法氏囊活性五肽BP5特异性结合肽的筛选及鉴定  

Screening and identification of specific binding peptides to Bursopentin by phage display peptide library

作　　者：王臣[1] 郭香玲[1] 李小康[1] 汪洋[1] 张春杰[1] 吴庭才[1] 李德元[2] 陈溥言[2] 

机构地区：[1]河南科技大学兽医肿瘤免疫学重点实验室,河南洛阳471003 [2]南京农业大学农业部动物细菌学重点实验室,江苏南京210095

出　　处：《中国预防兽医学报》2014年第12期966-970,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然科学青年基金项目(31101792);河南省高校青年骨干教师项目(2012GGJS-077)

摘　　要：为鉴定与具有免疫调节功能和抗肿瘤潜能的法氏囊活性五肽(BP5)特异性结合的多肽,本研究利用噬菌体展示技术,以BP5-BSA为靶分子,对噬菌体随机12肽库进行4轮亲和筛选,结合ELISA鉴定和竞争抑制试验,筛选出3个能够特异性与BP5结合的噬菌体阳性克隆。测序结果显示,其编码的12肽序列分别为:PINMQTLNCMAA(P2-12)、GCTLNPMSDLLG(P6-12)和MSSTLNGMLNSL(P7-12),其核心基序为TLNXM。通过人工合成P2-12、P6-12、P7-12多肽,并采用MTT法检测其对BP5抗肿瘤细胞增殖能力的影响,结果显示,P2-12和P7-12肽在0.2μg/m L^20μg/m L浓度下,P6-12肽在2μg/m L^20μg/m L浓度下均能够抑制BP5抗小鼠WEHI-231 B淋巴瘤细胞增殖作用,其中P2-12在20μg/m L浓度下抑制作用最为显著(p<0.01)。此外,p53荧光素酶活性检测结果显示,这3种BP5结合肽在实验浓度下均能够下调BP5促p53基因转录的活性。以上结果表明,这3种BP5特异性结合肽具有下调BP5抗肿瘤活性。本研究为进一步研究BP5抗肿瘤细胞增殖作用的机制提供了相关的实验依据。To identify the peptides that specifically binding to Bursopentin （BP5） which exhibits the functions of immune regulation and anti-tumor,BP5-BSA was used to screen its binding peptides from the 12-mer random phage display peptide library.After four rounds of biopanning,ELISA and competitive inhibition test,three positive phage clones were found and the sequencing results showed which encoded the peptides of PINMQTLNCMAA（P2-12）,GCTLNPMSDLLG（P6-12） and MSSTLNGMLNSL （P7-12）,respectively,with the core motif of TLNXM.In addition,the peptides P2-12,P6-12 and P7-12 were synthesized and tested in WEHI-231 cells detected by MTT assay.The results indicated that P2-12 （0.2 μg/mL to 20 μg/mL）,P6-12 （2 μg/mL to 20 μg/mL） and P7-12 （0.2 μg/mL to 20 μg/mL） were able to down-regulate the BP5 anti-proliferative activity of WEHI-231 cells,and the effect of P2-12 at 20 μg/mL was significantly higher than other groups.Moreover,the results of p53 luciferase activity assay indicated that all the three BP5 binding peptides had the abilities to down-regulate the activity that BP5 promoted p53 gene transcription.In conclusion,the three specific peptides binding to BP5 were identified in this study,which provide some reference data for further study on the mechanism of BP5 anti-tumor.

关 键 词：法氏囊 活性五肽 噬菌体随机肽库 抗肿瘤 结合肽 

分 类 号：S852.65[农业科学—基础兽医学]