文献类型：期刊文献

中文题名：猪IL-4稳定表达细胞系的建立

英文题名：Establishment of a cell line stably expressing porcine IL-4

作者：李俊敏[1,2,3];蔡彬[4];雍艳红[1];巩栋梁[5];冯园[3];何玉林[3];巨向红[1]

机构：[1]广东海洋大学动物医学系,广东湛江524088;[2]河源市人民医院,广东河源517000;[3]桂林医学院,广西桂林541004;[4]湛江出入境检验检疫局,广东湛江524022;[5]广东海洋大学动物科学系,广东湛江524088

年份：2017

卷号：47

期号：7

起止页码：897

中文期刊名：中国兽医科学

外文期刊名：Chinese Veterinary Science

收录：CSTPCD、、北大核心2014、CSCD_E2017_2018、北大核心、CSCD

基金：国家自然科学基金项目(31101862;31472243)

语种：中文

中文关键词：猪;IL-4;真核表达;稳定表达细胞系

外文关键词：pig; IL-4; eukaryotic expression; stable expression of cell lines

中文摘要：为了构建能稳定表达猪白细胞介素4(interleukin-4,IL-4)的细胞系,本研究对猪IL-4基因进行了克隆,发现该基因开放阅读框(ORF)长402 bp,编码133个氨基酸,分子质量为15.02 ku,PI为8.64。与牛、绵羊、马、猫、狗和人的同源性分别为80.5%、76.7%、72.7%、72.9%、72.0%和69.2%,而与小鼠的同源性仅为41.5%。通过Eco RⅠ和Bam HⅠ酶切后将p IL-4基因插入含EGFP标签的p IRES2-EGFP载体中,构建出重组质粒p IRES2-EGFP-p IL-4。该重组质粒转染猪肠上皮细胞(IPEC-J2)后,经G418阳性筛选,获得稳定表达猪IL-4的细胞系。p IRES2-EGFP-PIL-4重组质粒转染IPEC-J2细胞后第24小时,荧光定量PCR结果表明,IL-4 m RNA的表达量较对照组及空载体组极显著升高(P<0.01),荧光显微镜下亦可见大量绿色荧光表达。该细胞系在连续传代28次以后,仍可见IL-4在基因水平和蛋白水平的高表达。本研究成功构建了真核表达载体p IRES2-EGFP-p IL-4和能稳定表达猪IL-4的细胞系,为进一步研究其功能奠定了基础。

外文摘要：This study was conducted to establish cells lines stably expressing porcine IL-4.The cloned porcine IL-4 gene consists of 402 bp,and the ORF of porcine IL-4 encodes a protein of 133 amino acids.The IL-4 polypeptide had an estimated molecular mass of 15.02 ku,PI of 8.64 and displayed a high homology to that of Bos taurus（80.5%）,Ovis aries（76.7%）,Equus caballus（72.7%）,Felis catus（72.9%）,Canis lupus familiaris（72.0%）,Homo sapiens（69.2%） and Mus musculus（41.5%）,respectively.After the digestion of EGFP-tagged p IRES2-EGFP vector by Eco R Ⅰ and Bam H Ⅰ enzymes,the ORF of porcine IL-4 was inserted and named as p IRES2-EGFP-p IL-4.Then the p IRES2-EGFP-p IL-4 was transfected into porcine intestinal epithelial cell line（IPEC-J2）,which was then screened by G418 to obtain the cell line stably expressing porcine IL-4.Consequently,the expression of IL-4 m RNA in transfected cells was significantly increased compared to control cells at hour 24 after transfection（P〈0.01）and transfected cells also showed high expression of green fluorescent.After G418 selection and expansion（positive clone） for 30 passages,the expression of IL-4 m RNA was increased by 200 times to controlcompared cells.We concluded that the eukaryotic expression vector pIRES2-EGFP-p IL-4 was constructed successfully and a cell lines that show the stable expression of porcine IL-4 was established,which laid the foundation for further studies of its functions.