摘要
目的利用噬菌体展示技术构建巨细胞病毒基质蛋白PP65的抗原,初步评价其抗原性。方法PP65的基因片段经酶切后,与T7噬菌体的基因组相应酶切位点结合,借助包装蛋白组装成为完整的噬菌体。PP65目的蛋白以融合蛋白的形式表达在T7噬菌体的头蛋白部位,每个噬菌体颗粒表面可表达5～15个拷贝。噬菌体可以在宿主细胞中快速繁殖,并且能够迅速裂解宿主细胞,使目的蛋白的富集达到很高的效率。人巨细胞病毒基质蛋白PP65可以激发机体强烈的免疫反应,运用T7噬菌体载体展示系统表达PP65的部分肽段,扩增噬菌体,以SDS-PAGE电泳和western-blot鉴定所表达的抗原特性。结果获得了重组T7-PP65噬菌体,通过鉴定,噬菌体表达在其头蛋白上的PP65抗原可以被天然PP65的单克隆抗体所识别。结论噬菌体展示技术可以有效地进行病毒蛋白抗原的表达,为抗原筛选和疫苗的研制提供科学依据。将PP65表达在T7噬菌体表面,可以研究其免疫原性及评估其作为疫苗的可能性。
Objective A cloning and expression system that allowed displaying of proteins on the surface of T7 phages was exploited to display a PP65 protein of the human cytomegalovirus as the epitope. The character of the antigen was evaluated rudimentally. Methods The objective segment of the PP65 genosome was integrated with the genosome of the T7 phage. The PP65 protein was expressed on the surface of the T7 as head fusion protein. There were 5 to 15 copies on the each phage. Owning to the rapid propagation and speedy lysis of host cell,the gather of phage would be in efficient way. The matrix protein PP65 of the human cytomegalovirus was capable of stimulating the intense immune reaction. Resuits The recombine T7-PP65 was obtained, which was judged with the SDS-PAGE electrophoresis and west-ern-blot test. The protein that expressed on the surface of the head protein of the T7 phage could be identified by the PP65 mono clonal antibody. Conclusion The phage display technology can be used to simulate the expression of the virus protein. It offers the convenience for the antigen selection and vaccine research. The character of the antigen can be investigated and the probability of the vaccine construction is estimated, in the way which express the epitope of the PP65 on the surface of the T7 phage.
出处
《临床输血与检验》
CAS
2006年第1期1-4,共4页
Journal of Clinical Transfusion and Laboratory Medicine
基金
2004年上海市教委科研重点项目(04BA04)
上海市科委重大攻关项目(04D219114)资助
