Butyrylcholinesterase(BCHE)is a natural bioscavenger that protects humans against organophosphate toxicity.Due to the limited yield of human BCHE(hBCHE)when purifying from human plasma,it is necessary to find an alter...Butyrylcholinesterase(BCHE)is a natural bioscavenger that protects humans against organophosphate toxicity.Due to the limited yield of human BCHE(hBCHE)when purifying from human plasma,it is necessary to find an alternative method to produce this protein.One potential method is to produce transgenic livestock that make modified milk containing high concentration of hBCHE.In this study,we cloned the hBCHEgene into a human lactoferrin(hLF)bacterial artificial chromosome(BAC)construct to make a hLFhBCHE BAC construct.Subsequently,we injected the BAC construct into pronuclei of mouse fertilized embryos and generated transgenic mice.Expression analysis showed that recombinant hBCHE(rhBCHE)was expressed efficiently in the mammary gland of the transgenic mice and the concentration of rhBCHE in the milk of individual mice ranged from 7612 to 15928 mg·L^(–1).Protein function tests showed that rhBCHE has the same enzymatic activity as the native hBCHE.Our results pave the way for making transgenic livestock to produce large quantities of rhBCHE.展开更多
The mammary gland provides a novel method for producing recombinant proteins in milk of transgenic animals. A key component in the technology is the construction of an efficient milk expression vector. Here,we establi...The mammary gland provides a novel method for producing recombinant proteins in milk of transgenic animals. A key component in the technology is the construction of an efficient milk expression vector. Here,we established a simple method to construct a milk expression vector, by a combination of homologous recombination and digestion-ligation. Our methodology is expected to have the advantages of both plasmid and bacterial artificial chromosome(BAC) vectors. The BAC of mouse whey acidic protein gene(mWAP) was modified twice by homologous recombination to produce a universal expression vector, and the human lysozyme gene(hLZ) was then inserted into the vector by a digestionligation method. The final vector containing the 8.5 kb mWAP 5′ promoter, 4.8 kb h LZ genomic DNA, and 8.0 kb m WAP 3′ genomic DNA was microinjected into pronuclei of fertilized mouse embryos, to successfully generate two transgenic mouse lines that expressed recombinant human lysozyme(rhLZ) in milk. The highest expression level of rhLZ was 0.45 g$L–1, and rhLZ exhibited the same antibacterial activity as native h LZ. Our results have provided a simple approach to construct a universal milk expression vector, and demonstrated that the resulting vector regulates the expression of hLZ in milk.展开更多
文摘Butyrylcholinesterase(BCHE)is a natural bioscavenger that protects humans against organophosphate toxicity.Due to the limited yield of human BCHE(hBCHE)when purifying from human plasma,it is necessary to find an alternative method to produce this protein.One potential method is to produce transgenic livestock that make modified milk containing high concentration of hBCHE.In this study,we cloned the hBCHEgene into a human lactoferrin(hLF)bacterial artificial chromosome(BAC)construct to make a hLFhBCHE BAC construct.Subsequently,we injected the BAC construct into pronuclei of mouse fertilized embryos and generated transgenic mice.Expression analysis showed that recombinant hBCHE(rhBCHE)was expressed efficiently in the mammary gland of the transgenic mice and the concentration of rhBCHE in the milk of individual mice ranged from 7612 to 15928 mg·L^(–1).Protein function tests showed that rhBCHE has the same enzymatic activity as the native hBCHE.Our results pave the way for making transgenic livestock to produce large quantities of rhBCHE.
基金supported by High-Level Talent Project of Foshan University (GG040965)Science and Technology Project of Foshan City (GS54207)Science Foundation of Jiangsu Province (BK20160206)
文摘The mammary gland provides a novel method for producing recombinant proteins in milk of transgenic animals. A key component in the technology is the construction of an efficient milk expression vector. Here,we established a simple method to construct a milk expression vector, by a combination of homologous recombination and digestion-ligation. Our methodology is expected to have the advantages of both plasmid and bacterial artificial chromosome(BAC) vectors. The BAC of mouse whey acidic protein gene(mWAP) was modified twice by homologous recombination to produce a universal expression vector, and the human lysozyme gene(hLZ) was then inserted into the vector by a digestionligation method. The final vector containing the 8.5 kb mWAP 5′ promoter, 4.8 kb h LZ genomic DNA, and 8.0 kb m WAP 3′ genomic DNA was microinjected into pronuclei of fertilized mouse embryos, to successfully generate two transgenic mouse lines that expressed recombinant human lysozyme(rhLZ) in milk. The highest expression level of rhLZ was 0.45 g$L–1, and rhLZ exhibited the same antibacterial activity as native h LZ. Our results have provided a simple approach to construct a universal milk expression vector, and demonstrated that the resulting vector regulates the expression of hLZ in milk.
