HCV核心蛋白噬菌体随机展示肽库的构建  

Construction of phage random peptides library for HCV core protein

作　　者：潘卫[1] 戚中田[1] 贺祥[2] 陈秋莉[1] 潘欣[1] 

机构地区：[1]第二军医大学基础医学部微生物学教研室,上海200433 [2]第二军医大学科研部

出　　处：《第二军医大学学报》2000年第9期857-860,共4页Academic Journal of Second Military Medical University

基　　金：国家自然科学基金!资助项目 ( 1983 0 3 3 0 ) ;上海市科技发展基金!资助项目 ( 98JC14 0 2 3)

摘　　要：目的 :为了探讨噬菌体展示技术在筛选和鉴定病毒抗原表位研究中的应用前景。 方法 :利用随机引物 PCR法以HCV核心基因为模板合成随机 DNA片段 ,再经特定引物二次扩增后获得高丰度的 HCV核心蛋白随机 DNA片段 ,将该片段插入噬菌粒载体 p CANTAB5 X的 Xba 位点 ,转化大肠杆菌 TG1,辅助噬菌体拯救 ,建立噬菌体随机肽展示文库。结果 :所构建的随机文库含 1.2 3× 10 5个不同克隆 ,库容噬菌体滴度为 2× 10 1 2 TU/ m l(转导单位 / ml) ,PCR法检测插入阳性为 2 8.1% ,核酸杂交证实 40 %的克隆为 HCV核心基因阳性克隆。 结论 :所建的随机文库有足够大的库容和较好的多样性 ,可以满足HCV C抗原表位的筛选。Objective: To investigate the possibility of application of phage display technique in screening and identifying the virus antigenic epitopes. Methods: The random DNA fragments of hepatitis C virus (HCV) core protein were synthesized using PCR method with the random primer containing a specific sequences at 5′ terminal, and amplified by PCR with the specific primer. The random DNA fragments were inserted into XbaⅠ cloning site of phagemid pCANTAB5X, and transformed into E coli TG1. The phage display library for random HCV core peptides was constructed by rescuing transformed E coli TG1 with the help virus M13KO7. Results: The library contained about 1.23×10 5 different clones, and it′s size was about 2×10 12 transforming units per ml. Forty percent of transformed clones were positive for nucleotide hybridization detection with the HCV core gene probe, and 28.1% clones had visiable insertion of DNA fragments as detected by PCR. Conclusion: The data suggest that the library size and diversity may meet the need for mapping and screening of HCV core protein epitopes. [

关 键 词：噬菌体随机展示肽库 核心蛋白 丙型肝炎 HCV 

分 类 号：R512.63[医药卫生—内科学] Q785[医药卫生—临床医学]