猪lncRNA2099互作蛋白的筛选与分析  

Screening and analysis of interacting protein of porcine lncRNA2099

作　　者：张冬杰[1] 马守正 汪亮[1] 刘娣 ZHANG Dongjie;MA Shouzheng;WANG Liang;LIU Di(Institute of Animal Husbandry,Heilongjiang Academy of Agricultural Sciences,Harbin 150086,China)

机构地区：[1]黑龙江省农业科学院畜牧研究所,哈尔滨150086

出　　处：《黑龙江畜牧兽医》2025年第3期43-46,共4页Heilongjiang Animal Science And veterinary Medicine

基　　金：国家自然科学基金项目(32172696);国创中心先导项目(NCTIP-XD/B01);黑龙江省农业科学院创新工程重大项目(CX23ZD06)。

摘　　要：为了鉴定新发现的一个长链非编码RNA2099(lncRNA2099)的互作蛋白,试验使用体外培养的猪前脂肪细胞,PCR扩增获得带T7启动子的lncRNA2099体外转录用模板,通过RNA pull down、蛋白质银染及质谱分析的方法对可能与lncRNA2099互作的蛋白进行了筛选与分析。结果表明:PCR扩增和体外转录成功获得了带有生物素标记的lncRNA2099和反义链探针,以反义链探针为阴性对照,lncRNA2099在体外培养的脂肪细胞中共下拉出132个互作蛋白,去掉与阴性对照相同的86个蛋白后,获得46个特异蛋白。根据得分排名,与lncRNA2099互作的蛋白分别是膜联蛋白A2(ANXA2)、肌球蛋白重链2(MYH2)、Y-box结合蛋白(YBX1)和α微管蛋白(α-Tubulin)。说明RNA pull down技术联合质谱分析技术,可鉴定出新发现的lncRNA的互作蛋白。In order to identify a newly discovered long chain non-coding RNA2099(lncRNA2099)interacting protein,porcine adipose cells cultured in vitro were selected as the study objects.lncRNA2099 template with T7 promoter was obtained by PCR amplification.Proteins that might interact with lncRNA2099 were screened and analyzed by RNA pull down,protein silver staining and mass spectrometry.The results showed that lncRNA2099 labeled with biotin and antisense probe were successfully obtained by PCR amplification and in vitro transcription.Using the antisense chain probe as the negative control group,a total of 132 interacting proteins were pulled down in adipocytes cultured in vitro,and 46 specific proteins were obtained after removing the same 86 proteins as the negative control group.According to the ranking of scores,the proteins interacting with lncRNA2099 were membrane associated protein A2(ANXA2),myosin heavy chain 2(MYH2),Y-box binding protein(YBX1)and Tubulinαchain(α-Tubulin).The results indicated that the RNA pull down technique combined with mass spectrometry could identify the interacting proteins of newly discovered lncRNAs.

关 键 词：猪 长链非编码RNA2099 前脂肪细胞 RNA pull down 质谱分析 

分 类 号：S828.9[农业科学—畜牧学]