Study On Genetic Characterization Of Transgenic Mice And Antibacterial Activity Of RhLF In The Milk

This research focuses on the expression of hLF gene in transgenic mice, and establish a high expression transgenic mice by filterring homozygote.At the same time,the experiment is intended to detect the antibacterial activity of rhLF in transgenic mice,which is the basis to produce transgenic goat or cow of hLF gene.The bovine alphal-casein and goat beta-casein regulate sequence and hLF cDNA gene were used to construct AP and PCL25 expression vector. By microinjection , the original generation(G0)transgenic mice of PCL25 and AP vector were obtained ,2♀and 1♀. G0 transgenic mice are breed with normal mice. All transgenic mice were tested with Gene integration by PCR .Then three transgenic mice which were selected from the first generation(F1)at random are breed with normal mice. The F2,F3,F4 transgenic mice were obtained by the same way. Through hybridization and backcross, the homozygous transgenic mice were obtained by test cross assay and PCR.After collecting the milk of mice, mice whey is acquired by wiping off fat and casein. Using hLF as a standard reference, the rhLF quantity were analysed by ELISA assay and Western-blot assay .Through dialyse and gel filtration chromatography, the rhLF were obtained. We separately carry on bacteriostasis experiment to coliform and salmonella.The result showed that the contract integration rate was about 57.14%(12/21)in the first generation(F1)of AP expression vector. The gene integeration rate was about 50%(10/20) in the second generation(F2).In the third generation(F3),the gene integeration rate was 50%(17/34).In the fourth generation(F4),the gene integeration rate was 53.33%(24/45). The contract integration rate was about 53.85%(14/26)in the first generation(F1)of PCL25 expression vector. The gene integeration rate was about 39.58%(19/48)in the second generation(F2).In the third generation(F3),the gene integeration rate was 31.11%(14/45).In the fourth generation(F4),the gene integeration rate was 38%(19/50).The rhLF in the milk of every transgenic mice were tested with ELISA assay. The results showed that the expression of rhLF was inconsistent not only between the generations, but also between different individuals in the same generation. They have no difference(P>0.05).Through hybridization or backcross,we obtained 2 homozygous(1♀1♂)of AP transgenic mice and1 homozygous(1♀)of PCL25 transgenic mice.The level of rhLF expression was about 5.65g?L-1 and 5.98g?L-1 .The result showed that the expression of rhLF in homozygous transgenic mice was higher than heterozygote transgenic mice.The results of ELISA-assay and Western-Blot ,showed that rhLF of AP and PCL25 transgenic mice all have the standard hLF size(aboat 78kDa). The rhLF density is 7.52g?L-1 and 8.04g?L-1.The bacteriostasis experiment showed that both have the bacteriostasis function. Along with rhLF density's increasing, the antibacterial activity increases.The research indicated that AP and PCL25 expression vector not only can express target gene in the original generation transgenic mice and its offspring,but also can be steadily inherited to the posterity of transgenic mice.The rhLF has bacteriostasis function. All of them are the basis to produce transgenic goat mammary gland bioreactors in the next step.