冷诱导RNA结合蛋白(CIRP)慢病毒过表达载体的构建及病毒包装与滴度测定

Construction and Identification of Lentiviral Vector Over-expressing Cold Inducible RNA-binding Protein(CIRP)

构建冷诱导RNA结合蛋白(CIRP)过表达慢病毒载体,并对其进行病毒包装、鉴定与滴度测定,为进一步研究CIRP功能提供基础工具。本研究从4℃冷处理8 h的SD大鼠睾丸组织中获得CIRP的cDNA序列测序后,将其克隆入LV5质粒中,经双酶切及测序鉴定;利用脂质体将鉴定的阳性重组LV5-cirp表达载体与pGag/Pol、pRev、pVSV-G 3个质粒共转染到HEK-293T细胞,72 h收获上清,包装产生慢病毒。将所得病毒悬液梯度稀释后感染293T细胞,检测病毒滴度,以及采用Western blot方法验证基因表达情况。结果表明:经琼脂糖凝胶电泳鉴定、PCR鉴定、酶切及测序结果证明成功构建了LV5-cirp重组质粒,序列比对与设计序列符合率100%,并成功包装成慢病毒,病毒滴度为6.30×108TU/m L;将制备成功的CIRP慢病毒过表达载体感染293T细胞后,Western blot检测结果显示细胞中CIRP蛋白表达水平显著高于正常细胞以及慢病毒阴性对照组(P<0.01)。本实验成功构建CIRP慢病毒过表达载体并完成了慢病毒包装及滴度的测定,为今后的研究提供了基础工具。

To provide a basis tool for further research of CIRP function, a lentiviral vector for the over-expression of cold inducible RNA-binding protein （CIRP） was constructed in this study. The virus vector was packaged, identified and titer determined. After obtained CIRP cDNA sequencing of testicular tissue from SD rats with cold treated at the temperature of 4℃, the duration of cold stress were 8 h , the CIRP cDNA was inserted into LV5 vector, and then identified by double enzyme digestion and nucleotide sequencing. LV5-cirp, pGag/Pol, pRey and pVSV-G were co- transfected into HEK-293T cells for packaging of the lentivirus by liposome respectively, harvesting the supernatant in 72 hours. HEK-293T cells were then transduced with an appropriately diluted lentivirus supernatant for the titration of virus titer, and validated cirp＇s expression by western blot. The lentiviral vector pCDH-CMV-MCS-EF1-copGFP vector for CIRP was constructed successfully, and the virus in the supernatant reached a titer of 6. 30×10^8 TU/mL. Further more, the expression level of CIRP was very significantly higher than normal and lentivirus negative control groups in 293T cells identified by western blot when infected with the lentiviral vector for the over-expression of CIRP （P〈0.01）. This study indicated that over-expression lentiviral vector of CIRP was successfully constructed and packaged and the titer was determined, and this provide a basis tool for further research of CIRP.