| Milk and whey contained large amounts of lactose.There was 70%-90%adult,especial the Asian and African in the world who had lactose intolerance.Lactose intolerance largely limited the intake of milk and dairy products.β-galactosidase can hydrolyze the lactose of milk and whey.The reduction of lactose can solve the problem of the people who had lactose intolerance and improve the nutritional value of milk and dairy products.At the same time,the reduction of lactose can get rid of the inconvenience of lactose crystallization and separation when whey condensed in the dairy processing.The milk content between cow and human is different.The most proteins in human milk are whey proteins,about 70%of total proteins and the others are caseins.The most proteins in cow milk are caseins,about 78.8%of total proteins,and whey proteins are fewer,about 21.2%of total proteins.So the nutritional value of cow milk can be improved if the composition and content of milk protein can be reformed to be closed to human milk.It can bring a reformation to use the transgenic technology to change the milk composition to improve the nutritional value and fabrication character.Those include content improvement of some high value ingredient, elimination of no-wanted ingredient,producing of some ingredient of human milk and producing protein drugs by mammary gland bioreactor.Alpha lactalbumin is the main protein of whey proteins.It is the good source of essential amino-acid and branched-chain amino acid.It can regulate the producing of milk.And it is the only protein of whey proteins which can bind metals including calcium.It also has the abilities,such as bacteriostasis,apoptosis suppression and cerebrum reaction ability enhancement.This study was designed to construct the eukaryotic expression vector by gene recombination technology and express human alpha lactalbumin gene andβ-galactosidase gene in the bovine mammary epithelial cells to produce active human alpha lactalbumin andβ-galactosidase.Theβ-galactosidase can reduce the lactose content in milk.It aimed at the expression of human alpha lactalbumin gene andβ-galactosidase gene at a high level in the mammary glands of cows further.That would provide a viable technical means and credible theories on production of transgenic animal milk and improvement of the milk quality.In this study,the 5' flanking regulatory regions of bovineαS1 casein gene(1.2kb) was amplified by PCR and human alpha lactalbumin gene cDNA(0.76kb) was synthetized.The 1.2kb region of 5' flanking sequence of bovine casein gene and human alpha lactalbumin gene was combined with the PSV vector which has SV40 promotor andβ-galactosidase gene(LacZ gene) to construct the expression vectorαS1-LA-psv.At the same time,human alpha lactalbumin gene cDNA(0.76kb) was combined with the PSV vector behind the SV40 promotor(without LacZ gene) to construct the expression vector LA-cDNA-psv.Bovine mammary epithelial cell was cultured, purified and identified by the tissue-special expression of cytokeratin 18.Afterwards,the eukaryotic expression vectorαS1-LA-psv was transfered into bovine mammary epithelial cells by cationic liposome vector.Then the expression ofβ-galactosidase was detected by two ways in bovine mammary epithelial cells.The first way is by situ staining in the cells,and in which theβ-galactosidase could catalyze the disassociation of X-gal.The dark blue production in the cells was observed by optical microscope,which is the expression ofβ-galactosidase in the cells.Another way is by the Beta-Glo E2000 detection system produced by Promega.The activity ofβ-galactosidase from 24h to 120h in cell lysate and culture medium were detected.The results showed that in the cell lysate,the expression of lacZ gene could be detected at 24h after transfection,and had a gradually upward trend which declined at 72h,reached the minimum at 144h.In the cell culture medium,the expression of lacZ gene can be detected also at 24h after transfection,and had a gradually upward trend which declined at 48h,reached the minimum also at 144h.The cells which were transfected were passaged,the expression ofβ-galactosidase was also detected at the first and second generation,but from the third generation, the expression could not be detected again.The lactose content in the transfected cells were detected by HLPC.The results showed there was no change at 24h,and notable reduction appeared from 24h to 144h,the most reduction appeared from 48h to 72h.The expression of human alpha lactalbumin was detected at 72 hour by Western Blot after transfected The production of human alpha lactalbumin was 0.64mg/mL approximately.The eukaryotic expression vector LA-cDNA-psv was transfered into bovine mammary epithelial cells by cationic liposome vector too.The expression of human alpha lactalbumin was detected at 72 hour by Western Blot after transfected.The production of human alpha lactalbumin was 0.85 mg/mL approximately.All these results showed bovine mammary epithelial cell which was cultured and purified have the ability to express the exogenous genes,the 5' flanking regulatory regions of bovineαS1 casein gene(1.2kb) which was cloned can direct the expression of exogenous genes highly,and the constructed expression vector can expressβ-galactosidase and alpha lactalbumin in bovine mammary epithelial cells.In this study,the eukaryotic expression vectorαS1-LA-psv was also injected to normal bovine mammary gland.After transfected,the lactose content and milk production were detected from 24h to 120h The notable reduction of lactose content appeared from 24h to 120h,and there was no notable change in milk production.These results showed the constructed expression vector also can express the exogenous genes in bovine mammary gland and produce the diabetic milk without effect on the normal lactation.
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