Construction of expression vector of human lactoferrin and its expression in bovine mammary epithelial cells

Lactoferrin has multiple biological roles, including inflammation and immune modulation, iron absorption in newborns, non-iron-dependent antimicrobial activity, regulation of the level of retinoblastorna protein, protection against cancer development and metastasis, transactivation of the p53 tumor suppressor gene. Moreover, the ubiquitous expressed human lactoferrin (hLF) is species, tissue, and cell-type specific. To facilitate further studying of the biological function of hLF, we hereby constructed and expressed a recombinant vector containing exogenous hLF gene and bovine mammary gland B-lactoglobulin (BLG) gene. cDNAs of both hLF gene (2259bp) and 5' flank regulatory fragment of BLG (1449bp) consisting of the promoter region, exon 1 and intron 1 of the gene were obtained respectively. Then the translation initiation codon of 5' flank regulatory fragment was mutated from ATG to AAG and renamed as mbIg. hLF and mblg were recombined through PCR method and was named as BL, which was then cloned into pEGFP-Cl vector and renamed pMBL. Later on, the pMBL vector was transferred into a cell line of bovine mammary epithelia by liposomal transfection and cultured with G418 drug for 3 weeks to obtain positive transgene cell clones. Then the expression of hLF was detected in the positive cell clones by Immunocytochemistry and Western Blotting assays. The results showed that the exogenous hLF gene had been successfully integrated into the chromosome of the positive cell clones, which highly expressed hLF.