Study On BLG As Regulatory Sequence Direct Human-lactoferrin CDNA Expression In GMECs,SP2/0 And Hybridoma Cells

The mammary gland-specific expressional vector containing BLG/hLF cDNA (BLC-14) was transfected into goat mammary epithelial cells and myeloma cells by electroporation. Positive cells were selected by G418 and PCR and then were proliferated and induced by prolactin. The result of ELISA analysis on cultured cell supernatant showed that the transfected cells could express and se-crete recombinant human lactoferrin (rhLF).Normal myeloma cells (SP2/0) and spleen cells from hLF transgenic ICR mice were fused and selected by HAT, Positive cells were checked by PCR and were proliferated and induced by prolactin. The result of ELISA analysis of supernatant showed that the hybridoma cells could express and se-crete rhLF.1. The mammary gland-specific expressional vector were Constructed by BLG regulation sequence and human lactoferrin gene.The expressional vector included the goat BLG regulatory sequences containing the 4.2kb 5`end and 1.8 kb 3`end regulatory regions, the hLF cDNA, the enhancer sequences of human Cytomegalovirus(CMV),SV40 polyA sequences and neor -resistant gene.2. Establishment of goat mammary epithelial cell lines in vitro:Primary goat mammary cells were obtained from the lactating dairy goat; In the first time, primary goat mammary cells have been mix some fibroblast; Then the purified mammary epithelial cells were obtained by trypsin digestion in medium DMEM/F12 after 2-3 passages.3. By electroporation, BLC-14 containing neor gene as the selective marker was transfected into goat mammary epithelial cells and myeloma cells of mice. 59 clonies from goat mammary epithelial cells and 44 clonies from cells of mice were obtained. After selection of two weeks by 400μg/ml G418, positive clone cells were proliferated and induced by prolactin. The result of ELISA on cultured goat mammary epithelial cells supernatant showed that 8 colonies expressed rhLF.The expressional level of rhLF in GMEC and myeloma cells of mice was about 0.781μg.mL^-1 and 0.435μg.mL^-1 respectively.4. Establishment of hybridoma cell which express rhLF.Both of normal myeloma cells of mice and splenocyte which were from transgenic mice were fused by PEG;then hybridoma cell were selected by HAT;60 colonies were checked by PCR and detection of protein expressing , 5 of 60 colonies express rhLF at level of that 0.653μg.mL^-1.This study showed that mammary gland-specific expressional vector not only expression in specific goat mammary cells but also expression in the cells of myeloma cells of mice and hybridoma cell. According to result, we can infer that expression vector-BLC14 changes character of myeloma cells after transfected and induce to express rhLF.