摘要
目的构建慢病毒载体及观察其介导的RNA干扰猪卵巢颗粒细胞c-Fos基因表达情况。方法通过在线软件设计猪c-Fos基因shRNA序列,合成、退火形成双寡链核酸后克隆到PGMLV-SC1载体,测序。用构建的慢病毒表达载体和包装质粒共转染293T细胞,包装成4组病毒(shRNA c-Fos1、shRNA c-Fos2、shRNA c-Fos3、shRNA c-Fos4),并测定滴度。感染猪卵巢颗粒细胞,采用PCR和Western blot检测各组细胞c-Fos mRNA和蛋白表达。结果重组克隆中插入片段序列与设计的Oligo序列完全一致。滴度为1×108TU/mL,感染复数30时,阴性对照组、实验shRNA c-Fos4、shRNA c-Fos3、shRNA c-Fos2、shRNA c-Fos1组c-Fos mRNA的表达分别为对照组的0.842 7±0.065 6、0.797 6±0.073 8、0.694 1±0.048 9、0.653 7±0.047 0、0.312 3±0.010 6倍(P<0.05),c-Fos蛋白的表达下降。结论实验shRNA c-Fos1组c-Fos基因下调最明显,成功构建猪卵巢颗粒细胞c-Fos基因慢病毒RNAi表达载体并筛选出最佳siRNA序列。为c-Fos基因的功能研究提供了研究基础。
Objective To construct a lentiviral vector carrying swine c -Fos gene and investigate its effects on c - Fos gene expression in porcine ovarian granulosa cells （GCs）. Methods Porcine c - Fos gene short hairhin RNA （shRNA） sequence was designed using software available on -line. After synthesis and annealing, the double -stranded oligonucleotides were cloned into the pGMLV - SC1 plasmid followed by sequence analysis. The recombinant lentivirus was harvested from 293T cells cotransfected with the recombined plasmid and lentiviral packing materials. Porcine ovarian granulose cells were infected with the recombinant lentivirus, c - Fos gene expression in the cells was determined by PCR and Western blot. Results The constructed lentiviral expression vector pGMLV - SC1 was corresponded to the expected. In the condition that viral titer was 1 ~ lOs TU/mL and the MOI （multiplicity of infection） was 30, the expression of c - Fos were 0. 842 7 -＋0. 065 6, 0. 797 6 ＋0. 073 8, 0. 694 1 ＋-0. 048 9, 0. 653 7 ＋0. 047 0 and 0. 312 3 ＋0. 010 6 in con- trol group, scram group, shRNA c - Fos4 group, shRNA c - Fos3 group, shRNA c - Fos2 group and shRNA c - Fosl group, respectively; with significant down - regulation of c - Fos protein. Conclusion The lentivirus RNAi vector targe- ting c - Fos gene is successfully constructed, laying foundations for further study of c - Fos gene.
出处
《广东医学》
CAS
CSCD
北大核心
2014年第3期336-339,共4页
Guangdong Medical Journal
基金
国家自然科学基金资助项目(编号:81273786)
关键词
RNA干扰
慢病毒
猪卵巢颗粒细胞
c - Fos
RNA interference
lentivirus
porcine ovarian granulosa cell
