摘要
拟用pⅧ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。
The research was designed to study the expression of Zearalenone （ ZEN ） mimicking epitope by p ~ phage display system and verify its reactogenicity. An expression vector pC89-CZEN was constructed by linking the oligonucleotide contained ZEN mimicking epitope sequence and Enterokinase cleavage sites with the pC89S4 phagemid which had been double digested by EcoR I and BamH I. Engineered bacteria pC89-ek-zen was obtained after pC89-CZEN being transformed into competent XL1-Blue cells. The phage particles displaying Zearalenone mimicking 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. The results 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 the medium, induced expression temperature and time. The binding efficiency after enzyme digestion was higher than before.
出处
《生物技术通报》
CAS
CSCD
北大核心
2013年第1期144-150,共7页
Biotechnology Bulletin
基金
国家自然科学基金项目(30860240)
江西省自然科学基金项目(2010GZN0022)
