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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 explore the application prospect of phage display technology in the screening and identification of viral epitopes. Methods: A random DNA fragment was synthesized by random primer PCR using HCV core gene as a template. After amplification by specific primers, a highly abundant HCV core protein random DNA fragment was obtained. The fragment was inserted into the pCDNATAB5 X Of Xba sites, transformed into E. coli TG1, helper phage rescue, the establishment of phage random peptide display library. Results: The constructed random library contained 1.2 3 × 10 5 different clones. The titer of the phage library was 2 × 10 12 TU / ml (transduction unit / ml). The positive rate of insertion was 21.8% Forty percent of the clones were confirmed as HCV core positive clones. Conclusion: The constructed randomized library has a large enough capacity and a good diversity, which can meet the screening of HCV C epitopes.