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目的探讨携带大鼠细胞间黏附分子1(ICAM-1)核糖核酸干扰(RNAi)的重组慢病毒构建方法,筛选出基因抑制效率最高的重组慢病毒。方法(1)根据基因序列,设计并合成针对4个靶点的寡核苷酸,将其与双链DNAoligo进行连接反应;将连接产物转化DH5α大肠杆菌,PCR筛选获得阳性克隆并进行基因测序。(2)将重组载体与病毒包装质粒共转染人胚肾293T细胞,获得病毒颗粒。(3)重组慢病毒体外转染NRK细胞,以RT-PCR法筛选出基因抑制效率最高的重组慢病毒。结果(1)经过酶切、连接、转化后获得阳性克隆;基因测序结果完全正确。(2)经RT-PCR证实,3号病毒在感染复数(MOI)=50时,基因抑制率即达到88.4%。结论成功构建了含有绿色荧光蛋白(GFP)报告基因的大鼠ICAM-1RNAi慢病毒载体。
Objective To investigate the construction of recombinant lentivirus carrying ICAM-1 RNAi and to screen out the recombinant lentivirus with the highest gene inhibition efficiency. Methods (1) According to the gene sequence, oligonucleotides targeting at four targets were designed and synthesized, and ligated with double-stranded DNAoligo. The ligation product was transformed into DH5α E.coli. The positive clones were screened by PCR and sequenced. (2) Recombinant vector and virus packaging plasmid were cotransfected into human embryonic kidney 293T cells to obtain virus particles. (3) NRK cells were transfected with the recombinant lentivirus in vitro, and the recombinant lentivirus with the highest gene inhibition efficiency was screened by RT-PCR. Results (1) Positive clones were obtained after digestion, ligation and transformation. The result of gene sequencing was correct. (2) The result of RT-PCR confirmed that virus 3 reached 88.4% at MOI = 50. Conclusion Rat ICAM-1 RNAi lentiviral vector containing green fluorescent protein (GFP) reporter gene was successfully constructed.