Study On Expression Of Human Lactoferrin Gene Controlled By Different Regulatory Region Of Milk Protein In Transgenic Mice

The use of transgenic animals for mammary bioreactors is a rapidly developing and intensely interesting field due to the increased demand for high quality pharmaceuticals and medically diagnostic preparations. The manufacture of recombinant human protein is more profitable by mammary gland bioreactor than that by other biological system.The mammary gland specific expression is a major objective for producing hetergenous proteins in the milk of transgenic animals. Foreign protein have been expressed specifically in the mammary under the direction of particular milk protein regulatory sequences,such as:goatβ–lactoglobulin(BLG),goatβ–casein,bovineαs1–casein, and rat whey acid(WAP) protein. However,much of the evidences regarding the mammary as bioreacoter indicated that only a small percentage of the mammary gland of transgenic animals expressed functional protein,and most of them were found at low levels in milk.To obtain high and stable expression of hLF in transgenic animals,various promoters were used to construct expression vectors in this research.Three vectors were constructed to express human lactoferrin(hLF) gene in transgenic animals. Evaluating the efficiency of different regulatory region in transgenic expression,which establish the foundation for producing transgenic goat mammary gland bioreactors. We used theβ-lactoglobulin regulatory sequence and hLF cDNA gene to construct BCL expression vector. The goat beta-casein regulatory sequence and hLF cDNA gene were used to construct PMH expression vector. We used the bovine alphal-s1 casein regulatory sequence and hLF cDNA gene to construct ACF expression vector.The result of original generation(G0) BCL transgenic mice:1871 eggs were microinjected with the 10.6 kb fragment digested from the BCL expression vector with SalI. and NotI. 927 eggs were transferred into 31 forster mothers,13 of which were pregnant and 65 pups were obtained, among which 6 pups (4♀, 2♂) were transgenic mice, the rate of integration was 9.23%. 3 pups of No.11(♂) were transgenic mice, the rate of integration was 15.78% (3/19);6 pups of No.23(♂) were transgenic mice, the rate of integration was 37.5% (6/16). The result of original generation(G0)PMH transgenic mice:1658 eggs were microinjected with the 19.1 kb fragment digested from the PMH expression vector with SalI and NotI. 816 eggs were transferred into 26 forster mothers, 11 of which were pregnant and 56 pups were obtained, among which 5 pups (4♀, 1♂) were transgenic mice,the rate of integration was 8.93%. 3 pups of No.30(♂) were transgenic mice, the rate of integration was 21.43% (3/14). The result of original generation(G0)ACF transgenic mice:784 eggs were microinjected with the 10.8 kb fragment digested from the ACF expression vector with SalI and NotI. 352eggs were transferred into 13 forster mothers,4 of which were pregnant and 22 pups were obtained, among which 3 pups (2♀, 1♂) were transgenic mice,the rate of integration was 13.64%. But no pups of No.8(♂) were transgenic mice.The result of ELISA assay indicated that the recombinant human lactoferrin (rhLF) were secreted in the milk of BCL,PMH and ACF transgenic mice. The average level of the rhLF in the milk of BCL,PMH and ACF transgenic mice were respectively about 5.874,3.556 and 3.251mg/mL.Western blot result showed that the recombinant protein expressed in the milk of BCL,PMH and ACF transgenic mice had the same molecular weight as the native protein(about 78 kD).In this research,We construct BCL,PMH and ACF transgenic mammary gland expression vector. The modified hLF vector was highly expressed in mammary gland of resulting transgenic mice. The expressive specificity,stability,profile and bio-activity of this hLF were analyzed. All of them are the basis to produce transgenic goat mammary gland bioreactors in the next step.