应用核糖体展示技术筛选抗人p53单链抗体的效果  

Screening of anti-human p53 single chain antibody by ribosome display technology

作　　者：陈荫楠 罗婉妹 罗彩林 郑晨娜 郭娜燕 CHEN Yinnan;LUO Wanmei;LUO Cailin;ZHENG Chenna;GUONayan(Quanzhou Medical College,Quanzhou 362000,Fujian)

机构地区：[1]泉州医学高等专科学校,福建泉州362000

出　　处：《菏泽医学专科学校学报》2023年第1期1-5,13,共6页Journal of Heze Medical College

基　　金：泉州医学高等专科学校科研项目(XJK1617B)。

摘　　要：目的运用核糖体展示技术筛选抗人p53单链抗体,并进行初步分析。方法诱导表达p53蛋白,用其免疫小鼠。提取免疫小鼠脾脏组织的总RNA,通过反转录PCR和重叠延伸PCR分别扩增抗体重链可变区基因VH-Linker和轻链可变区基因VL-Linker,并合成VH-linker-VL型单链抗体基因。TNT T7 Quick for PCR DNA试剂盒对单链抗体基因进行体外转录与翻译,以p53蛋白为靶标,经过5轮筛选富集ScFv基因。将获取单链抗体片段进行原核表达后,采用ELISA法和细胞免疫学法检测。结果成功构建抗p53单链抗体库,并筛选出p53蛋白具有较强结合活性的单链抗体株p53-scFv1、p53-scFv2和p53-scFv3,可用以检测细胞中的p53蛋白。结论利用核糖体展示技术获得亲和力较好的抗人p53单链抗体,可为p53抗体的制备提供新思路。Objective To screen and analyze anti-human p53 single chain antibody by ribosome display technology.Methods Immunemicewere induced expression of p53 protein.Total RNA was extracted from spleen tissues of immunized mice.The VH-Linker and VL-Linker genes were amplified by reverse transcription PCR and overlapping extension PCR respectively,and VH-Linker-VL type SCFV genes were synthesized.TNT T7 Quick for PCR DNA kit was used in transcription and translation of single chain antibody gene,with p53 protein as the target SCFV gene was enriched after five rounds of screening.After prokaryotic expression,the SCFV fragments were detected by ELISA and cellular immunoassay.Results Anti-p53 single chain antibody library was successfully constructed.The single chain antibody strains p53-scFv1,p53-scFv2 and p53-scFv3 with strong binding activity were screened out to detect p53 protein in cells.Conclusion The ribosome display technology was used to obtain anti-human p53 SCFV with good affinity,which can provide a new idea for the preparation of p53 antibody.

关 键 词：P53蛋白 单链抗体 核糖体 

分 类 号：Q753[生物学—分子生物学]