植酸酶转基因猪成纤维细胞系的构建  

Establishment of a New Type Phytase Transgenic Swine Fibroblast Cell Line

作　　者：陈晓亮[1,2] 黄怡[2] 殷丽丽[3] 王士长[2] 林秀坤[1] 

机构地区：[1]中国农业科学院北京畜牧兽医研究所,北京100193 [2]广西大学动物科技学院,南宁530004 [3]中国农业大学农学与生物技术学院,北京100193

出　　处：《畜牧兽医学报》2013年第1期7-14,共8页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：转基因生物新品种培育重大专项(2009ZX08010-013B;2009ZX08010-024B)

摘　　要：本研究旨在获得对胃蛋白酶和胰蛋白酶具有耐受性的植酸酶转基因猪成纤维细胞系。本研究首先检测了转植酸酶基因的酵母发酵液分别经不同浓度、pH2.0的胃蛋白酶和不同浓度、pH7.0的胰蛋白酶处理后的酶活残余量;然后采用流式细胞术筛选了慢病毒转染的阳性猪成纤维细胞,并进行鉴定。结果表明,酵母粗酶液经胃蛋白酶和胰蛋白酶处理后,残留酶活分别保持在70%和80%以上;包装后的慢病毒滴度为3.75×107,经流式细胞术筛选的转基因细胞阳性率为1.2%,PCR和RT-PCR结果表明,植酸酶基因已整合到基因组上,且该基因在转录水平上表达。本研究为制备植酸酶转基因细胞系提供了一种快速有效的方法,为转基因猪的培育奠定了基础。The aim of the study was to generate a transgenic swine fibroblast cell line of pepsin- and trypsin-toleranceing. After culturing, transgenic yeast fermentation broth was collected to detect the enzyme activity under the condition of pH2.0 for pepsin treatments with different con- centrations and pHT. 0 for trypsin treatments with different concentrations, respectively. The expression vector containing phytase gene was packaged in lentivirus and then infected swine fi- broblasts. After subculturing, positive cells were screened with flow cytometric approach and cultured. The results showed that more than 70% of phytase activity remained after treatment with pepsin and more than 80% activity was remained when treated with trypsin. Lentivirus was packaged with 3.75 × 107 titer and transfected into porcine fibroblast cells, with 1.2% positive rate obtained. PCR assessment indicated that the exogenous gene was inserted into the chromo- some of the swine fibroblast cells successfully. Moreover, RT-PCR evaluation suggested that the phytase gene expressed in the transcript level. In short, the study provides a fast and effective approach for preparing phytase transgenic cell line, as well as a foundation for further breeding transgenic pig.

关 键 词：转基因 植酸酶 慢病毒 流式细胞分选 猪成纤维细胞 

分 类 号：S828[农业科学—畜牧学] S813.3[农业科学—畜牧兽医]