利用噬菌体随机肽库筛选人肝再生增强因子特异结合肽及其抗肝癌作用的研究

Screening of hALR-specific binding peptide from phage display peptide library and its biological activities of inhibiting liver cancer

目的利用噬菌体随机肽库筛选人肝再生增强因子(human augmenter of liver regeneration,hALR)特异结合肽并研究其抗肝癌作用。方法以hALR为靶分子,利用Ph.D.TM噬菌体肽展示系统筛选出与hALR特异性结合的单克隆噬菌体,ELISA法鉴定筛选出的单克隆噬菌体与hALR的结合性,对特异性结合噬菌体DNA中插入片段进行PCR扩增、测序及短肽合成,MTS法检测特异结合肽对人HepG2肝癌细胞增殖的影响,观察特异结合肽对裸鼠人HepG2肝癌移植瘤生长的影响。结果经过3轮生物淘筛,特异性结合噬菌体得到富集;筛选到的单克隆噬菌体与hALR结合具有特异性(与对照组比较P<0.05);特异结合肽在体外能抑制人HepG2肝癌细胞增殖(24 h抑制率为46.2%vs 6.5%,P<0.01;48 h抑制率为23.6%vs 5.9%,P<0.05),在体内能抑制裸鼠人HepG2肝癌移植瘤生长[(1.250±0.512)cm3vs(4.590±0.398)cm3,P<0.01;(1.100±0.453)g vs(3.794±0.299)g,P<0.01];该特异结合肽免疫小鼠后机体内无相应抗体检出,且对裸鼠的重要脏器组织结构及功能无明显损害。结论利用噬菌体肽库筛选到hALR特异结合肽,该短肽通过阻断hALR促肝癌细胞增殖作用,能抑制人HepG2肝癌细胞在体内外增殖,达到抗肝癌的作用,同时产生较低的免疫原性及毒性损害。

Objective To screen the human augmenter of liver regeneration （hALR）-specific binding peptide from phage display random peptide library and investigate its biological activities of inhibiting liver canc- er. Methods The phage clones that specifically bound with target protein hALR were selected from phage dis- play random peptide library. ELISA was used to identify the specificity of binding between the selected phage and target protein hALR. The acquired DNA inserts were amplified using PCR, and the specific binding pep-tides were synthesized after sequencing. Methyl tolyl sulfide （MTS） assay was employed to measure the biologi- cal activities of the specific binding peptides on the proliferation of human HepG2 liver cancer ceils. The biolog-ical activities of the specific binding peptides on the growth of human HepG2 cell xenograft tumor was studied through the tumor size in nude mice. The immunogenicity and toxicity of the specific binding peptide on nude mice were also tested. Results The specific binding phages were enriched after 3 rounds of biopanning. They showed the specificity on binding with target protein hALR. The specific binding peptide showed inhibition on proliferation of human HepG2 liver cancer cells in vitro （The inhibitory rate of 24 hours ：46.2% vs 6.5% ,P 〈 0. 01 ; The inhibitory rate of 48 hours ：23.6% vs 5.9% ,P 〈 0. 05 ） and on growth of human HepG2 cell xenograft tumor in vivo [ （ 1. 250 ± 0.512 ） cm3 us （4.590 ± 0,398 ）cm3 ; P 〈 0. 01 ; （ 1. 100 ± 0. 453 ） g vs （ 3.794 ± 0. 299 ） g, P 〈0. 01 ）]. There was no detectable antibody after being immunized by the specific binding peptide and no significant damages on important organs in mice. Oonclusion A hALR-specific binding peptide is selected from phage display peptide library. The peptide with low immunogenicity and toxicity shows inhibition on the proliferation of liver cancer ceils by blocking the effect of hALR in vitro and in vivo.