暂态表达重组蛋白鸡输卵管生物反应器的优化
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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
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
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
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
引文
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3. Charles R Allerson, Alan Martinez, Emine Yikilmaz, et al. A high-capacity RNA affinity col-umn for the purification of human IRP1 and IRP2 overexpressed in Pichia pastoris. RNA, 2003, 9: 364-374.
4. Upshall A, Kurnar A A, Bailey M C, et al. Bio /Technology, 1987, 5: 1301-1304.
5. Lynn D E. Comparative susceptibilities of insect cell lines to infection by the occlusion-body derived phenotype of baculoviruses. J Inverteb Pathol, 2003, 83(3): 215-222.
6. Lucas B K,Giere L M, DeMarco R A, et al. High-level production of recombinant proteins in CHO cells using a dicistronic DHFR intron expression vector. Nucleic Acids Res, 1996, 24(9): 1774.
7. 方洛云,邹晓庭,许梓荣. 转基因技术及转基因动物研究进展. 饲料研究,2001,12:9-12.
8. 钟军,易自力,蒋建雄等. 生物反应器在动植物基因工程中的应用. 河南科技大学学报(农学版),2003,23(4):21-24.
9. 陈学进,曾申明,李和平. 动物生物反应器. 国外畜牧科技,1998,25(1):39-43.
10. 曾溢滔. 转基因动物与医药产业,上海教育出版社,1999.
11. 常惠芸,谢庆阁. 转基因动物生物反应器生产药用蛋白的研究进展. 中国兽医科技,1998,28(5):40-43.
12. Houdebine, L-M. Transgenic animal bioreactors. Transgenic Res. 2000,9: 305-320.
13. HAN Yu-gang et al. Advance of Transgenic Animal Bioreactors. Animal Science Abroad,2002,29(1):30-33.
14. 尹春光,杜立新,岳永生. 转基因动物的生物反应器. 32-35.
15. 刘森,梁国栋. 利用动物乳腺生物反应器生产药用蛋白. 生物工程学报,2000,16(4): 421-424.
16. Wright G. High level expression of active human a-1-antitrypsin in the milk of transgenic sheep. Biotechnology,1991,9: 830-834.
17. Velander W H, Johnson J L, Page R L, et al. High-level expression of a heterologous protein in the milk of transgenic swine using the cDNA encoding human protein C. Proc Natl Acad Sci USA,1992,89(24) : 12003 - 12007.
18. Swanson ME, Martin MJ, O’Donnell JK et al. Production of functional human hemoglobin in transgenic swine. Bio/ Technology, 1992 ,10 :557.
19. 郑怀玉. 用膀胱作生物反应器生产人生长激素实验. 上海实验动物科学,1999 ,19 (1):66-67.
20. 尹春光,杜立新. 转基因鸡的研究策略. 生物学通报,2004,39(3):13-15.
21. 赵英会,杜立新,张念华. 用逆转录病毒法制作鸡输卵管生物反应器的研究. 动物科学与动物医学,2001,18(3):19-21.
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