摘要
目的 探讨噬菌体随机肽展示技术在筛选和鉴定病毒抗原序列研究中的应用前景。方法 用噬菌粒展示载体pCANTAB5X ,构建HCV核心蛋白噬菌体随机展示肽库。用抗 HCV核心单独阳性的血清对该库进行 4轮筛选 ,获得多个阳性克隆 ,测定和分析 8个杂交阳性克隆的DNA序列。结果 构建的随机文库含 1.2 3× 10 5个不同克隆 ,噬菌体滴度为 2× 10 12 TU/ml(转化单位 /ml)。所测定的 7个阳性序列中的 6个为HCV核心蛋白序列 ,这些序列均含有与免疫筛选结果相似的HCV核心抗原序列。另一个为大肠杆菌nrfa基因。结论 所建的噬菌体文库有足够大的库容和较好的随机性 ,可以从该文库中筛选出正确的HCVC抗原序列。噬菌体展示技术完全可应用于病毒蛋白抗原序列的筛选 ,并具有简便、快速。
Objective To screen out the antigenic peptides from HCV core random peptides libraries displayed on phage and to explore a new way to screen and identify the virus antigenic peptides. Methods The phage display library for HCV core random peptides was constructed by insertion of random DNA fragments of HCV core protein synthesized using PCR method into XbaⅠ cloning site of phagemid pCANTAB5X. The anti HCV core mono serum was used to screen the libraries displayed for 4 rounds for antigenic peptides. The DNA sequences of 7 selected clones with positive hybridization from numbers of selected clones were determined and analysed. Results The library contains about 1.23×10 5 different clones, and it's size was about 2×10 12 transforming units per ml. The sequences data showed that six out of 7 sequences are HCV core sequences which contain several major HCV core antigenic peptides similar to those obtained by immunoscreening. Only one was E.coli nrfa gene. Conclusion Our data suggested that the library size and diversity may meet the requirement for mapping and screening of HCV core protein epitopes. Comparing with immunoscreening, phage display technique can be applied to study the viral antigenic peptides with the advantages of easy operation, accuracy and less laborious.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2001年第4期359-363,共5页
Chinese Journal of Microbiology and Immunology
基金
国家自然科学基金资助项目 (No .39830 330 )
上海市科技发展基金资助项目 (No .98JC14 0 2 3)
