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目的 构建并稳定携带人p5 8.2基因的重组逆转录病毒载体及稳定的包装细胞系。方法 用PCR技术从pSPORT1 -p5 8.2扩增出编码p5 8.2N端 1～ 345个氨基酸的全长基因 ,定向克隆入逆转录病毒载体pMSCV neo中 ,酶切鉴定 ;脂质体法将重组逆转录病毒转入包装细胞系PT6 7,G4 1 8筛选建立稳定产病毒的细胞株。结果 双酶切鉴定 ,成功构建重组逆转录病毒载体 ;重线逆录病毒载体转入包装细胞 ,经G4 1 8筛选 ,形成了抗性克隆 ,并测得病毒滴度为 3× 1 0 5cfu/ml,提示构建携带人p5 8.2基因的逆转录病毒载体及稳定的包装细胞系成功。结论 构建人p5 8.2基因的逆转录病毒载体可行 ,并且可望成为一种很好的对血液系统疾病进行基因治疗的方法
Objective To construct and stabilize recombinant retroviral vector and stable packaging cell line carrying human p5 8.2 gene. Methods The full-length cDNA encoding a peptide of 1 ~ 345 amino acids at the N-terminus of p5 was amplified by PCR from pSPORT1 -p5 8.2 and cloned into the retroviral vector pMSCV neo. The recombinant plasmid was reverse transcribed The virus into the packaging cell lines PT6 7, G4 1 8 screen to establish stable virus-producing cell lines. Results Recombinant retroviral vector was successfully constructed by double enzyme digestion. The recombinant retroviral vector was transfected into packaging cells and screened by G418 to form resistant clones. The virus titer was 3 × 105cfu / ml, suggesting that the construction of a retroviral vector carrying a human p5 8.2 gene and a stable packaging cell line were successful. Conclusion The construction of a retroviral vector for human p5 8.2 gene is feasible and is expected to be a promising method for gene therapy of hematological diseases