绵羊NYD-SP27基因慢病毒干扰载体的构建与鉴定

Construction and Identification of Ovine NYD-SP27 gene Lentiviral Vector

构建并筛选针对干扰绵羊NYD-SP27基因的shRNA慢病毒载体。使用RNAi Target Sequence Selector软件设计并合成针对绵羊NYD-SP27基因的4条shRNA表达序列(shRNA1、shRNA2、shRNA3、shRNA4)及1条阴性对照序列(NC),分别将其连接到载体pLentiLox3.7(PLL3.7)中,得到4个shRNA干扰载体NYDSP27-shRNA及1条阴性对照载体NC-shRNA,同时将构建好的干扰载体及辅助质粒瞬时共转染至HEK-293FT细胞中,收集病毒液纯化后感染SP27-21细胞,48h后用实时荧光定量PCR(RT-qPCR)检测NYD-SP27相对表达量,将干扰效果最好的载体用于进一步的研究中。结果表明,构建了重组慢病毒干扰载体,并成功包装成干扰慢病毒NYDSP27-shRNA-LV以及用来感染BHK-SP27-21细胞,48h后用实时定量PCR筛选出高效干扰绵羊NYD-SP27的片段NYDSP27-shRNA-1。为进一步研究NYD-SP27基因表达下调对绵羊精子发育的影响奠定了基础。

In order to obtain interference of shRNA lentiviral vector of NYD-SP27 gene in ovine,in this research,the RNAi Target Sequence Selector software was used to design and synthesize 4 expressing sequences（shRNA1,shRNA2,shRNA3,shRNA4）and 1 control sequence（NC）for ovine NYD-SP27 gene,and then concatenating them into a blank plasmid vector pLentiLox3.7（PLL3.7）respectively,the constructions of 4 shRNA interfering vector NYDSP27-shRNAs and 1 negative control vector NC-shRNA were completed,meanwhile,co-transfecting interfering vector and helper plasmid into EK-293 FT cell instantaneously.the BHK-SP27-21 cells were infected with virus liquid that purified by ultrafiltration device after concentration,the relative expression of NYD-SP27 was detected by real-time quantitative PCR after 48 hinfection,and the carrier with the most efficiently interference vector was used to further experimental study.The results showed that the recombinant lentivirus vector was successfully constructed and packaged into lentivirus NYDSP27-shRNA-LV as well as BHK-SP27-21 cells were infected by recombinant lentivirus.The efficient interference ovine NYD-SP27 fragment-NYDSP27-shRNA-1 was screened by fluorescence quantitative PCR after 48 hinfection.This study laid the foundation for further research on the down-regulation of NYD-SP27 gene expression in ovine sperm development.