Expression Of LacZ Gene In Goat Mammary Epithelial Cells

Milk and whey, which contained large amounts of lactose,but there was 70%-90% of the Asianand African in the world who had lactose intolerance.Lactose intolerance largely limited the intakeof milk and dairy products,and it may bring about a series of adverse effects.Lactose intolerancecan be relieved by producting low-lactose milk,which producted byβ-galactosidase hydrolysis oflactose.And also this reaction would improve the nutritional value of milk and dairy products,at thesame time it got rid of the inconvenience of lactose crystallization and separation when wheycondensed in the dairy processing.In addition,the hydrolysates production of this reaction could beused in the producting of whey drinks,syrup,food additives.This study was designed to constructe the eukaryotic expression vector by gene recombinationtechnology, and expressβ-galactosidase gene in the goat mammary epithelial cells,then explored theexpression of different stages.It aimed at the expression ofβ-galactosidase gene at a high level inthe mammary glands of cows further, and providing a viable technical means and credible theorieson production of transgenie animals and transgenic milk,improvement of milk quality for thefuture.In addition,the eukaryotic expression vector constructed in this study contained the wholelacZ gene sequence,and its upstream regulatory sequence ended with restriction endonucleasesites,which provided a convenient,reliable model for the study and research on different eukaryoticgene regulation sequences.In this study, total DNA was extracted from the mammary tissue of Holstein cows.And the 5'flanking regulatory regions of 1195bp of bovineαS1 casein gene were amplified by PCR from cowmammary DNA and sequenced.The homology rate is 99.75%,which compared the sequences withthe corresponding sequences published in GenBank by computer software DNAMAN6.0.Itsuccessfully constructed the eukaryotic expression vector gαslp-psv, which gained by inserting the5'flanking sequence of bovineαS1-casein gene into the upstream of lacZ gene by the recombinantDNA technology.Then,the goat mammary epithelial cell could be isolated by collagenase digestionand cultured.Further, it identified for separation of cultured goat mammary epithelial cells usedimmunohistochemical staining method to determinate the expression of keratin 18 of mammaryepithelial cell.Afterwards,it was used cationic liposome vectors to transfer the gαslp-psv into goatmammary epithelial cells,thus the expression ofβ-galactosidase could be detect by twomethods.One is situ staining in the cells,in which theβ-galactosidase could catalyze the disassociation of X-gal,and the dark blue production in the cells would be obversed by commonoptical microscope,that is the expression ofβ-galactosidase in the cells.Another is the Beta-Glodetection system producted by Promega.lt detected the expression ofβ-galactosidase in thetransfected cell culture medium and the broken cells liquid at different stages.According to the testresults,it showed that in the cell culture medium the expression of lacZ gene can be detected at 24hours after transfection and expression had a gradually upward trend which declined at 72 hours,and 144 hours reached the minimum.ln the broken cells liquid,the expression of lacZ gene can bedetected at 24 hours after transfection and expression had a gradually downward trend which reachthe minimum at 144 hours.So it confirmed thatβ-galactosidase was secretory protein.On the basisof the expression of transfected cells passage,the first generation reached the highest level,theexpression reduced after cells passage,to the third generation the expression could not be detected.