Objective: To generate a hybrid locus that the transcription of human lysozyme(hLYZ) genomic se⁃quence is directed by the bovine αS1-casein gene locus. Methods: We described here a successive three-step gap repair method of our laboratory invented. The six homologous arms were inserted into pBR322 to construct pBR322-gaprepair vector. The gap repair method mediated by Red recombination system was applied to arrest the 9 kb 3′flanking region of the bovine αS1-casein gene, the 5 kb hLYZ genomic sequence and the 20 kb 5′flank⁃ing region of the bovine αS1-casein gene. These three DNA fragments were automatically combined together with⁃out any gap in the gap-repair vector, and a bovine αS1-casein-hLYZ hybrid locus was constructed. Results:Pre⁃cise replacement of the coding sequence of the bovine αS1-casein with the hLYZ genomic coding sequence. The result was successfully verified by PCR, restriction enzyme digestion and sequencing. Conclusion: This gap repair method provides a purposes of ideas and ways for the construction of large mammary-gland expression vector.% 目的：构建一个牛αS1酪蛋白调控序列指导人溶菌酶(hLYZ)基组序列的杂合基座。方法：采用本实验室发明的连续3步缺口修复技术。将6个无痕连接的同臂插入以 pBR322为载体的骨架中，构成能进行3次连续基抓捕的载体，利用 Red 同重组系统介导的缺口修复技术，分别抓捕牛αS1酪蛋白3'端调控序列(9 kb)、hLYZ 基座序列(5 kb)、牛αS1酪蛋白5'端调控序列(20 kb)，使这3个基片段自动无痕地连接在基抓捕载体上，形成牛αS1酪蛋白-hLYZ 杂合基座。结果：实验经过 PCR 扩增、限制性内切酶酶切验证和序列测定，验证了 hLYZ 的基组序列对牛αS1酪蛋白编码基组序列的精确置换。结论：这种修复技术为乳腺生物反应器高效表达大载体的制备提供了可行的思路及方法。
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