关键词: 基因打靶, 乳腺生物反应器, β-乳球蛋白, α-乳白蛋白, 山羊

Abstract:

【Objective】The targeting vector of β-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.

Key words: gene targeting, mammary gland bioreactor, β-lactoglobulin, α-lactalbumin, goat