连续3步缺口修复技术构建牛αS1酪蛋白-人溶菌酶杂合基因座  

A Successive Three-Step Gap Repair Method to Generate the Bo vine αS1-Casein-hLYZ Hybrid Gene Locus

作　　者：郑芸芸[1,2] 周艳荣[2] 吴晓洁[2] 林艳丽[2] 熊福银[2] 李力[1] 陈红星[2] 

机构地区：[1]陕西师范大学生命科学学院,陕西西安710062 [2]军事医学科学院生物工程研究所,北京100071

出　　处：《生物技术通讯》2013年第2期173-177,共5页Letters in Biotechnology

摘　　要：目的:构建一个牛αS1酪蛋白调控序列指导人溶菌酶(hLYZ)基因组序列的杂合基因座。方法:采用本实验室发明的连续3步缺口修复技术。将6个无痕连接的同源臂插入以pBR322为载体的骨架中,构成能进行3次连续基因抓捕的载体,利用Red同源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基因座序列(5 kb)、牛αS1酪蛋白5'端调控序列(20 kb),使这3个基因片段自动无痕地连接在基因抓捕载体上,形成牛αS1酪蛋白-hLYZ杂合基因座。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的基因组序列对牛αS1酪蛋白编码基因组序列的精确置换。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。Objective： To generate a hybrid locus that the transcription of human lysozyme（hLYZ） genomic se- quence is directed by the bovine ctSl-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 o^Sl-casein gene, the 5 kb hLYZ genomic sequence and the 20 kb 5＇flank- ing region of the bovine c^Sl-casein gene. These three DNA fragments were automatically combined together with- out any gap in the gap-repair vector, and a bovine c^Sl-casein-hLYZ hybrid locus was constructed. Results： Pre- cise replacement of the coding sequence of the bovine ctSl-easein 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.

关 键 词：牛αS1酪蛋白 人溶菌酶 杂合基因座 缺口修复技术 

分 类 号：Q78[生物学—分子生物学]