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首页> 外文期刊>BMC research notes >Rapid establishment of a HEK 293 cell line expressing FVIII-BDD using AAV site-specific integration plasmids
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Rapid establishment of a HEK 293 cell line expressing FVIII-BDD using AAV site-specific integration plasmids

机译：使用AAV位点特异性整合质粒快速建立表达FVIII-BDD的HEK 293细胞系

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Background Stable human cell lines have gradually become the preferred system for large scale production of recombinant proteins for clinical applications because of their capacity of proper protein post-translational modification and low immunogenicity. However, human cell line development technologies are commonly based on random genome integration of protein expressing genes. It is required to screen large numbers of cell clones to identify stable high producer cell clones and the cell line development process usually takes 6 to 12 months. Adeno-associated virus type 2 (AAV2) Rep protein is known to induce rAAV DNA integration into a specific site (AAVS1) of the human chromosome 19 and integrated transgenes can stably express proteins. We take advantage of this AAV unique feature to develop a rapid protocol to clone a stable recombinant protein expression human cell line. Findings We have constructed two plasmids. One plasmid, pSVAV2, contains the AAV rep gene for the synthesis of integrase; the second plasmid, pTRP5GFPFVIII-BDD, contains B-domain-deleted factor VIII (FVIII-BDD) and GFP gene flanked by AAV ITRs. Human embryonic kidney (HEK) 293 cells were co-transfected with the two plasmids and the cells were screened by green fluorescence to establish the recombinant FVIII-BDD cell line. PCR analysis showed that the FVIII-BDD gene has been integrated into the AAVS1 site of human chromosome 19. The FVIII-BDD protein secreted into the extracellular media exhibited coagulant activity. Conclusion We developed a method of rapid establishment of human HEK 293 cell line expressing recombinant FVIII-BDD protein with AAV site-specific integration plasmids.
机译：背景技术由于稳定的人细胞系具有适当的蛋白翻译后修饰能力和低免疫原性，因此它们已逐渐成为大规模生产用于临床应用的重组蛋白的优选系统。然而，人类细胞系开发技术通常基于蛋白质表达基因的随机基因组整合。需要筛选大量细胞克隆以鉴定稳定的高产细胞克隆，并且细胞系发育过程通常需要6至12个月。已知与腺相关的2型病毒（AAV2）Rep蛋白可诱导rAAV DNA整合入人类19号染色体的特定位点（AAVS1），并且整合的转基因可以稳定表达蛋白质。我们利用这一AAV独特的功能来开发快速方案，以克隆稳定的重组蛋白表达人类细胞系。结果我们构建了两个质粒。一种质粒，pSVAV2，含有用于合成整合酶的AAV rep基因。第二个质粒pTRP5GFPFVIII-BDD包含B结构域缺失的VIII因子（FVIII-BDD）和侧接AAV ITR的GFP基因。用这两个质粒共转染人胚胎肾（HEK）293细胞，并通过绿色荧光筛选细胞，以建立重组FVIII-BDD细胞系。 PCR分析表明，FVIII-BDD基因已经整合到19号染色体的AAVS1位点中。分泌到细胞外培养基中的FVIII-BDD蛋白表现出凝结活性。结论我们开发了一种通过AAV位点特异性整合质粒快速建立表达重组FVIII-BDD蛋白的人HEK 293细胞系的方法。

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