Cloning And Analysis Of Beta-casein Gene 5' Flanking Sequence From Xinong Saanen Dairy Goat And Construction Of Mammary Gland Specific Expressional Vector

Mammary gland bioreactor is a useful biological system which expresses foreign genes in animal mammary gland and produces functionally pharmaceutical protein in milk.This production route is appealing for it's advantages, such as the simplicity of access to the expressed protein, the high production of the mammary gland,as well as the capabilities to perform post-translational processing. As an alternative of cell culture, it is a new biotechnology.In fact, successful gene transfer has been described for all the major commercial livestock species including goats, pigs, and cows. In the present study, specific primers for goat ?-casein gene promoter had been designed and synthesized according to the published nucleic acid sequence of goat complete ?-casein gene. Xinong Saanen Dairy Goat β-casein 5' flanking sequence was obtained by long and accurate PCR. The PCR product was then cloned into pMD18-T vector and sequenced. The cloned fragment covered the 5' flanking sequence of the goat β-casein gene from –4359 to +2106 bp. Bioinformatic analysis indicated that putative promoter sequence of the cloned Xinong Saanen Dairy Goat β-casein 5' flanking sequence shared 96%~98% homology with other goat β-casein gene promoters. Xinong Saanen Dairy Goat β-casein 5' flanking sequence region contained promoter sequences and putative recognition sites for several transcriptional factors, including HOXF, Oct1, AP1, CEBP, STAT, NFKB, GR, ER, PR, ETS and the intron 1, exon 1 and exon 2 of the goat β-casein gene. Reconstructed reporter plasmid pGL3/B65 was established by inserting 6,465 nucleotides from the promoter region into the luciferase reporter vector pGL3-Basic. Finally, the pGL3/B65 was transfected in vitro via liposomes into the primary goat mammary epithelial cell. Transient transfection assay showed the β-casein 5' flanking sequence region had promoter function, and it responded to hormonal treatment. Then, the β-casein 5' flanking sequence was instead of the pCMV promoter in pcDNA3.1(-) vector to construction of a mammary gland specific expressional vector pcDNA3.1-65. And then hBMP2 cDNA was cloned into pcDNA3.1-65. The primary goat mammary epithelial cells(MEC) transiently transfected with vector pcDNA3.1(-), pcDNA3.1-65, and pcDNA3.1-65-hBMP2 were established and induced with prolactin. Immuocytochemical staining revealed that the expression of hBMP2 was increased significantly in MEC/pcDNA3.1-65-hBMP2 cells as compared with vector control cells under the same experimental condition. The similar change was also found in the level of hBMP2 protein through Western Blotting. Conclusion: 1.The Xinong Saanen Dairy Goat ?-casein gene 5' flanking sequence was cloned which is about 6.5kb fragment containing of the promoter, exon1, intron1 and exon 2. Experiment results showed it assuredly had the promoter and enhancer activities. 2.The mammary gland specific expressional vector pcDNA3.1-65 based on the ?-casein 5' flanking sequence was well constructed. The primary goat mammary epithelial cells transiently transfected with pcDNA-3.1-65 responsed to prolactin induction for target protein synthesis. The work has provided a basis for establishing transgenic mammary gland bioreactor.