iELISA) and Western blot,respectively.【Result】The extracellular domain of EGFR gene with length of 1 860 bp was cloned by PCR and prokaryotic expression vector pET-32a(+)-ECD was constructed successfully.The denatured recombinant protein in the form of inclusion body with weight of 94 ku was obtained under the induction condition of 0.8 mmol/L IPTG and 37 ℃ for 6 h.Indirect ELISA analysis showed that the titer of the antibody obtained was 1∶16 000.Specific bands of prokaryotic expressed ECD protein and EGFR protein in both mammary gland epithelial cell and HEK293 cell were detected by Western blot analysis.【Conclusion】The antibody against extracellular domain (ECD) of dairy goat EGFR was prepared successfully and is capable for scientific experiments.
Key words:  epidermal growth factor receptor (EGFR)  polyclonal antibody  prokaryotic expression