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目的 :构建能用于抗原表位筛选的庚型肝炎病毒 (HGV)全基因组 c DNA噬菌体随机展示肽库。 方法 :将 DNase 部分消化的 HGV全基因组 c DNA随机片段 ,克隆到噬菌粒载体 p CANTAB5 X中 ,使 HGV抗原以融合蛋白的形式随机展示于噬菌体表面 ,并用原位杂交、限制性酶切及 DNA测序等方法鉴定所建文库的随机性和完整性。 结果 :所建肽库的容量为5 .34× 10 4个菌落形成单位 ,滴度为 1.41× 10 1 4 TU/ ml。原位杂交结果证明在 HGV基因组的不同区域均有阳性菌落 ,酶切鉴定和 DNA测序结果显示插入片段的长度和片段来源区域具有随机性。结论 :所建肽库具有良好的完整性、随机性及实用性 ,为进一步研究奠定了基础。
OBJECTIVE: To construct a randomized peptide library of hepatitis G virus (GG) whole genome c DNA phage that can be used for antigen epitope screening. Methods: HGV genomic c DNA fragments partially digested with DNase were cloned into the phagemid vector pCANTAB5X. The HGV antigen was expressed on the surface of the phage in the form of fusion protein at random. The hybridization was performed by in situ hybridization and restriction enzyme digestion DNA sequencing and other methods to identify the randomization and integrity of the built library. Results: The constructed peptide library had a capacity of 5.34 × 10 4 colony forming units with a titer of 1.41 × 10 14 TU / ml. In situ hybridization results showed that there were positive colonies in different regions of HGV genome. The results of restriction enzyme digestion and DNA sequencing showed that the length of insert and the region of fragment origin were random. Conclusion: The constructed peptide library has good integrity, randomness and practicability, which lays the foundation for further research.