Production Of Efficiently Human Serum Albumin-expressing Transgenic Cattle

Human serum albumin (HSA), the most abundant protein in plasma, is a monomericmulti-domain macromolecule, and is also a non-glycosylated protein. HSA is widely used inmedical treatment and biopharmacy. HSA for clinical applications is mainly derived fromhuman plasma. Plasma-derived HSA (pdHSA) sources are limited, expensive, and have therisk of infecting with pathogenic microorganisms (e.g. viruses and prions). So, recombinanthuman serum albumin (rHSA) is the ideal substitute of pdHSA. Non-glycosylated feature ofHSA makes it can be efficient expressed and correct folded in many hosts. Currently, rHSAhas been produced in E. coli, yeast, and plant seeds (e.g. rice) expression systems.Nevertheless, for the production of natural active eukaryotic proteins, animal mammary glandbioreactor has more advantages. The purpose of this study is to optimize the vectorconstruction, screen more efficient HSA mammary-specific expression systems, and produceHSA transgenic cattle. We constructed HSA bovine mammary specific expression vector byusing phiC31integrase enzyme systems, cHS4insulators and other regulatory elements,screened out efficiently HSA-expressing vector by verification experiments in vitro and invivo. Then we transfected bovine fetal fibroblast cells with the vector, used the transfecedclonal cells as donor cells and produced HSA transgenic cattle by somatic cell nucleartransfer.1. Using PCR and RT-PCR to amplify HSA genomic and cDNA sequences, through HSAfirst exon of BstE Ⅱ and7th exon of Nco Ⅰ sites to integrate these two sequences fragmentsto construct HSA minigene including the first14exons, introns and before6, Section15exons. The results of comparative analysis showed that the homology between HSA minigeneand template DNA sequence is99.5%, the homology of amino acid sequences is99.8%, andthat the secondary and tertiary structure are not significant different from template. Weconstructed a HSA gene and EGFP fusion expression vector pN-H16, and transfected thisvector into293cells. Observing EGFP expression under a fluorescence microscope anddetecting HSA mRNA expression by RT-PCR to confirm the HSA gene has been correctlytranscripted.2. Sequence analysis of bovine β-casein promoter and cHS4insulator core genesequence showed their homology compared with template sequence was higher than99%. The homologies of LoxP sequence, attB sequence and BGHpolyA with their templates were100%. Transfected plasmid p-C (containing the β-casein promoter-EGFP expression cassette)into HC11cells and293cells to detect the tissue-specific activity of bovine β-casein promoterby observation of EGFP expression; Comparing the number of G418-cell clones fromtransfected pC1-E (insulator inserted between neo gene expression promoter and enhancer)and transfected pC1-EP (there is no insulator between neo gene expression promoter andenhancer) cells to analyze insulator activity; Detecting the integration site of pC1-A(including attB sequence) in fibroblasts to test whether attB sequences is able to be mediatedby phiC31integrase; Detecting whether loxP sequences are able to be Cre-mediated to deletegene flanked by them by PCR amplification of neo gene in G418neo gene cloned cellstreated by Cre enzyme; Comparing the efficiency of the293cells transfected by plasmidscontaining or uncontaining by flow cytometry to test whether BGHpolyA has the activity ofenhancing gene expression. Functional verification tests showed that all regulatory elementshave their corresponding functions.3. Using the above-mentioned regulatory elements, we constructed four tissue-specificHSA expression vectors pACH (including attB sequences), pIACH (including attB sequenceand three forward insulators), pIACH (-)(containing attB sequence and a pair of reverseinsulators) and pIACH (+)(containing attB and a pair of forward insulators), used them withintegrase expression vector to co-transfect bovine mammary epithelial cells (BMECs) andscreened positive transgenic cells. As the same time, we also transfected the cells withoutintegrase as a nagetive control. We detected the effects of phiC31and insulator on transfectionefficiency and gene expression by reverse nested PCR, RT-PCR and western-blotting. Theresults show the transfection efficiency of group transfected by pACH and pCMVint wassignificantly higher than other groups; HSA expression level of group transfected by pIACH(-) and pCMVint was obviously higher than other groups. The results showed that theintegrase system can improve the efficiency of gene transfection efficiency and expressionlevel of foreign genes, and the combination of phiC31integrase and insulator can furtherenhance the level of HSA expression.4. We transfected mammary glands of lactating mice by electroporation, detected theactivity of β-casine promoter-HSA expression cassette and effects of phiC31integrase onHSA expression level in vivo by RT-PCR, western-blotting and ELISA. The results showedthat mammary-specific HSA expression vector was capable of expressing HSA in mousemammary glands and phiC31integrase significantly improved the level of HSA expressionthan the control group without integase in vivo.5. We used plasmids pIACH (-) and pCMVint co-transfected bovine fetal fibroblasts, screened positive clones by G418, identified integration of exgious gene by PCR, tested theintegration sites of transgenic cells by neverse PCR, and chose well-integrated cells as nucleardonor cells for somatic cell nuclear transfer. Then we had2transgenic cattle by somatic cellnuclear transfer technology and confirmed the cattle were transfected with HSA gene by PCRand southern-blotting. The results of detection of integration site, which were performed byreal-time quantity PCR and nested-reverse PCR, showed the copy number of inserted-genewas one and the integration site was BF19.