【摘要】 【目的】构建山羊β-乳球蛋白(BLG)基因打靶载体,以期用人乳白蛋白(hALA)基因的编码区置换山羊BLG基因的编码区,借助BLG的内源性调控序列指导hALA的表达。【方法】以山羊血液基因组为模板PCR扩增获得BLG基因的5′端侧翼序列(BLG5)、第5外显子和第5内含子的部分序列(E5)和3′端侧翼序列(BLG3),同时以人血液基因组为模板克隆了hALA基因的基因组序列(ALA),并将上述片段连接到克隆载体pMD18 T-simple或pBluescript KS(-),得到载体pB5T,pB3T,pAKS和pAEKS。对pB5T进行亚克隆,将BLG5连接到pAEKS中,构建载体pBAE;最后将BLG5-hALA-E5和BLG3分别连接到打靶载体ploxpII中,标记为pBAT。【结果】经酶切和/或测序等鉴定,以上载体均正确。【结论】构建得到了旨在将hALA定向插入到山羊乳蛋白基因座位的打靶载体。更多还原

【Abstract】 【Objective】The targeting vector of ?-lactoglobulin(BLG) in goat was constructed to replace the coding region of BLG with genomic DNA of human α-lactalbumin and to direct the expression of hALA under the internal regulatory elements of BLG..【Method】 Firstly,5’ flanking region of BLG(BLG5),3’ flanking region of BLG(BLG3),partial sequence of the 5-exon and 5-intron(E5) and hALA were amplified from serum genomic DNA,and then were inserted into the cloning vector pMD18-T simple or pBluescript KS(-) named pB5T,pB3T,pAKS and pAEKS.Secondly,BLG5 was subcloned and ligated into pAEKS forming pBAE.Finally,the targeting vector was constructed after integrating the sequence of BLG5-hALA-E5 and BLG3 into the general targeting vector ploxpII.【Result】The vectors were identified with the methods of cutting with responding endonucleases and/or sequencing,all of which showed that the vector was constructed successfully.【Conclusion】 The targeting vector that can target the hALA into the locus of BLG in goat was got.更多还原