连续三步‘Gap-repair’构建小鼠WAP--人LF杂合基因座

A Successive Three-step ‘Gap-repair’ Method to Generate the mWAP-hLF Hybrid Gene Locus

为了构建一个利用小鼠乳清酸蛋白(mWAP)基因座完整的上下游调控序列指导人乳铁蛋白(hLF)基因组序列在乳腺特异性高效表达的mWAP-hLF杂合基因座,我们采用了连续三步‘Gap-repair’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,构成能连续进行三次gap-repair的基因抓捕载体。然后在大肠杆菌内利用Red同源重组系统介导的gap-repair技术,第一步从含mWAP基因座的细菌人工染色体(mWAPBAC)上亚克隆了8kb的mWAP基因3'端完整侧翼序列到抓捕载体上;第二步,从hLFBAC上亚克隆29kb的从起始密码子(ATG)到终止密码子(TAA)的hLF基因组序列;第三步,从mWAPBAC上亚克隆12kb的mWAP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,形成一个全长约49kb的mWAP-hLF杂合基因座。经过PCR扩增、限制性内切酶消化和序列测定验证,我们构建的这个杂合基因座,达到了原来mWAP基因座中mWAP基因组编码序列从起始密码子到终止密码子被hLF基因组序列精确置换的目的。这种连续三步gap-repair构建杂合基因座乳腺表达载体的技术,将为乳腺生物反应器高效表达大载体的制备提供一种全新的思路和方法。

To generate a mWAP-hLF hybrid locus that the transcription of human lactoferrin（hLF） genomic sequence is directed by the up＆down stream regulatory sequence of murine whey acidic protein （mWAP） gene locus, we describe 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 8 kb 3＇ flanking region of the mWAP gene was subcloned from the Bacterial artificial chromosome which harbors the mWAP gene locus（mWAP BAC） into the gap-repair vector; in the second step, the 29 kb hLF genomic sequence from the ATG code to the TAA code was subcloned from the hLF BAC; in the third step, the 12 kb 5＇ flanking region of the mWAP gene was subcloned from the mWAP BAC. Finally, all these three DNA fragments were automatically combined together without any gap in the gap-repair vector, and a 49 kb mWAP-hLF hybrid locus that the hLF genomic sequence was flanked by the 5＇ ＆ 3＇ flanking region of mWAP gene locus was constructed. The result was confirmed by PCR, restriction enzyme digestion and sequencing. Our method provide a new way for the construction of large mammary-gland expression vector.