中华大蟾蜍EDF-1重组蛋白的原核表达、纯化及抗血清的制备

doi:10.13560/j.cnki.biotech.bull.1985.2018-0291

生物技术通报 ›› 2018, Vol. 34 ›› Issue (10): 129-134.doi: 10.13560/j.cnki.biotech.bull.1985.2018-0291

中华大蟾蜍EDF-1重组蛋白的原核表达、纯化及抗血清的制备

刘怡君, 贾宇坤, 王玲芳, 刘虹杏, 杨仙玉

浙江农林大学动物科技学院,临安 311300

收稿日期:2018-03-29 出版日期:2018-10-26 发布日期:2018-11-07
作者简介:刘怡君,女,硕士研究生,研究方向：中药材多肽类有效成分的基因克隆与研发;E-mail：2441220499@qq.com;贾宇坤同为本文第一作者
基金资助:
国家自然科学基金项目（31372149,31772409）

Prokaryotic Expression,Purification and Antiserum Preparation of Recombinant EDF-1 of Bufo gargarizans

LIU Yi-jun, JIA Yu-kun, WANG Ling-fang, LIU Hong-xing, YANG Xian-yu

College of Animal Science and Technology,Zhejiang Agriculture and Forestry University,Lin’an 311300

Received:2018-03-29 Published:2018-10-26 Online:2018-11-07

摘要/Abstract

摘要： 通过实验获得中华大蟾蜍（Bufo gargarizans）内皮分化相关因子-1（Endothelial differentiation related factor-1,EDF-1）重组蛋白及高效价抗血清。在前期研究工作中已克隆中华大蟾蜍EDF-1的开放阅读框（Open reading frame,ORF）（GenBank登录号K F769459）,但因密码子的偏好性使中华大蟾蜍原有密码子在原核细胞中发生识别困难,重组蛋白原核表达未能成功。本文对中华大蟾蜍的EDF-1的ORF进行密码子优化,委托公司合成并插入原核表达载体,构建重组质粒pET-28b-EDF-1-Bufo。将重组质粒转入大肠杆菌（E. coli）感受态细胞BL21（DE3）,使用IPTG诱导重组蛋白表达,钴胶纯化重组蛋白,纯化的重组蛋白免疫小鼠制备鼠抗重组中华大蟾蜍EDF-1的抗血清,然后通过Western-blot和ELISA检测抗血清的特异性及其效价。重组中华大蟾蜍EDF-1得到良好表达,抗血清特异性良好,效价为1∶8 000。为进一步研究EDF-1的生物学功能奠定基础,同时为研发抑制内皮细胞分化相关的抗肿瘤药物提供重要参考。

Abstract: This study aims to acquire the recombinant protein of endothelial differentiation related factor-1（EDF-1）of Bufo gargarizans and high-titer antiserum. The open reading frame（ORF）of EDF-1 of B. gargarizans has been cloned（GenBank accession number：K F769459）in previous studies. However,the recombinant protein using original ORF was not expressed in prokaryotic system due to the codon bias that might make the codons of B. gargarizans difficult to be recognized in prokaryotic cells. The codon of EDF-1 ORF of B. gargarizans was optimized,synthesized chemically,and inserted into prokaryotic expression vector,thus the recombinant plasmid pET-28b-EDF-1-Bufo for prokaryotic expression was constructed. The recombinant plasmid was transformed into Escherichia coli competent cells BL21（DE3）. The expression of recombinant EDF-1 of B. gargarizans was induced by IPTG,and the EDF-1 was purified with cobalt agarose. Then mice were immunized with the purified recombinant protein to prepare the antiserum against EDF-1. The specificity and titer of the antiserum was determined by Western blot and ELISA,respectively. As results,the recombinant EDF-1 of B. gargarizans was well expressed and the antiserum was highly specific,and the titer of the antiserum against recombinant EDF-1 of B. gargarizans was 1∶8 000. These results lay a foundation for the further study of biological function of EDF-1 and provide important reference for development of antitumor drugs for inhibiting endothelia cell differentiation.

Key words: Bufo gargarizans, EDF-1, recombinant protein expression, codon optimization

刘怡君, 贾宇坤, 王玲芳, 刘虹杏, 杨仙玉. 中华大蟾蜍EDF-1重组蛋白的原核表达、纯化及抗血清的制备[J]. 生物技术通报, 2018, 34(10): 129-134.

LIU Yi-jun, JIA Yu-kun, WANG Ling-fang, LIU Hong-xing, YANG Xian-yu. Prokaryotic Expression,Purification and Antiserum Preparation of Recombinant EDF-1 of Bufo gargarizans[J]. Biotechnology Bulletin, 2018, 34(10): 129-134.

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中华大蟾蜍EDF-1重组蛋白的原核表达、纯化及抗血清的制备

Prokaryotic Expression,Purification and Antiserum Preparation of Recombinant EDF-1 of Bufo gargarizans

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