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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 ,汉坦病毒很可能还有别的受体
Objective To clone and express the β3 integrin gene, and to prepare the antibody for the isolation and enrichment of β3 integrin-negative Vero cells. Methods The β3 integrin gene was amplified by RT PCR, subcloned into pQE30 expression plasmid and expressed in prokaryotic cells. Western blotting was used to confirm the expression and immunoreactivity. The purified protein was used to immunize rabbits to prepare antibody, and the complement-mediated antibody-dependent cytotoxicity assay was used to isolate and enrich the target cells. Immunofluorescence and Western blotting were used to detect the screening results. Results The β3 integrin gene was successfully cloned and expressed in pQE30 expression system. Immunoblotting confirmed that the expressed β3 integrin protein has good immunoreactivity. High-performance antibodies were prepared. Immunofluorescence and immunoblotting did not detect the expression of β3 integrin on the surface of Hantaan virus-sensitive cells such as Vero, VeroE6, Hep 2, 2BS and 293 cells. Conclusion In addition to β3 integrins, Hantaan virus is likely to have other receptors