摘要
目的:构建一个利用牛αS1酪蛋白基因座完整的上下游调控序列指导人组织型纤溶酶原激活剂(tPA)基因组序列在乳腺特异性高效表达的牛αS1酪蛋白-人tPA杂合基因座。方法:采用连续3步基因抓捕的方法。首先,以pBR322载体作为骨架,插入预先无痕连接在一起的6个同源臂,构成能连续进行3次基因抓捕的抓捕载体;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,第一步从含牛αS1酪蛋白基因座的细菌人工染色体(BAC)上亚克隆9 kb的牛αS1酪蛋白基因3'端侧翼序列到抓捕载体上,第二步从人tPA BAC上亚克隆17 kb的从起始密码子(ATG)到终止密码子(TGA)的人tPA基因组序列,第三步从牛αS1酪蛋白BAC上亚克隆20 kb的牛αS1酪蛋白基因5'端完整侧翼序列,并使这3个基因片段在抓捕载体上自动无痕地连接在一起,形成一个长约46kb的牛αS1酪蛋白-人tPA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,构建的杂合基因座中,原牛αS1酪蛋白基因组编码序列从起始密码子到终止密码子被人tPA基因组序列精确置换。结论:连续三步基因抓捕构建杂合基因座乳腺表达载体的技术,为乳腺生物反应器高效表达大载体的制备提供了探索性研究。
Objective： To generate a bovine αS1-casein-htPA hybrid locus that the transcription of human tissue plasminogen activator（htPA） genomic sequence is directed by the updown stream regulatory sequence of bovine αS1-casein gene locus.Methods Results： We described here a successive three-step gap-repair method.First,a gap-repair vector based on pBR322 vector backbone by inserting six joint homologous arms was constructed.Then using gap-repair method mediated by Red recombination system of λ-prophage in Escherichia coli,in the first step,the 9 kb 3＇ flanking region of the bovine αS1-casein gene was subcloned from the bacterial artificial chromosome（BAC） which harbors the bovine αS1-casein gene locus into the gap-repair vector;in the second step,the 17 kb htPA genomic sequence from the ATG code to the TGA code was subcloned from the htPA BAC;in the third step,the 20 kb 5＇ flanking region of the bovine αS1-casein gene was subcloned from the bovine αS1-casein gene BAC.All these three DNA fragments were automatically combined together without any gap in the gap-repair vector,and a 46 kb bovine αS1-casein-htPA hybrid locus was constructed.The result was confirmed by PCR,restriction enzyme digestion and sequencing.Conclusion： Our method provide a new way for the construction of large mammary-gland expression vector.
出处
《生物技术通讯》
CAS
2011年第6期796-800,共5页
Letters in Biotechnology
