噬菌体展示技术筛选羊小肠上皮细胞穿透肽  

Screening of Sheep Small Intestine Epithelial Cell Penetrating Peptide by Phage Display Technique

作　　者：刘思佳 徐晓东 姜宁[1] 潘春媛[1] 宋军 赵磊 赵芳芳[1] 李沐阳 张爱忠[1] LIU Sijia;XU Xiaodong;JIANG Ning;PAN Chunyuan;SONG Jun;ZHAO Lei;ZHAO Fangfang;LI Muyang;ZHANG Aizhong(Key Laboratory of Low-Carbon Green Agriculture in Northeastern China,Ministry of Agriculture and Rural Affairs,Key Laboratory of Efficient Utilization of Feed Resources and Nutrition manipulation in Cold Region of Heilongjiang Province,College of Animal Science and Veterinary Medicine,Heilongjiang Bayi Agricultural University,Daqing 163319,China)

机构地区：[1]黑龙江八一农垦大学动物科技学院,黑龙江省寒区饲料资源高效利用与营养调控重点实验室,农业农村部东北平原农业绿色低碳重点实验室,大庆163319

出　　处：《动物营养学报》2023年第9期6033-6041,共9页CHINESE JOURNAL OF ANIMAL NUTRITION

基　　金：国家自然科学基金项目(32072759);黑龙江省肉羊协同创新与推广体系专项资助。

摘　　要：为解决抗菌药物不能穿透细胞膜杀伤胞内致病菌的问题,本试验以羊小肠(空肠段)上皮细胞为靶细胞,利用噬菌体展示技术4轮体外生物淘洗,系统地筛选羊小肠上皮细胞的特异性穿透肽,将其作为抗菌肽的载体从而提高抗菌肽的穿透能力。试验采用噬菌体展示技术,经过4轮体外生物淘洗,得到了能与羊小肠上皮细胞结合的多肽;4轮减数筛选后,噬菌体的回收率逐渐提高,特异性的噬菌体克隆得到了有效富集,酶联免疫吸附法(ELISA)测定单克隆噬菌体对羊小肠上皮细胞的亲和力,亲和力≥2的为阳性,筛选阳性噬菌体克隆,提取DNA进行测序,选择高重复率序列,合成细胞穿透肽,同时合成与细胞穿透肽的氨基酸序列及结构相同但组合顺序不同的对照多肽,并与异硫氰酸荧光素(FITC)标签连接。用细胞增殖/毒性检测试剂盒(CCK8)测定细胞穿透肽和对照肽对细胞活力的影响,用荧光显微镜初步观察穿透肽的穿透性,用激光共聚焦显微镜及流式细胞仪检测穿透肽在胞内的定位及穿透强度。结果表明:经4轮体外减数筛选噬菌体的回收率增长了226倍,随着第1轮至第4轮筛选轮次增加,与羊小肠上皮细胞结合的噬菌体依次增加;使用ELISA进行检测,结果显示,在随机挑选的30个单克隆噬菌体中,22个检测为阳性克隆(亲合力≥2);提取阳性克隆噬菌体进行DNA测序分析后,得到2条重复率高的氨基酸序列,VLNSLID命名为SCPP1,HSTTAQF命名为SCPP2;细胞活力结果表明,在浓度低于256μmol/mL时2条细胞穿透肽及其对照多肽的细胞活力仍在90%以上,表明对细胞无毒;荧光显微镜下观察及激光共聚焦结果表明,SCPP1和SCPP2可以特异性穿透羊小肠上皮细胞膜,且SCPP1可以穿透细胞核表现出比SCPP2更强的穿透性,2条对照多肽无穿透能力;流式细胞仪结果显示,SCPP1的胞内平均荧光强度明显高于SCPP2,且呈时间剂量依赖。本试验筛选出了In order to solve the problem that antibacterial drugs cannot penetrate the cell membrane to kill intra-cellular pathogenic bacteria,this experiment used sheep small intestinal epithelial cells(empty intestine seg-ment)as target cells and four rounds of in vitro bioelution by phage display technology to systematically screen the specific penetrating peptides of sheep small intestinal epithelial cells and use them as carriers of antimicrobi-al peptides so as to improve the penetrating ability of antimicrobial peptides.The experiment used phage display technology,and after four rounds of in vitro bioelution,the peptide that could bind to sheep small intestine epi-thelial cells was obtained;after four rounds of subtraction screening,the recovery rate of phage gradually in-creased,and the specific phage clones were effectively enriched,and the affinity of monoclonal phage to sheep small intestine epithelial cells was determined by enzyme-linked immunosorbent assay(ELISA),and those with affinity≥2 were positive,and positive phage were screened clones were extracted,DNA was sequenced,high repetition rate sequences were selected,cell-penetrating peptides were synthesized,and control peptides with the same amino acid sequence and structure but in different combination order as the cell-penetrating pep-tides were synthesized and ligated with fluorescent FITC tags.The effects of cell penetrating peptide and control peptide on cell viability was measured by CCK8,and the penetration of penetrating peptide was initially ob-served by fluorescence microscopy,and the intracellular localization and penetration intensity of penetrating peptide were detected by laser confocal microscopy and flow cytometry.The results showed as follows:the re-covery of phage increased 226-fold after four rounds of in vitro subtraction screening,and the phage bound to sheep small intestinal epithelial cells increased sequentially as the number of screening rounds increased from the first to the fourth round;detection using ELISA showed that 22 of 3

关 键 词：噬菌体展示技术 肽库 羊小肠上皮细胞 细胞穿透肽 

分 类 号：S826[农业科学—畜牧学]