摘要
为了建立人凝血Ⅸ因子乳腺生物反应器，以MCK增强子，β－actin启动于控制hFIX小基因（minigene）表达顺序，反向插入G1Na框架，构建复制缺陷反转录病毒载体，并制备重组反转录病毒颗粒；以CMV启动子，hFIXcDNA和腺病毒框架构建腺病毒载体pAdCMVhFIX，与质粒pJM17共转染包装细胞制备合hFIX基因的重组腺病毒颗粒．两种病毒颗粒分别通过直接转染活体奶山羊乳腺组织，建立了hFIX蛋白的乳腺表达系统和快速检测系统．另外，利用小鼠MAR（matrixattachmentregion）元件，牛β－Casein基因，人凝血Ⅸ因子小基因和cDNA构建了乳腺组织特异性表达载体．以Stearylamine（SA）阳离子脂质体介导、尾静脉注射的方法转染哺乳期雌性小鼠的活体组织，建立Ⅸ因子表达的快速检测系统，并研究了β－casein基因，hFIX基因intron1对hFIX基因在乳腺组织中表达调控的作用．在此基础上将筛选得到的pMCIXm表达载体通过显微注射技术注人小鼠和奶山羊受精卵细胞，制备6只转基因小鼠和5只转基因奶山羊．其中在2只转基因小鼠和1头奶山羊乳汁中测得hFIX蛋白的表达并具有凝血活性．
For the purpose of establishing mammary gland bioreator for human clotting factor Ⅸ (hFIX), the human clotting factor Ⅸ (hFIX) minigene, driven by MCK (muscle creatine kinase) enhancer and β-actin promoter, was inserted into retroviral backbone of G1Na with the opposite direction to obtain the replication-defective retroviral vector. Adenoviral vector pAdCMVhFIX containing CMV promoter and human clotting factor Ⅸ(hFIX) cDNA were constructed and co-transfected 293 packaging cells with plasmid pJM17. Afterward,the recombinant retroviral and adenoviral virons infected the mammary gland of lactation goat, and the expression system and rapid detective system for hFIX gene in vivo were constructed successfully. In addition, mammary gland specific expression vectors for hFIX minigene, cDNA and LacZ reporter gene containing mouse MAR(matrix attachment region)element, bovine β-casein gene promoter were constructed. Vectors were packaged by stearylamine(SA) liposome and transferred into lactating mice via tail vein. The results suggested that the 2. 0 kb sequence of β-casein gene and the nitron 1 of hFIX gene had obvious roles on expression and regulation of hFIX gene in mammary gland. Six transgenic mice and five transgenic goats were generated by microinjection of pMCIXm. The hFIX protein in milk of two transgenic mice and one transgenic goat had been detected and appeared to clinic activity.
出处
《复旦学报（自然科学版）》
CAS
CSCD
北大核心
1998年第4期365-371,共7页
Journal of Fudan University：Natural Science
基金
国家科委八六三高科技项目
上海市科委科技攻关项目
关键词
人凝血因子Ⅸ
乳腺生物反应器
转基因动物
human clotting factor Ⅸ (hFIX)
mammary gland bioreactor
transgenic animal
