Cattle αS1casein- People LYZ Heterozygous Loci Construction And Identification Of Expression In The Mouse Mammary Gland

Human lysozyme (human lysozyme, hLYZ), also known as muramidase, which can hydrolyze the mucopolysaccharidesβ(1→4) glycosidic bond of bacterial cell wall,and is a very important non-specific defense factor among human body taking part in kinds of human immunological reactions.Human lysozyme widely present in the human body, cells and tissues, and are up to750mg/L in human milk, hLYZ play an vital role in the prevention of intestinal diseases and invigorate baby’s health effectively.However, the resources are limited,and the cost is high.In this study, we use transgenic animals producing the recombinant protein, and efficient expression vectors,give us a chance to obtain high-quality and low-cost hLYZ.We choose the bovine aSl-casein to construct the bovine a S1-casein-hLYZ hybrid locus.First of all, bovine α S1-casein accounts for39%-46%of the total amount of protein in the milk and its concentration is the highest.Secondly, U.S. CenPharning company nurtured the world’s first transgenic cattle containing human lactoferrin.The company use the bovine α S1-casein gene5’end,and the3’end to develop the expression vector, and using which to construct the expression cassette containing human lactoferrin cDNA.Transgenic cow produce milk10tons per year.This successful case shows that the bovine aS1-casein genes mammary gland expression vector has a good practical value.So the bovine aSl-casein gene locus was chosen to be regulatory elements to direct the expression of hLYZ genomic coding sequencee, and a aSl-casein-hLYZ hybrid locus was constructed. Tne bovine αS1casein-hLYZ hybrid locus was constructed.In order to get this hybrid locus,we use the successive three-step "Gap-repair"method invented by our lab to maximize the natuaral.we constructed a gap-repair vector based on pBR322vector backbone by inserting six joint homologous arms. Then using "Gap-repair "method mediated by Red recombination system of λ-prophage in Escherichia coli.To achieve the purpose of bovine aSl-casein gene locus of bovine α S1casein sequence from the initiation codon to the termination codon precisely replaced the hLYZ genome sequence, successfully obtained a bovine aSl-casein-hLYZ hybrid locus.The first step,homologous arms T5and T6were dissociated by restriction enzyme digestion to catch the9Kb3’flanking region of bovine aSl casein gene locus with bovine aSl casein BAC as template;The second step,the first product as a carrier,homologous arms T3and T4were dissociated by restriction enzyme digestion to catch the4.3Kb3’flanking region of hLYZ gene locus with hLYZ BAC as template;The third step,the second product as a carrier,homologous arms T1and T2were dissociated by restriction enzyme digestion to catch the8.6Kb5’flanking region of bovine aSl-casein gene locus with bovine aSl casein BAC as template.After PCR amplification, restriction endonuclease digestion and sequencing verified,we get about the50Kb αS1casein-hLYA hybrid gene locus.Using the constructed heterozygous locus both ends with Pvu I sites will carrier part removed,the microinjection of the purified carrier integrated into the fertilized eggs of mice prokaryotic, injection and then have a good fertilized eggs transplanted into false pregnancy female mice to produce transgenic mice.Analysis by PCR and southern blot, three of them are transgenic. Then we use SDS-PAGE and western-blot analyse of the milk of transgenic mice expressed human lysozyme in the milk of transgenic mice,and recombinant human lysozyme specificity.last we based on ELISA method to measured expression amount and calculate the single-copy locus expression, the highest level attained as to6.06g/L.We build bovine aSlcasein-hLYZ heterozygous locus can effectively in a transgenic mouse model expressing recombinant human lysozyme,And our motheld provide a new way for the construction of such as cattle and sheep large mammary glands expression vectors.