高滴度的含有目的基因IL-RA的逆转录病毒的包装和浓缩

Packaging and concentrating of high titer of retrovirus containing IL-4RA

摘要:

目的：构建具有高效转染率且含有标记基因的携带白介素4受体拮抗剂（IL-4RA）基因的重组逆转录病毒载体pLNC-IL-4RA。方法：将IL-4RA基因插入pLNC-Laz逆转录病毒载体获得重组的逆转录病毒表达载体pLNC-IL-4RA，重组的逆转录病毒表达载体pLNC-IL-4RA通过质脂体转染PA317细胞进行包装，PA317抗性克隆经过G418的抗性筛选产生并扩增，将含有病毒的PA317细胞培养液上清收集后转染NIH3T3细胞进行病毒滴度的测定。结果：扩增后的G418抗性克隆经过检测和滴度测定结果显示含有目的基因的逆转录病毒最高的病毒滴度为1×104CFU/mL。选择含有最高病毒滴度的PA317细胞抗性克隆进行基因组的提取和鉴定，结果显示目的基因IL-4RA成功地整合到了宿主的基因组中。结论：含有目的基因IL-4RA的逆转录病毒载体经过PA317细胞包装后获得了较高的病毒滴度，为下一步哮喘模型的基因治疗打下了基础。［中国当代儿科杂志，2009，11（9）：761-764］

Abstract:

OBJECTIVE: To construct a recombination retroviral expression vector pLNC-IL-4RA with high efficiency transfection and carrying a screening label. METHODS: IL-4RA was inserted into retroviral vector pLNC-Laz to get recombination retroviral expression vector pLNC-IL-4RA and then transfected into packaging cell line PA317 by liposome transfection. The transfected PA317 cells were obtained and amplified by G418 pressure screening. The cell culture supernatants containing viruses were harvested and the viral titer was determined by NIH3T3 cells infection. RESULTS: The G418 resistant clones were titrated and checked for the presence of replication virus. The results showed that the highest titer of viral supernatant was 1×104 CFU/mL. Genome DNA isolated from the cell clone of the highest titer showed the function gene, IL-4RA cDNA, had integrated into the genome of host cells verified by PCR. CONCLUSIONS: The recombination retroviral vector pLNC-IL-4RA encoding IL-4RA after packaging PA317 cells have higher viral titer. This provides a basis for gene treatment of asthma.［Chin J Contemp Pediatr, 2009, 11 (9):761-764］