The Optimization Study Of Chicken Oviduct Specific Promoters

The study of using transgenic animals producing pharmaceutical proteins has made great achievements, broad application prospects. Compared with mammalian mammary gland bioreactor, using poultry fallopian tubes as a bioreactor has more superiority that individual small, high egg production, and generation interval smaller, easier to mass production, relatively high protein content in eggs and other advantages. But based on their reproductive physiology characteristics of poultry, chicken oviduct bioreactor research is lagging behind, because lacking of access to get specific and efficient means of expression and regulation. In recent years, transgenic poultry preparation research institutions at home and abroad, has achieved success. How to make transgenic poultry which efficiently product recombinant proteins, has become a key to decide whether poultry bioreactor can enter application fields. In this study, based on the results the laboratory had, chicken oviduct ovalbumin gene upstream regulatory sequence and the first intron were selected as the research object, and its expression vector was constructed. Some aspects of this research are as following:1. The construction of a series of promoter-reporter vectoranalysis on upstream regulation sequence based on the result lab has had,-921～38region has high expression activity, but lost organization special specific, upstream2.1kb has high of transPCRiption activity and organization special specific,3.1kb region has more tight organization special specific, longer than3.1kb expression activity significantly reduced. Based on an analysis of the results,-922Kb--2073Kb and-2801Kb--3100Kb region is devided into12parts of different lengths, then inserted into the plasmid pGL4-921. Sequencing results show that12different expression vectors was constructed successfully.2. The construction of a series of mini intron-reporter vectorThe ovalbumin gene-1338-+1640fragment (A:Intron-contain) and a-1338-38fragment (B:Intron-less) were successfully amplified by the method of PCR, then insert the luciferase report vector, named pGL41640, pGL438; design in a carrier on the basis of removing the first intron, only keep the donor site, and receptor sites of luciferase reporter vector (C:Intron-delation), named pGL4synthesis; and then Insert mini intron (approximately300bp) fragments to C vePCRor, The eukaryotic expression vectors of pGL4-synthesis+57, pGL4-synthesis+368, pGL4-mini intron-3, pGL4-synthesis+988, pGL4-synthesis+1298were constructed by using pGL4-synthesis plasmid as backbone. Sequencing showed that these carriers build success.3. The establishment of primary chicken oviduct epithelial cell culture system.We separated chicken oviduct epithelial cells from laying hens, exploration of the technical aspects,such as Organizational separation, trypsin digestion, cell purification and culture before transfection was need. The successful separation of tubal primary epithelial cells provide the test platform to detect tissue-specific expression of ovalbumin gene promoter4. The activity and specificity dectection of expression of ovalbumin promoter fragmentIn order to test the gene expression activity and specificity under control of different ovalbumin promoter fragments, DF-1cells were transfected with the twenty promoter-reporter plasmids and fluorescent enzyme activity was detected,to find out the plasmid pGL4-UP-1412of strongest activity and intron-recombind plasmid pGL4-mini-intron-3; at the same time infers some regions contain enhancer.The fallopian tube epithelial cells was transfected with twenty promoter-reporter plasmids, the fluorescent enzyme activity test was no significant difference with the control results, pending further experiments.