从人肝癌细胞cDNA表达文库筛选人肝再生增强因子相互作用蛋白及其活性初步鉴定

Screening the hALR-interacting protein from the cDNA library of hepatocarcinoma cells and studying its biological functions

目的利用噬菌体表面展示技术筛选人肝再生增强因子(hALR)相互作用蛋白并初步鉴定噬菌体展示肽的生物学活性。方法以hALR为靶蛋白,采用T7噬菌体表面展示技术从人肝癌细胞cDNA表达文库筛选与 hALR相互作用的特异噬菌体克隆;对获得的特异噬菌体克隆cDNA插入片段进行测序及生物信息学分析;利用3H- TdR掺入法测定噬菌体展示肽及联合hALR对QGY肝癌细胞的增殖效应。结果经过四轮生物淘洗,特异噬菌体克隆富集,获得212 bp的噬菌体cDNA插入序列,生物信息学分析与人Citron激酶同源性达100％;该噬菌体展示肽及联合应用hALR对QGY细胞具有促增殖效应。结论利用噬菌体表面展示技术可以筛选出与hALR具有相互作用的多肽,该序列与人Citron激酶完全同源,Citron激酶可能参与了hALR促肝癌细胞增殖的过程。

Objectives To screen the hALR-interacting protein by phage-displayed technique and identify its biological activities. Methods The specific phage clones that interacted with target protein hALR from a cDNA library of hepatocarcinoma cells were selected using the T7 phage-displayed technique. The acquired cDNA inserts were sequenced and analyzed by bioinformatic tools. The biological activities of the phage-displayed peptide affecting QGY hepatocarcinoma cells were studied using ^3H-TdR method. Results The cDNA inserts with 212 bp were acquired after 4 rounds of biopanning, They showed 100% homology with citron kinase. The phage-displayed peptide and the peptide combined with hALR affected QGY cells proliferation. Conclusions hALR-interacting peptide can be specifically screened by phage-displayed technique. Ciaron kinase that interacted with hALR potentially plays an important role in the proliferation of hepatocarcinoma cells.