稳定表达CD163蛋白的猪肺泡巨噬细胞系的建立  

Establishment of stable PAM(porcine alveolar macrophage) cell lines expressing CD163 protein

作　　者：赵凯[1,2] 冯磊 郑其升 侯继波 陈溥言[1] 

机构地区：[1]南京农业大学动物医学院,江苏南京210095 [2]国家兽用生物制品工程技术研究中心,江苏南京210014

出　　处：《江苏农业学报》2012年第3期565-570,共6页Jiangsu Journal of Agricultural Sciences

基　　金：国家高技术研究发展计划("863"计划)项目(2011AA10A213);江苏省农业自主创新基金项目[CX(09)4065;CX(10)2049]

摘　　要：以RT-PCR方法从原代猪肺泡巨噬细胞中获得CD163 cDNA序列,然后测序鉴定,对突变碱基进行修正,将修正后碱基序列克隆入真核表达载体pcDNA4.0中,构建重组真核表达质粒pcDNA4.0-CD163,通过BamHⅠ/NotⅠ双酶切及测序鉴定其正确性。使用脂质体2000将pcDNA4.0-CD163转染猪肺泡巨噬细胞系3D4/21(CRL-2843),通过Zeocin抗性筛选,建立稳定表达CD163的猪肺泡巨噬细胞系。RT-PCR、间接免疫荧光(IFA)检测结果表明CD163在mRNA和蛋白质水平均已表达。通过Zeocin抗性筛选,共获得8株表达CD163的重组细胞株,IFA检测结果表明表达的CD163定位于重组细胞的细胞膜表面。去除筛选抗性后连续传代10代,间接免疫荧光检测结果确定重组细胞株在10代内性状稳定,说明稳定表达CD163的猪肺泡巨噬细胞系构建成功。The gene of CD163 was cloned by RT-PCR from porcine alveolar macrophage （ PAM）, and was then se- quenced. The mutations of gene were executed by the site-directed mutagenesis kit from TaKaRa company according to the submitted GenBank sequence. The correct gene fragment was cloned into the eukaryotie expression vector pcDNA4.0 and i- dentified by enzyme digestion and sequencing. The recombinant plasmid was transfeeted into PAM cell line 3D4/21 （ CRL- 2843） by lipofeetamine 2000. Eight positive cell clones were screened by culture media with zeoein. IFA result indicated that CD163 protein was located on cell membrane. The recombinant cell line was stable after 10 continuous passages. The recombinant PAM cell line with stable-expression of porcine CD163 protein was constructed successfully.

关 键 词：CD163 猪肺泡巨噬细胞 重组细胞系 

分 类 号：Q78[生物学—分子生物学]