"mwap - Hlyz" Hybrid Milk Protein Loci Construct And Its Expression In The Transgenic Mice Mammary Gland

Lysozyme is an important defensive factor presented in the body liquid and tissues of human and plays a significant role in the non-specific immunity; it will be widely used in many fields. But it is very expensive to purify the hLYZ from the human colostrums, which limited by supply. Our study tried to use bioreactors to express recombinant hLYZ. To generate a mWAP-hLYZ hybrid locus that the transcription of human lysozyme (hLYZ) genomic sequence is directed by the up&down stream regulatory sequence of murine whey acidic protein (mWAP) gene locus, we describe here a successive three-step’Gap-repair’method. First, 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, in the first step, the7.2kb3’flanking region of the mWAP gene was subcloned from the Bacterial artificial chromosome which harbors the mWAP gene locus(mWAP BAC) into the gap-repair vector; in the second step, the4.3kb hLYZ genomic sequence from translation initiation site(ATG) to termination site(TAA) was subcloned from the hLYZ BAC; in the third step, the8.6kb5’flanking region of the mWAP gene was subcloned from the mWAP BAC. Finally, all these three DNA fragments were automatically combined together without any gap in the gap-repair vector, and a22kb "mWAP-hLYZ" hybrid locus was constructed, in which the3-kb mWAP genomic coding sequence in the mWAP gene locus was substituted with that of5kb hLYZ exactly from ATG to TAA.The result was confirmed by PCR、restriction enzyme digestion and sequencing. Our method provides a new way for the construction of large mammary-gland expression vector, and can also provide an alternative way to hLYZ.Then we use the mWAP-hLYZ hybrid locus as transgenic vector to produce transgenic mice. The DNA was microinjected into mouse zygote and transferred into recipients, at last27pups born. Analysis by PCR and southern blot, seven of them are transgenic. SDG-PAGE and Western Blot analysis results show that all seven transgenic mice line high-level expressed recombinant hLYZ in the milk. Take together these data stress the usefulness of the mWAP-hLYZ hybrid locus expression vector for a position-independent high level expression. And our motheld provide a new way for the construction of large mammary-glands expression vectors.