摘要
目的: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溶菌酶(hLYZ)基因组序列在乳腺内特异性高效表达的mWAP-hLYZ杂合基因座,实现人溶菌酶的高效表达。方法:采用连续3步缺口修复的方法。首先,以pBR322载体作为骨架,插入预先合成的6个同源臂序列,构成能够连续进行3次缺口修复的基因抓捕载体。然后在大肠杆菌内利用λ噬菌体Red同源重组系统介导的同源重组方法:第一步,从含mWAP基因座的细菌人工染色体(BAC)上亚克隆8 kb的mWAP基因3'端完整侧翼序列到抓捕载体上;第二步,从含hLYZ基因的BAC上亚克隆5 kb的从起始密码子(ATG)到终止密码子(TAA)的hLYZ基因组序列;第三步,从mWAP BAC上亚克隆9kb的mWAP基因5'端完整侧翼序列,并使上述3个片段在抓捕载体上自动无痕地连接在一起。结果:构建了全长约22 kb的mWAP-hLYZ杂合基因座,经PCR扩增、限制性内切酶酶切和序列测定验证,构建的杂合基因座达到原mWAP基因座中mWAP基因组编码序列从起始密码子(ATG)到终止密码子(TAA)完全被hLYZ基因组序列精确置换的目的。结论:通过连续3步缺口修复构建杂合mWAP-hLYZ基因座乳腺表达载体,为乳腺生物反应器高效表达人溶菌酶提供了可行的思路和方法。
Objective： To generate a mWAP-hLYZ hybrid locus that the transcription of human lysozyme（hLYZ） genomic sequence is directed by the flank regulatory sequence of murine whey acidic protein（mWAP） gene locus.Methods： We described here a successive three-step gap-repair method.First,we constructed a gap-repair vector based on pBR322 vector backbone by inserting six-joint-homologous-recombinant-primers.Then using gap-repair method mediated by Red recombination system of λ-prophage in Escherichia coli.First,the 8 kb 3′ flanking re gion of the mWAP gene was subcloned into the gap-repair vector from the bacterial artificial chromosome（BAC） which harbors the mWAP gene locus;second,the 5 kb hLYZ genomic sequence from translation initiation site（ATG） to termination site（TAA） was subcloned into the gap-repair vector from the hLYZ BAC;third,the 9 kb 5′ flanking region of the mWAP gene was subcloned into the gap-repair vector from the mWAP BAC.Finally,all these three DNA fragments were automatically combined together without any gap in the gap-repair vector.Re sults： A 22 kb mWAP-hLYZ hybrid locus was constructed,in which the 3 kb mWAP genomic coding sequence in the mWAP gene locus was substituted with 5 kb hLYZ genomic coding sequence exactly from ATG to TAA.The the gap-repair vector was confirmed by PCR,restriction enzyme digestion and sequencing.Conclusion： The method provides a new way for the construction of large mammary-gland expression vector,and provides an alter native production way to hLYZ.
出处
《生物技术通讯》
CAS
2012年第5期648-652,657,共6页
Letters in Biotechnology
关键词
细菌人工染色体
缺口修复
乳清酸蛋白
人溶菌酶
bacterial artificial chromosome
gap-repair
whey acidic protein
human lysozyme
