Chicken Ovalbumin Gene Regulatory Sequences, Regulation Of Human ¦Á1-antitrypsin Gene Expression In Cho Cells

α1-Antitrypsin (α1AT) is an important protease inhibitor in mammalian blood, its major physiological function appears to be the inhibition of neutrophil elastase , a potent protease that hydrolyzes structural proteins. It also inhibits many other serine proteases, including trypsin, chymotrypsin, collagenase, thrombin, kallikrein, and plasmin. α1AT is a glycoprotein synthesized in the liver and consists of a single polypeptic chain 394 aa ,with three carbohydrate side chaining, giving an overall molecular weight of 52,000 .The α1AT gene has a size of 12.2Kb on chromosome 14q32.1,consists of 4 exons and 6 introns, and α1AT mRNA has a size of 1434 bp including 5' non-coding region of 49bp and coding region of 1254bp, an end codon, 3'non-coding region of 79bp and poly (A). Clinical trials have shown thatα1-antitrypsin deficiency can be treated by replacement therapy ,researchers have used the techniques of genetic engineering to produce humanα1-AT in a microorganism. To direct the regulatory elements of chicken ovalbumin gene and make a preliminary ananlysis of 5'upstream regions , the transcription initiation site of ovalbumin gene was confirmed by 5'RACE method, at the same time the regulatory elements of chicken ovalbumin gene was determined by sequence analysis .To investigate the ability of regulatory elements to direct the exogenous gene expression ,1.5Kb fragment and 2.9Kb fragment were amplicated by PCR method , two fragments were subcloned to manmalian expression vector pGFP-N2 by recombinant DNA technology, the CMV promoter was cut off from pGFP-N2 ,so two expression vectors were constructed , one is the P2. 9koval-GFP including promoter, first exon, first intron of chicken ovalbumin gene , the other is the P1. 5koval-GFP including first intron of chicken ovalbumin gene. Restriction enzyme digestion and DNA sequence analysis revealed that 5'upstream regions of ovalbumin gene were not only identical to those of the published chicken ovalbumin gene, but also were contained in the recombinant vector. They were transfected into the CHO cell and the primary cell cultures of chicken oviduct by Lipofectin, they were used for fluorescence detection . GFP protein existed in GFP transfected the CHO cell and the primary cell cultures of chicken oviduct. It is demonstrated that GFP reporter gene under the direction of chick ovalbumin gene promoter could be expressed in the CHO cell and in the primary cell cultures of chicken oviduct. humanα1AT cDNA was inserted into the second exon of chick ovalbumin gene, the P5'UTR-AT-3'UTR expression vector contained 5'UTR,exon 1 and intron 1of the chick ovalbumin gene with 2.9Kb and full-length of humanα1AT cDNA as well as 3'UTR of the chick ovalbumin gene, so that the chick ovalbumin code regions were replaced with humanα1AT cDNA .Restriction enzyme digestion DNA sequence analysis and PCR implication revealed that the encoding sequence of signal peptide were not only identical to those of the published humanα1AT cDNA sequence, but also were contained in the recombinant vector. The recombinant plasmid was transfected into CHO cell with liposome transfection reagent for transient expression .The recombinant vector could express human in CHO cell through PCR, ELISA and Western blot analysis. The results demonstrated that chick ovalbumin gene regulatory elements can direct the humanα1AT cDNA successful expression in the CHO cell, it lays great foundation for the research on which expressed in chick oviduct as bioreactor .