摘要
本研究构建携带同源盒基因hoxA10的重组逆转录病毒载体,并建立稳定产毒的包装细胞株。通过PCR扩增获得hoxA10基因编码区全长序列,克隆至逆转录病毒载体MSCVneo,并测序鉴定插入的hoxA10基因。将重组载体及空载体经脂质体分别转染包装细胞系PT67,以G418筛选稳定产毒细胞株。收集病毒悬液并测定病毒滴度。结果表明:重组逆转录病毒载体MSCVneo插入的hoxA10基因序列正确。将筛选所得的高效产毒细胞株命名为PT67/MSCVneo、PT67/MSCVneo-hoxA10。测定病毒滴度分别为5×105CFU/ml、4×104 CFU/ml。结论:成功构建了同源盒基因hoxA10的重组逆转录病毒载体,建立了稳定、高效、准确产生逆转录病毒的细胞株,为探讨hoxA10基因在造血干细胞的增殖和分化功能提供了实验基础。
This study was purposed to construct the recombinant retroviral vector containing human homeobox gene hoxA10 and to establish the packaging cell lines which stably produce viruses. The whole coding region of hoxA10 gene was amplified by PCR and inserted into the retroviral vector MSCVneo. The recombinant vector was identified by DNA sequencing, The recombinant and control retroviral vectors were transfected into the packaging cell line PT67 by liposome Lipofectaminean^TM2000. These cell lines stably producing retrovirus were isolated following G418 selection. The viral suspension was harvested and the viral titer was determined by N1H3T3 cells. The results showed that the recombinant retroviral vector was proved to encode hoxA10 genes by sequencing. The cell lines efficiently producing virus were screened by CJ418 and designated as PT67/MSCVneo and PT67/MSCVneo-hoxA10. The titers of them were 5 × 10^5 CFU/ml and 4 × 10^4 CFU/ml respectively. It is concluded that the recombinant retroviral vector containing homeobox gene hoxA10 and the stably packaging cell lines which efficiently and correctly produce viruses are successfully constructed, which provides a basis for further exploration of the hoxA10 gene function in the regulation of hematopoiesis.
出处
《中国实验血液学杂志》
CAS
CSCD
2010年第3期683-685,共3页
Journal of Experimental Hematology
基金
浙江省医药卫生科学研究基金(2006A020
2009A046)
