摘要
目的 克隆表达 β3 整合素基因 ,制备抗体 ,并用之进行 β3 整合素表达阴性Vero细胞的分离和富集。方法 采用RT PCR扩增克隆 β3 整合素基因 ,亚克隆至pQE30表达质粒 ,在原核细胞中表达 ,免疫印迹实验确证其表达及免疫反应性。纯化蛋白免疫接种家兔制备抗体 ,补体介导的抗体依赖性的细胞毒实验分离富集目的细胞 ,免疫荧光及免疫印迹检测筛选结果。结果 成功克隆 β3 整合素基因 ,并在pQE30表达系统中得到高效表达。免疫印迹证实所表达的 β3 整合素蛋白具有良好的免疫反应性。制备了高效抗体 ,免疫荧光及免疫印迹均未检测出 β3 整合素在Vero、VeroE6、Hep 2 ,2BS和 2 93等汉坦病毒敏感细胞表面的表达。结论 除 β3 整合素外 。
Objective To express and purify human β-3-integrin to serve as the antigen to prepare its antibody and to separate the Vero cell clones without expression of β-3-integrin.Methods The human β-3-integrin gene was amplified by using RT-PCR,then subcloned into a pQE30 expression vector and expressed in E.coli.The gene expression was confirmed by Western blot assay.Rabbit was inoculated with purified antigen to stimulate the antibody generation.The target Vero cells were separated by negative selection using antibody plus complement mediated cytolysis.The separated cell clones were confirmed by immunofluorescence and Western blot assay.Results The β-3-integrin gene was cloned and expressed effetively,Western blot assay revealed that the expressed protein held good immune reactivity.High titer antibody was generated.However the expression of β-3-integrin was not detected on Vero,VeroE6,Hep-2,2BS and 293 cells.Conclusion The results of the study suggested that hantavirus has other receptors on Vero cells beside β-3-integrin.
出处
《中华实验和临床病毒学杂志》
CAS
CSCD
北大核心
2002年第1期40-43,共4页
Chinese Journal of Experimental and Clinical Virology
