东北梅花鹿Galectin-1基因RNAi载体的构建  

Construction of vector for northeast China deer Galectin-1 gene RNAi

作　　者：秦欣欣[1,2] 孙红梅[2] 褚文辉[2] 王大涛[2] 郭倩倩[1,2] 章秀婷[2] 李春义[2] 

机构地区：[1]江苏科技大学生物与化学工程学院,江苏镇江212018 [2]中国农业科学院特产研究所,吉林省特种经济动物分子生物学国家重点实验室培育基地,长春130112

出　　处：《东北农业大学学报》2013年第9期57-62,F0002,共7页Journal of Northeast Agricultural University

基　　金：国家自然科学基金(31070878);973前期研究专项(2011CB111515)

摘　　要：构建东北梅花鹿galectin-1基因特异小分子干扰RNA(shRNAs)真核干扰载体,并获得稳定感染生茸区骨膜细胞系。设计2对galectin-1 mRNA特异性shRNAs,两端分别带有ClaⅠ/MluⅠ酶切位点和一个9 nt发夹结构,通过基因克隆技术将其插入真核表达质粒pLVTHM中,构建galectin-1 shRNA表达载体,利用PCR和测序筛选出阳性质粒。将重组质粒与PPAX及pMD2.G共转染到293 t细胞中,在倒置显微镜下观察转染效果并收集、浓缩病毒质粒,感染生茸区骨膜细胞。结果表明,成功构建东北梅花鹿galectin-1基因RNAi载体,获得稳定传代被感染生茸区骨膜细胞系,为进一步研究galectin-1基因在鹿茸再生中的作用奠定基础。To construct the RNA interference eukaryotic expression vectors for the deer galectin-1, and establish the antlerogenic pedosteal cell lines infected by RNAi vectors. Two pairs of small interfering RNAs targeting galectin-1 mRNA were designed. With both ends are equipped with Clal/Mlu l enzyme site and a 9 nt hairpin structure, through the gene cloning technology, constructing galectin-1 shRNA expression vectors were inserted into the eukaryotic expression plasmid pLVrHM, The positive plasmids were screened by PCR and then sequenced. The recombinant plasmid with PPAX and pMD2.G was co-transfected into 293 t cells, the transfection effects were observed under an inverted microscope, the virus plasmids were collected and concentrated, and used to infect antlerogenic periosteal cells. RNAi eukaryotic expression vector of the deer galectin-1 mRNA was successfully constructed. Establishment of the stably transfected anUerogenic pedosteal cell lines may lay a foundation for the exploration of the roles of galectin-1 in the antler regeneration.

关 键 词：鹿茸再生 galectin-1基因 RNAI 生茸骨膜细胞 

分 类 号：S767.5[农业科学—森林保护学] X172[农业科学—林学]