摘要
目的:构建赭曲霉毒素A模拟表位pⅧ噬菌体表达载体。方法:从pⅢ噬菌体随机肽库筛选到的多个赭曲霉毒素A模拟表位中取亲和力最高的模拟表位,通过化学方法合成两条包含该模拟表位及核酸内切酶位点的核苷酸序列,退火成双链后与经过相应酶切的pC89S4噬菌体质粒相连,即插入该载体pⅧ前导肽和成熟氨基酸之间,转化进XL1-Blue感受态细胞中,经XbaⅠ、EcoRⅠ和BamHⅠ三种酶分别酶切初步筛选,测序鉴定。结果:质粒测序与合成的序列一致。结论:即得到高密度赭曲霉毒素A的模拟表位噬菌体展示载体。
Objective：To construct a novel vector with the nucleotide sequence of ochratoxin A （OTA） mimotope based on M13 phage with pⅧ gene,which can give a highly efficient amplification of high-density OTA mimotope so as to provide a basis for preparing an alternative to OTA competitive antigen.Methods：The mimotope with the highest affinity was selected out of OTA mimotopes from the random peptide library of phase pⅢ.Two nucleotide sequences containing the nucleotide sequence of this mimotope and endonuclease sites were chemically synthesized,formed into a double-chain sequence by annealing,inserted into the gap between the genes of pⅧ leader peptides and mature amino acids for connection with vector pC89S4 that had been doubly digested with EcoRⅠ and BamHⅠand transformed into XL1-Blue competent cells.The transformed XL1-Blue competent cells were incubated in liquid SOC medium with shaking and then spreaded on solid LB medium in order to provide single colonies for cell amplification before plasmid extraction.The extracted plasmids were separately digested with XbaⅠ,EcoR Ⅰ and BamHⅠ,preliminarily screened and identified by sequencing.Results：The results of plasmid sequencing were in good accordance with the synthetic sequence.Conclusion：A phase display vector that can efficiently amplify high-density OTA mimotope has been successfully constructed.
出处
《食品科学》
EI
CAS
CSCD
北大核心
2010年第17期307-309,共3页
Food Science
基金
国家自然科学基金项目(30860240)
国家"863"计划项目(2007AA10Z427)
