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 aSl-casein-htPA hybrid locus that the transcription of human tissue plasminogen activator (htPA) genomic sequence is directed by the up&down stream regulatory sequence of bovine aSl-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 -prophage in Escherichia coli, in the first step, the 9 kb 3' flanking region of the bovine aSl-casein gene was subcloned from the bacterial artificial chromosome (B AC) which harbors the bovine aSl-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 aSl-casein gene was subcloned from the bovine aSl-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 aSl-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.
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