7个shRNA分子对绵羊BMPR-1B基因的干扰作用分析

为获得有效干扰绵羊BMPR-1B基因的shRNA干扰分子,从绵羊卵巢组织中扩增了1 515 bp BMPR-1B基因全长编码区cDNA序列,添加HA标签序列后,插入Plex-mcs慢病毒质粒,构建Plex-BMPR-1B慢病毒表达载体,转染HEK293细胞,并与2个包装质粒共转染293T细胞进行病毒包装,用获得的重组慢病毒感染HEK293细胞;同时,将7个干扰分子与Pll-LentiLox 3.7载体重组,并与3个包装质粒共转染293T细胞进行病毒包装,获得干扰分子的重组病毒颗粒。最后用干扰分子重组的病毒颗粒感染整合Plex-BMPR-1B的HEK293细胞,进行qRT-PCR和Western blot检测。结果显示:重组BMPR-1B蛋白质在稳定整合Plex-BMPR-1B的HEK293细胞系中获得了表达;研究中设计的7个shRNA分子能抑制绵羊BMPR-1B基因表达水平68.30%～99.86%,其中PLL-BMPR-1B-1306和PLL-BMPR-1B-1475干扰分子的干扰效果最好。

Interference of seven shRNA molecules on ovine BMPR-1B gene

In order to get the effective shRNA molecules of interfering sheep BMPR-1B gene,1 515 bp coding sequence cDNA of BMPR-1B gene was amplified from sheep ovaries and was added with HA tag.The cDNA with HA tag was inserted into Plex-mcs to build Plex-BMPR-1B lentiviral expression vector.HEK293 cells were transfected with Plex-BMPR-1B and co-transfected 293T cells with two packaging plasmids.HEK293 cells were then infected with above recombinant lentivirus.At the same time,7 shRNA molecules were recombined with Pll-LentiLox 3.7 vector,and co-transfected 293T cells with 3 packaging plasmids to package virus.The recombinant lentivirus with shRNA molecules were obtained.At last,HEK293 cells with Plex-BMPR-1B were infected with recombinant lentivirus,and qRT-PCR and Western blot were performed.The results showed that recombinant BMPR-1B protein was expressed in HEK293 cells recombined with Plex-BMPR-1B.All 7 shRNA molecules in this experiment could inhibit the expression level of sheep BMPR-1B gene by 68.30%-99.86%.PLL-BMPR-1B-1306 and PLL-BMPR-1B-147 shRNA molecules were the most effective shRNA molecules.