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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 a hybrid locus of human serum albumin (hSA) and mouse whey acid protein (mWAP) regulatory sequences. Methods and Results: Three pairs of homology arms were pre-grafted to pBR322 vector. The 3 ’regulatory region of 8kb mWAP locus was amplified by gap-repair technique based on Red homologous recombination system. 16 kb of hSA genome coding sequence and 13 kb of mWAP locus 5 ’regulatory region for three consecutive gene capture, and finally the full length of 37 kb of the milk protein heterozygous locus into pBR322 vector; by PCR amplification, limiting Endonuclease digestion and sequence validation demonstrated that the coding sequence in the mWAP locus was precisely replaced with the genomic coding sequence of hSA. CONCLUSION: The pBR322 vector with mWAP-hSA hybrid locus has been constructed and provided data for studying the expression effect of heterozygous loci in mammary gland and the feasibility of substitutional locus expression.