视黄醇类核内受体α基因慢病毒表达载体构建

目的 通过构建大鼠视黄醇类核内受体-α(RXR-α)基因慢病毒表达载体,获得可供转染的滴度.方法 大鼠RXR-α基因序列进行聚合酶链反应(PCR)扩增,与经AgeI酶切后的pCC-FU-3FLAG载体连接产生慢病毒载体表达质粒pGC-fu-3flag-Rxra,转化DH5α,PCR筛选阳性克隆,片段长为411bp.测序并转入293T细胞Western blot鉴定,90 KDr处有特征条带.将pGC-fu-3flag-Rxra、pHelper 1.0、pHelper 2.0三质粒共转染293T细胞,包装成慢病毒,测病毒滴度,1.00E-04μl组和Control组的Ct值存在差异(2.775).结果 DNA测序及Western blot鉴定证实构建的大鼠RXR-α基因慢病毒表达载体pGC-fu-3flag-Rxra正确,浓缩慢病毒悬液滴度为2×108TU/ml.结论 成功构建携带大鼠RXR-α基因的重组慢病毒表达载体.

Construction and detection of lentiviral expression vector of retinoid X receptor alpha gene

Objective To construct recombinant lentiviral expression vector carrying rat retinoid X receptor alpha (RXR-α) gene,and to obtain the titer of the lentiviral stock.Methods Rat RAR-α encoding sequence was amplified,and ligated with pGC-FU-3FLAG lentivirol vector to produce the lentiviral expression vector named pGC-fu-3flag-Rxra.It was identified by polymerase chain reaction ( PCR,the fragment length of 411 bp),DNA sequencing and Western blotting.There was a characteristic band of about 90 kD.Virus packaging cells (293T cells) were cotransfected with lentivirol vector pGC-fu-3flag-Rxra,pHelper 1.0 and Helper 2.0 to produce lentivirus.The titers of recombinant viruses were determined by real time qPCR.There was significant difference in the Ct data between 1.00E-04 μl group and control group (2.775).Results Lentiviral expression vector of rat RXR-α gene proved by sequencing and Western blotting was successfully constructed.The titer of recombinant viruses was 2 × 108 TU/ml.Conclusion Rat RAR-α lentiviral expression vector was successfully constructed.