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目的:利用RNAi表达载体法构建并筛选携带针对CD3ζ基因的pSUPER retro RNAi逆转录病毒载体及稳定产毒的细胞克隆。方法:利用DNA重组技术,设计3条60 bp能转录产生靶向CD3ζ小发卡RNA(shRNA)的寡核苷酸序列,以pSUPER.retro.neo+gfp质粒作为空载体经限制性核酸内切酶酶切后与RNAi序列进行重组,构建CD3ζ-pSUPER retroRNAi逆转录病毒载体,脂质体法转染包装细胞系PA317,建立产生逆转录病毒的细胞克隆。结果:重组载体经限制性核酸内切酶酶切、PCR和测序鉴定表明CD3ζ-pSUPER retroRNAi重组质粒构建成功;脂质体法将重组载体转染包装细胞系PA317,表达绿色荧光蛋白,表明包装成功。结论:成功地构建了特异性沉默CD3ζ基因的pSUPER retroRNAi逆转录病毒载体及稳定产毒的细胞系,为后续研究CD59与CD3ζ在T细胞内信号转导的相互作用提供理论和实验依据。
OBJECTIVE: To construct and screen pSUPER retro RNAi retroviral vectors and stable virus-producing cell clones carrying CD3ζ gene by RNAi expression vector. Methods: Three oligonucleotides encoding 60 bp of CD3ζ targeting small hairpin RNA (shRNA) were designed and synthesized by using DNA recombination technology. The pSUPER.retro.neo + gfp plasmid was used as an empty vector and restriction endonuclease After digested with RNAi sequence, the CD3ζ-pSUPER retroRNAi retroviral vector was constructed, and the packaging cell line PA317 was transfected by lipofectamine to establish a retrovirus-producing cell clone. Results: Restriction endonuclease digestion, PCR and sequencing showed that the recombinant plasmid of CD3ζ-pSUPER retroRNAi was constructed successfully. The recombinant vector was transfected into the packaging cell line PA317 by liposome to express green fluorescent protein, indicating that the packaging was successful . CONCLUSION: The pSUPER retroRNAi retroviral vector with specific silencing of CD3ζ gene and stable cell-producing virus-producing cell line were successfully constructed, which provided theoretical and experimental evidences for further study on the interaction between CD59 and CD3ζ in T cells.