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目的:为了获得稳定高效表达凝血因子Ⅶ的哺乳动物细胞株,构建一个利用人β肌动蛋白(hACTB)基因座完整的上下游调控序列指导人凝血因子Ⅶ(hFⅦ)基因组序列在人胚胎肾细胞特异性高效表达的hACTB-hFⅦ杂合基因座。方法:采用3步连续缺口修复的方法。首先,以pBR322载体作为骨架,插入预先合成的6个同源臂,构成能进行3次连续基因抓捕的载体。然后在大肠杆菌内利用Red同源重组系统介导的缺口修复技术:第一步,从含hACTB基因座的细菌人工染色体(BAC)上亚克隆10 kb的hACTB基因3′端完整侧翼序列;第二步,从hFⅦBAC上亚克隆13 kb的从起始密码子(ATG)到终止密码子(TAG)的hFⅦ基因组序列;第三步,从hACTB BAC上亚克隆20kb的hACTB基因5′端完整侧翼序列,并使这3个基因片段自动无痕地连接在基因抓捕载体上,形成全长约50 kb的hACTB-hFⅦ杂合基因座。结果:经过PCR扩增、限制性内切酶消化和序列测定验证,构建的杂合基因座达到了原来hACTB基因座中hACTB基因组编码序列从起始密码子到终止密码子被hFⅦ从起始密码子到终止密码子的基因组序列基因组序列精确置换的目的。结论:连续3步缺口修复构建杂合基因座细胞表达载体的技术,将为细胞高效表达大载体的制备提供一种全新的思路和方法。
OBJECTIVE: To construct a mammalian cell line stably and efficiently expressing factor Ⅶ, a complete upstream and downstream regulatory sequence of human actin β (hACTB) gene was constructed to direct human hFⅦ genomic sequence in human embryonic kidney Specific and efficient expression of hACTB-hFⅦ heterozygous loci. Methods: 3-step continuous gap repair method. First, a pBR322 vector was used as a backbone, and 6 pre-synthesized homology arms were inserted to construct a vector capable of 3 successive gene seizures. Then, the red repair system of gap-mediated homologous recombination system was used in E.coli. In the first step, the 3’-end flanking sequence of hACTB gene was subcloned from bacterial artificial chromosome (BAC) containing hACTB locus. The In two steps, a 13 kb hFVII genomic sequence from the start codon (ATG) to the stop codon (TAG) was subcloned from hFVII BAC. The third step was to subclone the 20 kb full-length 5’-end of hACTB gene from hACTB BAC Sequence, and the three gene fragments automatically and seamlessly connected to the gene capture vector to form a total length of about 50 kb hACTB-hFⅦ heterozygous loci. Results: After PCR amplification, restriction endonuclease digestion and sequence analysis, the constructed heterozygous locus reached the original hACTB locus hACTB genome coding sequence from the start codon to the stop codon hF VII from the start code The purpose of the exact replacement of the genomic sequence of the sub-stop codon genomic sequence. CONCLUSION: The technique of constructing three-step gap repair constructs of heterozygous locus cell expression vectors will provide a new idea and method for the preparation of cell large efficient expression vector.