噬菌体展示系统表达玉米赤霉烯酮模拟表位肽

生物技术通报 ›› 2013, Vol. 0 ›› Issue (1): 144-150.

噬菌体展示系统表达玉米赤霉烯酮模拟表位肽

徐富勇¹ ,徐玲² ,刘仁荣² ,裘雪梅² ,朱立鑫²

1. 南昌大学生命科学与食品工程学院，南昌 330029 ； 2. 江西科技师范大学生命科学学院，南昌 330013

收稿日期:2012-06-11 修回日期:2013-01-31 出版日期:2013-01-30 发布日期:2013-01-30
作者简介:徐富勇，男，硕士，研究方向：生物转化与加工; E-mail: xfy218@163.com

Expression of Zearalenone Mimicking Epitope Peptide by Phage Display System

Xu Fuyong¹ ,Xu Ling², Liu Renrong², Qiu Xuemei², Zhu Lixin²

1. School of Life Sciences and Food Engineering，Nanchang University，Nanchang 330029;
2. School of Life Science，Jiangxi Science & Technology Normal University，Nanchang 330013

Received:2012-06-11 Revised:2013-01-31 Published:2013-01-30 Online:2013-01-30

摘要/Abstract

摘要： 拟用p Ⅷ 噬菌体展示系统表达玉米赤霉烯酮（Zearalenone ，ZEN）模拟表位肽，并验证其反应原性。通过将含有ZEN 模拟表位序列以及肠激酶酶切位点的寡聚核苷酸与经过EcoR I 和BamH I 双酶切的pC89S4 噬菌粒连接，构建表达载体pC89-CZEN。pC89-CZEN 转化感受态XL1-Blue 得到工程菌pC89-ek-zen，然后用KM13 超感染、IPTG 诱导表达，纯化后得到展示有玉米赤霉烯酮模拟表位的噬菌体颗粒。对KM13 超感染时菌体浓度OD600、IPTG 诱导时菌体浓度OD600、IPTG 加入量以及诱导表达温度和时间进行优化，以探索最佳表达条件；通过ELISA 测定反应原性，对比肠激酶酶切前后模拟表位肽与ZEN 抗体的结合效果。结果显示，成功构建了表达载体pC89-CZEN，最优表达条件为KM13 超感染时菌体浓度OD600 为0.4、IPTG 诱导时菌体浓度OD600 为0.6 以及IPTG 加入量为终浓度1.0 mmol/L、诱导表达温度为24℃、时间8 h，酶切后结合效果明显高于酶切前。

关键词: 噬菌体展示系统 , 玉米赤霉烯酮 , 模拟表位 , 肠激酶

Abstract: The research was designed to study the expression of Zearalenone(ZEN)mimicking epitope by p Ⅷ phage display systemand verify its reactogenicity. An expression vector pC89-CZEN was constructed by linking the oligonucleotide contained ZEN mimicking epitopesequence and Enterokinase cleavage sites with the pC89S4 phagemid which had been double digested by EcoR I and BamH I. Engineered bacteriapC89-ek-zen was obtained after pC89-CZEN being transformed into competent XL1-Blue cells. The phage particles displaying Zearalenonemimicking peptide were acquired after pC89-ek-zen being super infected by KM13 phage. The optimal expression conditions were also explored.The reactogenicity was detected by ELISA. ZEN antibody binding capacity(before and after Enterokinase digestion)were also compared. Theresults show that the expression vector pC89-CZEN was constructed successfully. The optimal expression conditions were explored by ELISA.The binding effect is influenced by bacteria concentration when helper phage KM13 and IPTG added, and the final concentration of IPTG in themedium, induced expression temperature and time. The binding efficiency after enzyme digestion was higher than before.

Key words: Phage display system , Zearalenone , Mimicking epitope , Enterokinase

徐富勇, 徐玲, 刘仁荣, 裘雪梅, 朱立鑫. 噬菌体展示系统表达玉米赤霉烯酮模拟表位肽[J]. 生物技术通报, 2013, 0(1): 144-150.

Xu Fuyong, Xu Ling, Liu Renrong, Qiu Xuemei, Zhu Lixin. Expression of Zearalenone Mimicking Epitope Peptide by Phage Display System[J]. Biotechnology Bulletin, 2013, 0(1): 144-150.

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