Abstract: 【Objective】In order to obtain a high efficiency mammary gland specific promoter, in the present study, promote efficiency of β-casein gene promoters derived from Holstein cow, Jersey cow and water buffalo will be comparatively studied.【Method】β-casein gene promoter sequences of Holstein cow, Jersey cow and water buffalo in Genbank were comparatively analyzed, primers of promoter sequences and 3′ployA sequences were designed according to the conservative fragment respectively, primers of the human IFN-2b gene and EGFP-Neo selective marker fragment were also designed. To facilitate the constructing expression vectors, appropriate restriction enzyme digestion sites were introduced into all the primers. Genome DNA was isolated from the leukocytes in venous blood samples. β-casein gene promoter fragments and 3′ployA fragments were amplified by PCR using the genome DNA as templates. Human IFNα-2b gene and EGFP-Neo selective marker fragments were also amplified by PCR using the plasmids conserved in our lab. Sequencing analyses were carried out; making sure that all the fragments were correct. Then, all fragments were inserted into the pMD18-T backbone in the designed order. Finally, 3 mammary gland expression vectors, pHSTBCNp-IFN, pJSBCNp-IFN and pSNBCNp-IFN were constructed. Transgenic cell lines were generated by transfected 3 recombinant plasmids into Bacp-37 cells and G418 selected. Transgenic cell lines were induced by combined hormones including Insulin (1 mg·L^-1), Transferrin factor (1 mg·L^-1), Hydrocortisone (1 mg·L^-1) and PRL (250 IU·L^-1). IFNα-2b expression levels were analyzed at mRNA and protein level by PCR, Western blot, QRT-PCR and ELISA.【Result】β-casein gene promoter and 3′ployA fragments of Holstein cow, Jersey cow and water buffalo were obtained by PCR. The fragments lengths were 5 219, 5 244 and 5 216 bp respectively. In the promoter fragments, the first exon, first intron and the partial second exon were included. 51 nucleotides in the partial second exon encoding 17 amino acid signal peptide. For the 3′ployA fragments were all 1 166 bp. After 3 transgenic expression vectors were generated, and transfected into Bcap-37 cells. After G418 screening, 3 positive transgenic cell lines were obtained successfully. PCR, Western blot, QRT-PCR and ELISA results showed that IFNα-2b was successfully expressed in these 3 transgenic cell lines when hormone induced. Transgenic cell lines contain pJSBCNp-IFN expression vector showed a significantly higher expression level than the other two at mRNA and protein levels (P＜0.05).【Conclusion】Jersey cow β-casein gene promoter possesses a higher efficiency in regulating foreign gene expression, and it is a considerable mammary gland specific promoter in transgenic research. Expression vectors constructed in the present study laid a foundation for constructing IFNα-2b transgenic mammary gland bioreactor.

Key words: promoter, Jersey Cow, interferon α-2b, Bcap-37, transgene