连续3次缺口修复构建小鼠乳清酸蛋白-人血清白蛋白杂合基因座  

A Successive Three-Step Gap-Repair Method to Generate the mWAP-hSA Hybrid Gene Locus

作　　者：吴晓洁[1] 林艳丽[1] 施庚寿[1] 熊福银[1] 周艳荣[1] 吕月蒙[1] 邓继先[1] 陈昭烈[1] 陈红星[1] 

机构地区：[1]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》2012年第3期375-379,共5页Letters in Biotechnology

摘　　要：目的:构建人血清白蛋白(hSA)与小鼠乳清酸蛋白(mWAP)调控序列的杂合基因座。方法与结果:在pBR322载体上连入预先无痕连接的3对同源臂,利用基于Red同源重组系统的缺口修复(gap-repair)技术,分别对8kb的mWAP基因座3′调控区、16 kb的hSA基因组编码序列及13 kb的mWAP基因座5′调控区进行连续3次基因抓捕,最终将全长37 kb的乳蛋白杂合基因座构入pBR322载体;通过PCR扩增、限制性内切酶消化和序列测定验证,确定mWAP基因座中的编码序列被精确地置换为hSA的基因组编码序列。结论:构建了整合有mWAP-hSA杂合基因座的pBR322载体,为研究杂合基因座在乳腺中的表达效果及置换型基因座表达的可行性提供了数据。Objective： To construct the hybrid gene locus region of mouse whey acid protein（mWAP） locus. Methods of human serum albumin（hSA） fused with regulatory ＆ Results： The three pairs of h.omologous arms were inserted into pBR322 to construct pBR322-gaprepair vector. Then the gap-repair technique based on Red homologous recombination system was applied to arrest the 3＇ regulatory region of mWAP locus（8 kb）, hSA genomic coding sequence（16 kb） and the 5＇ regulatory region of mWAP locus（13 kb） successively, resulted in 37 kb-length heterozygous gene locus emerged in the pBR322. Precise replacement of the coding sequence of mWAP with the hSA genomic coding sequence was finally verified by PCR amplification, restriction enzyme digestion and sequencing. Conclusion： We have obtained the pBR322 vector integration with mWAP-hSA heterozygous locus. The constructed heterozygous locus will be further studied on its expression efficiency in mammary gland and provided as the data support to the feasibility of expression of replaceable heterozygous locus.

关 键 词：小鼠乳清酸蛋白 人血清白蛋白 缺口修复 细菌人工染色体 

分 类 号：Q78[生物学—分子生物学]