奶山羊EGFR蛋白胞外区的原核表达及其多克隆抗体的制备

发布时间：2018-06-08 21:39

本文选题：表皮生长因子受体(EGFR) + 多克隆抗体　；参考：《西北农林科技大学学报(自然科学版)》2017年12期

【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 860bp的EGFR胞外区基因序列,成功构建pET-32a(+)-ECD载体。加入终浓度为0.8mmol/L的IPTG,于37℃诱导表达6h成功得到重组蛋白,该蛋白分子质量约94ku,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。
[Abstract]:[Objective] to express epidermal growth factor receptor (EGFR) receptor (ECD sequence) in the outer region of the epidermal growth factor receptor (factor receptor, EGFR) protein of milk goat, to purify the protein and immunize the rabbit, to prepare the specific polyclonal antibody against the goat EGFR protein, and to lay the foundation for the study of the function of goat EGFR protein. PMD19-T-EGFR was used as a template to amplify the EGFR extracellular domain (ECD) gene sequence by PCR method. The ECD sequence was connected to pET-32a (+) plasmid to construct pET-32a (+) -ECD prokaryotic expression vector, and the recombinant vector was introduced into the Escherichia coli BL21 (DE3) and induced expression. 2 month old White Rex rabbits were immunized with the purified recombinant protein by Ni-Charged MagBeads. The antibody titer was measured by ELISA (iELISA) method and the specificity of the antibody was detected by Western blot. [results] PCR amplified to 1 860bp EGFR extracellular domain gene sequence and successfully constructed pET-32a (+) -ECD carrier. The antibody titer of the modified protein.IELISA in the form was 1: 16000, and the Western blot detection showed that the prepared polyclonal antibody could specifically identify the ECD protein expressed in the prokaryotic cell and the EGFR protein in the goat mammary epithelial cells and HEK293 cells. [Conclusion] the goat EGFR polyclonal antibody was successfully prepared, and the antibody could be used for scientific research. Test.
【作者单位】：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;
【基金】：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)
【分类号】：Q78;S827

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