摘要
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： In order to obtain stable and efficient expression of coagulation factor WlI of mammalian cell lines, generate a hACTB-hFYII hybrid locus that the transcription of human coagulation I（hF＇VII） genomic se- quence is directed by the up and down stream regulatory sequence of human 13-aetin（hACTB） gene locus. Meth- ods： We described here a successive three-step gap-repair method. Firstly, a gap-repair vector based on pBR322 vector backbone by inserting six joint homologous arms was constructed. Secondly, using gap-repair method mediat- ed by Red recombination system of k-prophage in Escherichia coli, in the first step, the 10 kb 3＇ flanking region of the hACTB gene was subcloned from the bacterial artificial chromosome（BAC） which harbors the hACTB gene locus into the gap-repair vector; in the second step, the 13 kb hFWII genomic sequence from the ATG code to the TAG code was subcloned from the bFVII BAC; in the third step, the 20 kb 5＇ flanking region of the hACTB gene was subeloned from the hACTB BAC. Finally, all these three DNA fragments were automatically combined to- gether without any gap in the gap-repair vector, and a 50 kb hACTB-hFVII hybrid locus that the hFW~ genomic sequence was flanked by the 5＇ and 3＇ flanking region of hACTB gene locus was constructed. Results： The result was confirmed by PCR, restriction enzyme digestion and sequencing. Conclusion： Our method provide a new way for the construction of large cell efficient expression vectors.
出处
《生物技术通讯》
CAS
2012年第5期653-657,共5页
Letters in Biotechnology
