鸡耳蜗EFNA2基因RNAi慢病毒载体的构建及其靶基因离体沉默效率

Construction of RNAi Lentiviral Vector Targeting EFNA2 Gene and Its Effect of Gene Silencing in PAN of Chicken

目的构建鸡耳蜗EFNA2基因的RNA干扰(RNAi)慢病毒载体,在离体培养原代鸡听神经细胞中鉴定其沉默效率。方法针对鸡EFNA2基因序列设计特异性小干扰RNA(small interfering RNA,siRNA)靶点,构建慢病毒pFU-GW-iRNA载体,将筛选所得有效短发卡RNA(short hairpin RNA,shRNA)慢病毒载体在293T细胞中包装成病毒颗粒,随后将其感染离体培养原代鸡听神经细胞,最后采用Real-time PCR检测靶基因的沉默效率。结果 PCR克隆鉴定及测序结果显示所构建的shRNA慢病毒载体正确无误,包装病毒后的滴度为2.0×109 TU/ml,分别命名为LV-GFP-EFNA2-shRNA-l、LV-GFP-EFNA2-shRNA-2和LV-GFP-EF-NA2-shRNA-3。ShRNA慢病毒颗粒感染目的细胞后,EFNA2基因的mRNA表达量较阴性对照载体慢病毒感染组分别下降了76.11%、61.87%和68.44%。结论本研究成功构建了鸡EFNA2基因的RNAi慢病毒表达载体,并能在离体培养的原代鸡听神经细胞中有效沉默靶基因。

Objective To construct a RNAi lentiviral vector targeting chicken EFNA2 gene and detect its effect of gene silencing in PAN of chicken. Methods The specific siRNA sequences targeting chicken EFNA2 gene were cloned into pFU--GW--iRNA lentiviral vector. After screening for the valid siRNA, the lentivirus particles were packaged and EFNA2 specific shRNA was transmitted into PAN of chicken. Then real--time PCR was per- formed to determine the expression ievel of EFNA2. Results PCR and sequencing results revealed that shRNA plas- raids were correctly constructed. The virus with a titer of 2.0 109 TU/ml was successfully packaged and named LV -- GFP-- EFNA2 -- shRNA-- 1, LV-- GFP -- EFNA2 -- shRNA -- 2, LV-- GFP-- EFNA2 -- shRNA -- 3, respectively.： After transfection by virus, EFNA2 mRNA expression in PAN was decreased by 76.11%, 61.87%, 68.44% re- spectively compared with those of in negative control. Conclusion The efficient recombinant lentiviral RNAi vector of EFNA2 was constructed successfully and could cause target gene silencing in PAN of chicken.