【作者】 房浩霞；

【导师】 孙怀昌；

【作者基本信息】 扬州大学 ， 动物学， 2006， 硕士

【摘要】 随着重组DNA技术的飞速发展,已经出现了多种生产重组蛋白的表达系统,其中以转基因动物生物反应器,尤其是转基因哺乳动物的乳腺生物反应器和转基因家禽的输卵管生物反应器的优点最为突出,能在不损伤机体正常生理功能的条件下源源不断地获得表达产品。家禽具有繁殖周期短、生产性能高、研究成本较低等优点,其输卵管是生产蛋白质的天然“发酵罐”,更加适合作为生产重组蛋白的生物反应器。但由于家禽特殊的生殖生理特点,其转基因技术一直落后于其它动物。虽然近几年来相关技术有所突破,但家禽转基因研究仍有许多理论和技术难题有待突破,距离产业化开发仍有很远的路要走。为此,寻求禽类的转基因技术的突破和开辟新的研究方法已成为当前研究工作的重点。本课题组用自行克隆的鸡卵清蛋白(OV)基因表达调控序列构建成鸡输卵管定位表达载体,以人溶菌酶和组织激肽释放酶(hK1)基因为目的基因的鸡输卵管暂态表达试验结果表明,通过翅静脉注射一定剂量的重组载体后,能表达一定水平的重组酶并分泌到试验鸡的蛋清中,具有一定的开发利用价值。本研究在前期研究的基础上,以hK1基因为目的基因,通过对先前构建的鸡输卵管特异表达载体进行优化改造,旨在获得结构更为合理、表达水平更高、维持时间更长的表达载体。在先前构建的pOV3K载体的基础上,分别通过酶切消化或PCR扩增等方法,将其中3.0 kb的OV基因5′-调控序列的长度分步缩减,获得三个新的重组载体分别命名为pOV4K、pOV5K和pOV6K。将此重组载体与25 kDa聚乙烯亚胺(polyethylenimine,PEI)混合,经翅静脉注射产蛋鸡,用标准方法检测蛋清中的重组酶活性,结果表明,含1.1 kb OV基因5′-调控序列的pOV6K表达水平和表达时间均不如含3.0 kb OV基因5′-调控序列的pOV3K,含2.1 kb OV基因5′-调控序列的pOV4K和含1.7 kb OV基因5′-调控序列的pOV5K与含3.0 kb OV基因5′-调控序列的pOV3K表达水平相当,说明1.7 kb OV基因5′-调控序列足以指导外源基因更多还原

【Abstract】 Many gene expression systems have been established for production of large quantities of biologically active proteins for clinical and scientific research use, trans-genic animal bioreactors in particular. Among animal bioreactors, transgenic animal mammary gland and avian oviduct are expected to be excellent systems for production of recombinant proteins. Transgenic avian oviduct bioreactors have advantages of high expression levels and low cost. However, research on avian transgenesis is greatly lagged behind other animals due to the complexity of avian physiology.To develop chicken bioreactors for transient expression of recombinant proteins, oviduct-specific expression vectors have been constructed using the 3.0 kb 5′- and 3′- regulatory regions of chicken ovalbumin (ov) gene, and have been shown to express relatively high levels of recombinant enzymes using the genes of interest of human lysozyme and human tissue kallikrein (hK1). In this study, we optimized the structure of the previously constructed vector pOV3K by restriction digestion or PCR amplifica-tion to shorten the 5′-regulatory region to 2.1, 1.7 and 1.1 kb, and the modified vectors were named pOV4K, pOV5K and pOV6K, respectively. The new vectors and the con-trol vector pOV3K were injected into laying hens via wing vein route following mixing with 25 kDa polyethyleneimine (PEI). The eggs were collected and the enzymatic ac-tivity in egg white was detected. Among the four vectors tested, pOV5K with the 1.7 kb 5′-regulatory region showed an expression level similar to that of pOV3K. To further improve the transfection efficiency and expression levels, the expression cassette con-sisting of the 1.7 kb 5′- regulatory region of ov gene, hK1 cDNA and bovine growth hormone gene poly (A) was cut from the pOV5K vector and subcloned into the pITRLRep vector containing the inversed terminal region (ITR) and Rep gene of avian更多还原