噬菌体随机环7肽库筛选恶性疟原虫EBA-175的结合肽

Screening and identification of the binding peptides of Plasmodium falciparum erythrocyte binding antigen-175

目的从噬菌体随机环7肽库中筛选恶性疟原虫EBA175抗原的结合肽。方法以EBA175重组蛋白为靶筛选噬菌体随机环7肽库,通过ELISA、竞争抑制试验、Westernblot等方法鉴定获得的噬菌体短肽与EBA175之间的结合特性。对阳性克隆进行DNA序列测定,推导其氨基酸序列并与GPA氨基酸全序列进行了同源性比较。结果获得9株可与EBA175结合的阳性噬菌体克隆,序列分析显示为3种氨基酸序列,P1(MLLITIR)、P2(TRKLPRT)、P3(KRLMPLK)。其中出现频率最高的P1序列中LLI与EBA175的受体GPA的108110位氨基酸同源。竞争性ELISA显示展示序列P1的噬菌体能竞争抑制EBA175与其单抗的结合。结论获得了可与EBA175特异结合的阳性噬菌体短肽,·LLI··几位氨基酸可能对EBA175与GPA的结合起重要作用。

To screen the binding peptides of erythrocyte binding antigen-175 （EBA-175） of Plasmodium falciparum, the recombinant protein of EBA-175 was used to screen a disulfide constrained heptapeptide library. Three rounds of biopanning were carried out and then ELISA, competitive ELISA, and Western blot were used to evaluate the binding character between phage-borne peptides and EBA-175. The insert DNA sequences of positive clones were determined and their amino acid sequences were deduced. Results Showed that 20 clones from the third round were randomly selected, and nine ones among them could bind to EBA-175. Three sequences, PI（MLLITIR） ,P2（TRKLPRT） ,and P3 （KRLMPLK）, were obtained by analysis of DNA and amino acid sequences, and P1（MLLITIR） showed high homogeneity with the 108-110 aa of GPA. The competitive ELISA tests proved that anti-EBA-175 McAb could competitively inhibit the binding of P1 with EBA-175. It is concluded that these peptides displayed by phage can bind with EBA-175, and LLI probably play a significant role on the binding reaction of EBA-175 and GPA.