人凝血因子Ⅸ乳腺特异表达载体的构建及细胞转染  

Construction of Human CoaguLation Factor Ⅸ Mammary Expression Vector and Transfection

作　　者：韩雪洁[1] 萨日娜[1] 梁浩[1] 李雪玲[1] 李荣凤[1] 

机构地区：[1]内蒙古大学哺乳动物生殖生物学及生物技术教育部重点实验室,呼和浩特010021

出　　处：《中国农业科学》2014年第4期769-778,共10页Scientia Agricultura Sinica

基　　金：国家"863"计划项目(2009AA10Z111);农业部转基因生物新品种培育重大专项(2008ZX08007-002);内蒙古自治区自然科学基因资助项目(2009ZD02)

摘　　要：【目的】人凝血因子Ⅸ(human coagulation factorⅨ,hFIX)是凝血过程中的关键因子,对临床治疗血友病有着极其重要的意义。研究旨在构建人凝血因子Ⅸ乳腺表达载体,在猪乳腺上皮细胞中验证其乳腺特异表达能力,获得hFIX转基因雌性乳腺上皮细胞和猪胎儿成纤维细胞,为克隆乳腺特异表达人凝血因子Ⅸ的转基因猪做准备。【方法】按照TaKaRa公司RNAiso Reagent和Prime Script RT-PCR试剂盒的说明书,从人胎儿肝脏中提取RNA并扩增出人凝血因子Ⅸ(human coagulation factorⅨ,hFIX)cDNA序列,利用PCR方法扩增牛生长激素(BGH)polyA序列,将hFIX的cDNA序列与BGH polyA序列分别连接到表达载体pbCSN2-RC的牛酪蛋白(CSN2)启动子之后,构建带有新霉素抗性和红色荧光蛋白双筛选标记的乳腺特异性表达载体pbCSN2-hFIX-pA-RC。取本地屠宰的泌乳期猪乳房组织,利用组织块种植法培养猪乳腺细胞,并通过控时消化分离纯化猪乳腺上皮细胞,进行染色体分析后,挑选含有正常染色体数目的细胞,通过脂质体法将表达载体导入,经过G418筛选获得稳定表达红色荧光蛋白的转基因细胞,反转录、实时定量PCR验证乳腺表达载体构建的合理性和有效性。为了只对雌性胎儿成纤维细胞进行转基因操作,首先对培养的猪胎儿成纤维细胞进行SRY基因PCR扩增,确定其性别。之后,利用脂质体将证明有效的表达载体导入细胞,并进行G418和红色荧光蛋白表达筛选,对经过鉴定的阳性细胞进行扩大培养和冷冻保存,为下一步的体细胞克隆做准备。【结果】经PCR和酶切鉴定,人凝血因子ⅨcDNA和BGH PolyA成功克隆到载体pbCSN2-RC中,获得具有双筛选标记的pbCSN2-hFIX-pA-RC。转染后G418和红色荧光蛋白表达阳性的猪乳腺上皮细胞,经实时定量PCR鉴定,hFIX有高效表达,证明所构建载体具有指导人FIX在乳腺特异表达的能力,可以用于生产乳腺特异表达的动物。将构建�[ Objective ] Human coagulation factor IX （hFIX） plays a key role in blood coagulation and is important for clinical treatment of hemophilia. The objective of this study is to construct human coagulation factor IX （hFIX） mammary expression vector and test the expression of hFIX in porcine mammary epithelial, and obtain hFIX-transgenic porcine mammary epithelial and female porcine fetal fibroblast cells to prepare to produce hFIX mammary specific expression transgenic pigs. [ Method] Total RNA was extracted from human fetal liver tissues and human coagulation factor IX （hFIX） cDNA was amplified by RT-PCR followed the instructions of RNAiso Reagent and Prime Script RT-PCR Kit from TAKARA. PCR was performed to amplify bovine growth hormone （BGH） polyA fragment. Both hFIX cDNA and BGH polyA fragments were cloned to pbCSN2-RC plasmid, located after the bovine beta-Casein （CSN2） promoter, to achieve mammary specific expression vector pbCSN2-hFIX-pA-RC with neomycin- resistance and red fluorescence genes. Porcine mammary epithelial cells were obtained by culturing 1 mm3 mammary tissue cubes from the breast tissue of lactation pigs that slaughtered in the local slaughterhouse and purifying with time-controlled trypsinization. The chromosome analysis was performed on the derived cells, and the cells with normal number of chromosomes were transfected with pbCSN2-hFIX-pA-RC by lipofection technology. The transfected porcine mammary epithelial cells were screened by neomycin-resistance and red fluorescent expression. The expression of hFIX was further confirmed by real-time PCR. To only transfect the female fibroblasts, the SRY PCR was performed on the porcine fibroblasts cultured in the lab to determine the sex of the cells. Finally, the confirmed vector was transferred to porcine fetal fibroblast cells by lipofection technology. [ Result ] The results of PCR and restricted endonucleases digestion analysis showed that the hFIX cDNA and BGH PolyA were cloned into pbCSN2-RC, and the pbCSN2-hFIX-pA-RC with

关 键 词：人凝血因子IX 乳腺特异性表达载体 猪乳腺上皮细胞 猪胎儿成纤维细胞 转染 

分 类 号：R554.1[医药卫生—血液循环系统疾病] Q78[医药卫生—内科学]