| Through transgenic technology, animal mammary gland bioreactor could produce recombinant heterogenous protein for human medicine under the control of mammary gland special regulatory elements in mammary gland. It has the advantages of low expense, high yield, processivity, and especially the post-translational processing and modification of the exogenous protein for natural construction and activity. Thus nowadays it becomes a hot subject of bioreactor research at home and abroad. The key of the research is the construction of gland special high-efficient expression vector, including the usage of expression regulatory elements and cis-action factors which might improve expression level of the exogenous gene. Make sure that the expression vector would be available before making transgenic animal models. Meanwhile, mammary gland epithelial cells cultured in vitro have the superiorities as follows: the operation in vitro is simple and convenient, the experimental conditions could be controlled artificially, and the cell also secretes relatively pure and stable foreign protein by artificial hormone induction. Additionally, productions could be detected briefly. No doubt that the epithelial cells would be an ideal experimental material.A study, basing on the above principles, was designed to aim at testifying the effect of enhancer and insulator et al cis-regulatory factors on the expression of human lactoferrin (hLF) gene under the control of goat ?-lactoglobulin (BLG) regulatory elements. Finally the high-secreteing expression vectors were screened as sources for the further transgenic animal research.The gene elements with the goat BLG regulatory sequences containing the 4.2 kb 5`end and 1.8 kb 3`end regulatory regions, the Hlf cDNA, the enhancer sequences of human Cytomegalovirus(CMV), SV40 polyA sequences and neo-resistant gene, the chicken ?-globin insulator gene were used to construct into two mammary gland special expression vectors of human LF—BLC14 and BLCDI. Primary goat mammary cells were isolated from healthy lactating dairy goat by adopting collegenase I and hyaluronidase co-digestion, then pure mammary epithelial cells were obtained by trypsin digestion and repeatly-anchoring in medium DMEM/F12 through 2-3 passages. Using electroporation the purified mammary epithelial cells were transfected with BLC14 and BLCDI gene fragments. After selection of G418 for about 10d, 63 and 41 colonies of BLC-14 and BLCDI transfected cells were obtained independently, of which were picked out into another tissue culture dish to continue subculture. DNA samples of these colonies were screened by PCR, and 21, 13 colonies had been successfully integrated with foreign genes respectively. The PCR-positive strains were then induced with prolactin, and after 48 h, the supernate were collected for ELISA. 15 colonies expressing hLTF from BLC-14 and 8 colonies from BLCDI were obtained. The outcomes obtained from ELISA suggested that expression level of transfected BLC14 gene cell colonies was higher but not stable, and that of transfected BLCDI gene was relatively lower but more stable.The results demonstrate that the constructions of BLC14 and BLCDI expression vectors are valid, the hLF cDNA could be expressed in the epithelial cells under the control of the goat BLG regulatory elements; and the CMV enhancer could prompt its higher expression level, 5` and 3` flanking regions with the double-copy chicken ?-globin insulators could eliminate the"position effect"of exogenous gene, and stabilize its expression.
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