The Construction Of MWAP-hLTF Hybrid Locus Vector And Its' High-level Expression In Milk Of Transgenic Mice

Lactoferrin is an iron-binding glycoprotein that mainly exists in human colostrums. It not only has broad spectrum of antimicrobial properties, but also has the ability to improve antiviral activity, anticancer activity, and immunoregulatory activity of the body to prevent and cure infectious disease. But it is very expensive to purify the hLTF from the human colostrums, which limited by supply. So the mammary gland bioreactor appears to be capable of expressing Lactoferrin with high efficiency and low cost, and carrying out complex posttranslational modifications. But only very few recombinant protein have been obtained at levels sufficient for an industrial exploitation, one main obstacle is site-effect. This is clearly due to the fact that the expression vectors are not constructed in an appropriate manner. One strategy is using long genomic DNA fragments and whole up&down stream regulatory sequence of lactoprotein gene contain in BACs to construct hybrid locus expression vectors.To generate a mammary-gland expression vector that the transcription of human lactoferrin(hLTF) genomic sequence is directed by the up&down stream regulatory sequence of murine whey acidic protein(mWAP) gene locus, we constructed a gap-repair vector based on pBR322 vector backbone by inserting six joint homologous arms. Then using'Gap-repair'method mediated by Red recombination system ofλ-prophage in Escherichia coli, the 8Kb 3' flanking region of mWAP gene , the 29Kb hLTF genomic sequence from the ATG code to the TAA code, and the 12Kb 5' flanking region of mWAP gene was retrieved successively from the Bacterial artificial chromosome (BAC) which harbors the mWAP gene and the BAC which harbors the hLTF gene respectively. and automatically combined together without any gap. Finally, a 49Kb mWAP-hLTF hybrid locus was constructed, in which the hLTF genomic sequence was flanked by the 5'&3' flanking region of mWAP gene locus. The result was confirmed by PCR,restriction enzymes digestion and sequencing.Then we use the mWAP-hLTF hybrid locus as transgenic vector to produce transgenic mice. The DNA was microinjected into mouse zygote and transferred into recipients, at last 32 pups born. Analysis by PCR and southern blot, five of them are transgenic. SDG-PAGE and Western Blot analysis results show that all five transgenic mice line high-level expressed recombinant hLTF in the milk ,at an average level of 18.1mg/ml, vary from 16.0-19.6mg/ml.Take together these data stress the usefulness of the mWAP-hLTF 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.