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目的构建携带人RPS14基因重组慢病毒载体获得稳定产毒的细胞,并观察其在人MDS细胞株MUTZ-8细胞中的表达。方法通过反转录方法,获得目的基因RPS14;将目的基因克隆入慢病毒表达质粒pGC-FU Vector,构建含有RPS14基因的重组慢病毒载体pGC-FU RPS14,经PCR、酶切及测序鉴定其正确性;在Lipofectamine 2000介导下,将成功构建的质粒pGC-FU RPS14与慢病毒包装质粒pHelper 1.0、pHelper 2.0共转染293T细胞,通过实时定量PCR法测定病毒滴度,并将获得的重组慢病毒载体pGC-FU RPS14转染人MDS细胞株MUTZ-8,用流式细胞仪检测转染效率,RT-PCR检测靶细胞内RPS14 mRNA的表达。结果含有RPS14基因的重组慢病毒载体经PCR、酶切和测序鉴定构建正确,且测定的病毒滴度为2.0×109 TU/ml。流式细胞仪检测其转染效率为68.41%,RT-PCR检测到转染后RPS14 mRNA在靶细胞中的稳定表达。结论构建了携带人RPS14基因的慢病毒载体,转染人MUTZ-8细胞株后能够稳定表达RPS14基因。
Objective To construct a stable lentiviral vector carrying human RPS14 gene and obtain the stable gene-producing cells and observe its expression in human MDS cell line MUTZ-8. Methods The target gene RPS14 was obtained by reverse transcription method. The target gene was cloned into lentiviral vector pGC-FU Vector to construct recombinant lentiviral vector pGC-FU RPS14 containing RPS14 gene. The recombinant plasmid pGC-FU RPS14 was identified by PCR, restriction enzyme digestion and sequencing The recombinant plasmid pGC-FU RPS14 and pHelper 1.0 and pHelper 2.0 were co-transfected into 293T cells by Lipofectamine 2000. The virus titer was determined by real-time quantitative PCR. The vector pGC-FU RPS14 was transfected into human MDS cell line MUTZ-8. The transfection efficiency was detected by flow cytometry. The expression of RPS14 mRNA in target cells was detected by RT-PCR. Results The recombinant lentiviral vector containing RPS14 gene was correctly constructed by PCR, restriction enzyme digestion and sequencing, and the virus titer was 2.0 × 109 TU / ml. The transfection efficiency was 68.41% by flow cytometry. The stable expression of RPS14 mRNA in target cells was detected by RT-PCR. Conclusion A lentiviral vector carrying human RPS14 gene was constructed and transfected into human MUTZ-8 cell line stably expressing RPS14 gene.