摘要
为获得稳定表达CYP3A29基因的猪肺泡巨噬细胞系,利用RT-PCR方法获取猪CYP3A29基因序列后与克隆载体PMD18-T相连,得到PMD18-T-CYP3A29重组质粒并测序验证,利用Xho I和Not I双酶切重组质粒及pcDNA3.1(+)/zeo,回收后连接,构建重组真核表达载体pcDNA3.1(+)/zeo-CYP3A29,双酶切及测序验证其正确性,并以脂质体2000将其转染至猪肺泡巨噬细胞系3D4/21,经Zeocin抗性筛选,借助RT-PCR及Wesrern-blot鉴定该基因的表达情况。结果表明,成功获得表达CYP3A29的细胞株,且连续培养10代后,RT-PCR及Wesrern-blot仍能检测到该基因表达。说明,稳定表达CYP3A29基因的猪肺泡巨噬细胞系构建成功,该细胞系的成功构建为进一步研究分析猪CYP3A29基因的功能提供了可能。
The cDNA of CYP3A29 gene was amplified by RT-PCR and was then subcloned into plasmid PMD18-T vector to obtain a recombinant plasmid PMD18-T-CYP3A29, which was verified by DNA sequencing. CYP3A29 gene frag-ments and pcDNA3. 1（＋）/zeo vector fragments purified after digestion with Xho I and Not I were ligated together to gener-ate a pcDNA3. 1（＋）/zeo-CYP3A29 plasmid, which was verified by restriction enzyme digestion and DNA sequencing. The porcine alveolar macrophages cell （ PAM ） lines 3D4/21 was transfected by recombinant plasmid pcDNA3. 1 （＋）/zeo-CYP3A29 using lipofectamine 2000. RT-PCR and Western blot detected the successful expression of CYP3A29 in PAM cells. After 10 generations, CYP3A29 gene was detectable by RT-PCR and Wsetern-blot. The establishment of PAM cell lines stably expressing CYP3A29 gene enables the functions analysis of CYP3A29 gene.
出处
《江苏农业学报》
CSCD
北大核心
2014年第3期554-559,共6页
Jiangsu Journal of Agricultural Sciences
基金
国家自然科学基金项目(31301953)
国家生猪现代产业技术体系项目(nycytx-009)
江苏省自然科学基金项目(BK20131332)
江苏省农业科技自主创新基金项目[CX(13)2037]
