噬菌体展示技术筛选结核分枝杆菌PPE17蛋白的特异性结合肽

Screening specific binding peptides of Mycobacterium tuberculosis PPE17 protein by Phage display technique

目的运用噬菌体展示技术,对结核分枝杆菌PPE17蛋白特异性结合肽进行了初步筛选。方法从结核分枝杆菌基因组中扩增PPE17基因,克隆到pET28a中,在大肠杆菌BL21中表达,Ni2+柱纯化,并用SDS-PAGE和Western blot鉴定。将纯化的PPE17蛋白包被到ELISA板中,用噬菌体7肽库进行筛选,经4轮淘选后,随机选取噬菌斑进行测序,并用DNAMAN对阳性克隆所编码的多肽氨基酸序列进行分析比较。结果成功构建并表达了PPE17抗原,获得分子量约为37kD的可溶性蛋白。从第4轮的洗脱物中,随机挑选20个噬菌斑。测序结果可翻译成8种多肽分子,其中重复6次的多肽序列为LKWGHVY。结论通过噬菌体展示技术筛选到PPE17蛋白的特异性结合肽,有望成为鉴定该抗原的小分子诊断制剂。

Objective The specific binding peptide of Mycobacterium tuberculosis PPE17 protein was screened by phage display technique.Methods PPE17 gene was amplified from Mycobacterium tuberculosis genome,cloned into pET28a,expressed in E.coli BL21,purified by Ni2+column,and identified by SDS-PAGE and Western blot.The purified PPE17 protein was coated into an ELISA plate and screened by phage 7 peptide library.After three rounds of panning,phage plaques were randomly selected for sequencing.DNAMAN was used to analyze and compare the amino acid sequences of the polypeptide encoded by the positive clones.Results PPE17 gene was successfully constructed and expressed,and soluble protein with molecular weight of about 37kD was obtained.From the third round of eluents,20 plaque were randomly selected.The sequencing results could be translated into 8 polypeptide molecules,among which the polypeptide sequence repeated for 6 times was LKWGHVY.Conclusion The specific binding peptide of PPE17 protein is screened by phage display technology,which is expected to be a small molecular diagnostic reagent for the identification of this antigen.