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目的构建携带β-arrestin 2抗基因RNA的慢病毒表达载体并对其进行鉴定。方法将线性化的慢病毒载体pGC-FU与抗基因RNA在In-Fusion交换酶作用下构建目的质粒pGC-agRNA,转化感受态细胞,对长出的克隆应用菌落PCR鉴定,再对PCR鉴定阳性的克隆进行测序和比对分析。重组病毒质粒与另外两种辅助包装原件载体质粒通过LipofectamineTM2000共转染293T细胞,培养48 h后,收集细胞培养上清液,将病毒浓缩后在293T细胞中测定病毒滴度,并检测慢病毒载体在工具细胞NG108-15细胞的转染效率。结果抗基因RNA被成功构建入慢病毒表达载体pGC-FU,病毒滴度为2×109TU/mL。用该慢病毒感染NG108-15细胞,当感染复数(MOI)为100时,感染效率大于95%。结论成功构建了携带β-arrestin 2抗基因RNA的慢病毒表达载体,为研究β-arrestin 2在μ阿片受体调控机制中的作用提供了有效的研究工具,而且为抗基因RNA技术应用于体内外实验研究奠定了基础。
Objective To construct lentivirus vector carrying β-arrestin 2 anti-gene RNA and identify it. Methods The linearized lentiviral vector pGC-FU and anti-gene RNA were constructed by In-Fusion exchange enzyme and the recombinant plasmid pGC-agRNA was transformed into competent cells. The colonies were identified by colony PCR and the positive PCR identification The clones were sequenced and aligned. 293T cells were co-transfected with the vector plasmid of the other two kinds of auxiliary packaging by the recombinant plasmid and LipofectamineTM2000. After culturing for 48 hours, the cell culture supernatants were collected and the virus titer was determined in 293T cells after concentration of the virus and the lentiviral vector Transfection efficiency in instrumental cells NG108-15 cells. Results Anti-gene RNA was successfully constructed into lentiviral vector pGC-FU with a titer of 2 × 109 TU / mL. Infection of NG108-15 cells with this lentivirus resulted in an infection efficiency of more than 95% at an MOI of 100. Conclusion The lentiviral vector carrying β-arrestin 2 anti-gene RNA was successfully constructed, which provided an effective research tool for studying the role of β-arrestin 2 in the regulation mechanism of mu opioid receptor. Moreover, Internal and external experimental research laid the foundation.