Studies On Establishment Of Transgenic Vectors Of Human Alpha Lactalbumin With Regulatory Sequences And Modified Dairy Goat Fibroblast Cell Lines

Alpha-lactalbumin (α-LA) is the major calcium-binding protein in the whey proteins,and it is one of the lactose synthetase subunits and it participates in lactose biosynthesis. a-LA is nutritious and it could enhance immunity. a-LA is the major protein in human milk, about2.9mg/mL, but low in goat milk, so we can improve the quality of goat milk by transgenic technology. In this study, techniques of gene clone, cell culture, Lipofectamine transfection, PCR and Western-blot were used to establish the transgenic dairy goat fetal fibroblast cell lines(gFFCs) as the nucleus donor cells for transgenic dairy goat. And the transgenic dairy goats of a-LA in our lab were also verified by using RT-PCR, TAIL-PCR and Western-blot.(1) The gene cloning of a-LA regulatory sequence and construction of expression vector.Human genome DNA was used as template to amplify the whole human a-LA sequence including the5’flank regulation sequence and part of3’flank regulation sequence which was then cloned into pMD19-T vector to get vector PLAT. Sequence NI3A (neo-IRES-EGFP co-expression sequence as selection mark gene,1.7kb3’ PLG as3’flank regulation region sequence) from vector B9PLA from PLAT by enzyme digestion were then connected to get vector PLAN. The goat fetal fibroblast cells were transiently transfected with PLAN by Lipofectamine. The results showed that the transfected cells can express the green fluorescent protein.(2) The induced expression of vector PLAN in dairy goat mammary gland epithelium cells and detection of a-LA. Vector PLAN was transfected into the dairy goat mammary gland epithelium cells by LipofectamineTM LTX plus PLUSTM Reagent, at the same time transgenic vector5A and NI3A from our laboratory were used as controls. The transgenic cells were cultured in induction medium containing serum-free DMEM-F12with10μg/mL prolactin,5μg/mL insulin and and10μg/mL hydrocortisone. The culture supernatant were collected after24h,48h and72h of transfection to detect the expression of α-LA by Western-blot.The results showed that human a-LA were expressed in the culture supernatant with a molecular weight of about14kD, indicating that the constructed mammary gland specific vector PLAN have the bioactivity to induce the expression of human a-LA in the mammary gland cells. This study provided a firm foundation for the further study of mammary gland bioreactor(3) The establishment and sifting of gFFCs integrated with human a-LA gene including the regulatory region.The dairy goat fetal fibroblasts were transfected with linearized PLAN by LipofectamineTM LTX plus PLUSTM Reagent. Based on the Neo and EGFP gene, we cultured the cells with1000μg/mL G418for1week and500μg/mL G418for next2weeks, then we selected and expanded the cells which expressed the green fluorescent protein. The result of Nested-PCR showed that the transgene was stably integrated into the cells, and the cells could be the nucleus donor cell for transgenic dairy goats.(4) Identification of Human α-LA transgenic dairy goat.The unknown sequence near the5’flank and3’flank known sequence was successfully obtained by using Thermal Asymmetric Interlaced PCR (TAIL-PCR).We detected the mRNA and protein expression of a-LA in the mammary epithelial cells by RT-PCR and Western-blot, respectively. The results showed that the inserted sequence is homologous recombination by comparing the TAIL-PCR products with the known sequences and dairy goat genome sequences. The human a-LA can be detected in transgenic dairy goat milk and the purity was close to that of human milk. All of these indicate that the human a-LA gene has been integrated into genome successfully and the the transgenic goat mammary gland can express human a-LA efficiently.