蛋白激酶B2慢病毒载体的构建及其沉默效果的验证

目的:利用RNA干扰技术,以蛋白激酶B 2(AKT2)的mRNA为靶点,设计、构建AKT-2的RNA干扰慢病毒载体,并对其沉默效果进行验证.方法:根据目的基因设计3个小干扰RNA(siRNA)靶点,进行引物合成.将单链的引物退火成双链oligo序列,连接入双酶切线性化的RNA干扰载体.测序验证正确的转化子,进行高纯度质...

Construction of AKT2 lentivirus vector and validation of its silencing effect

Objective To design and construct an AKT2 lentivirus vector with RNA interference targeting AKT2mRNA， and verify its silencing effect. Methods Three siRNA targets were designed according to the target gene.Single-stranded primers were annealed into double-stranded oligo sequences and joined into double-enzymaticlinearized RNA interference vectors. Sequencing was performed to verify correct inverters and high-purity plasmidextraction was carried out. Three positive cloned plasmids were sequenced and transfected into HEK293-T cells toproduce lentivirus plasmids. The experimental rats were randomly divided into 5 groups with 10 rats in each group.The blank control group was not injected with lentivirus. The rats in the NC-shRNA negative infection group，PL-AKT2-shRNA-1 infection group， PL-AKT2-shRNA-2 infection group and PL-AKT2-shRNA-3 infection groupwere injected in the epididymis with 200 μL vectors respectively. Western blotting was used to detect the proteinsilencing effect of AKT2 in the epididymis of rats. Results The sequencing results showed that the three lentivirusvectors were packaged successfully. After infection with epididymis， AKT2 protein expression was significantly lowerthan that in the negative infection group and the normal control group， and the PL-AKT2-shRNA-3 plasmid groupwas more effective in interfering with AKT2. Conclusion The RNA interference target of AKT2 gene is successfullydesigned and the lentivirus vector of AKT2 gene is prepared. The gene silencing effect is obvious.