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Construction of Chicken Oviduct Specifical Expression Vector to Express Human Erythropoietin Gene

Author: YuXiaoLiang
Tutor: XieDaPing；YanHaiFeng
School: Hunan Agricultural University
Course: Biochemistry and Molecular Biology
Keywords: Ovalbumin Erythropoietin Coexpression Bioreactor Transfection
CLC: Q78
Type: Master's thesis
Year: 2009
Downloads: 40
Quote: 0
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Abstract


Mammalian bioreactor research has made great achievements, at present, scientists have been established cattle, sheep and other bioreactor to produce exogenous protein. Chicken oviduct bioreactor has become the mammalian mammary gland bioreactor development prospects of most animal bioreactors, if there is a breakthrough, it will be able to take advantage of the many valuable and inexpensive protein. Ovalbumin is the most abundant protein in egg white, and its 5 'end with a tissue-specific regulatory elements, and by hormones induced by almost all researchers utilized. Wherein the upstream vector prerequisite to achieve at home exogenous genes in the chicken oviduct tissue-specific expression of the promoter must be organized characteristics involved. In this study, the above principle, build chicken ovalbumin 5 'flanking region regulate human erythropoietin and green fluorescent protein co-expression vector with a green fluorescent protein gene as a reporter gene directs gene expression. In this study, mainly from the following aspects were studied. A chicken oviduct 5 'flanking sequence and the experimental hEPO cloning genomic DNA from the egg in the fallopian tube amplified approximately 1.3kb ovalbumin 5' regulatory region (OV), and from phEBS-HB amplified plasmid human erythropoietin (hEPO) genomic DNA, the two were subcloned into pGEM-T Easy vector, named pOV-T and phEPO-T, correct by restriction analysis and sequencing. 2 intermediate vector pEGFP-hEPO and specificity of the expression vector pOV-GFP-hEPO Construction phEPO-T cut from the hEPO fragment was subcloned into vector pEGFP-C1 cloning sites, named pEGFP-hEPO, by restriction endonuclease is correct. Finally pOV-T OV excised promoter fragment was subcloned into by Ase I and Vsp I double digestion of the incision, replace the CMV promoter, the restriction endonuclease properly constructed successfully chicken ovalbumin 5 'flanking region regulate human erythropoietin coexpression vector pOV-GFP-hEPO. 3 three kinds of expression vectors in primary chicken oviduct epithelial cells in primary chicken oviduct epithelial cells for the detection platform, using liposome transfection method, respectively, the two recombinant plasmids, pEGFP-C1 plasmid (positive control) and negative control transfected primary chicken oviduct epithelial cells and induce growth with hormones. An inverted microscope with a fluorescent green fluorescent protein. The results show that co-expression vector pOV-GFP-hEPO chicken oviduct epithelial cells can be positioned in the expression of the other two carriers in primary chicken oviduct epithelial cells can also be expressed but the expression different results. 48h of transfection, all three vectors expressing GFP-positive cells have reached the maximum number; using Image-Pro Plus 6.0 Analysis Software Analysis fluorescence photographs, found that the fluorescence intensity of the positive control highest expression, pEGFP-hEPO followed, pOV- GFP-hEPO Min. In short, pOV-GFP-hEPO successfully constructed, for the preparation of production of human erythropoietin chicken oviduct bioreactor foundation.

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