携带同源盒基因HOXA9的逆转录病毒载体的构建和鉴定

Construction and identification of recombinant retrovirus vector containing human homeobox gene HOXA9

目的构建携带同源盒基因(homeobox gene)HOXA9的逆转录病毒载体,并建立稳定产毒的包装细胞株。方法 PCR扩增HOXA9基因编码区全长序列克隆至逆转录病毒载体MSCVneo,经酶切、测序鉴定。将重组载体脂质体转染包装细胞系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。结果重组逆转录病毒载体MSCVneo经酶切、测序鉴定正确。将筛选所得的高效产毒细胞株命名为PT67/MSCVneo-HOXA9,测定病毒滴度为5×105CFU/ml。结论成功构建了携带HOXA9基因的逆转录病毒载体,建立了稳定、高效、准确产生逆转录病毒的细胞株。

Objective To construct a recombinant retrovirus vector containing human homeobox gene HOXA9 and to obtain the packaging cell clones which produce viruses stably.Methods The whole coding region of HOXA9 gene was amplified by polymerase chain reaction（PCR）,and then cloned to the retroviral vector MSCVneo.After restriction endonuclease digestion and DNA sequencing,the recombinant vector was identified and transfected into the packaging cell line PT67 by liposome LipofectamineTM2000.The cell clones producing retrovirus stably were isolated by G418 selection.The viral suspension was harvested and the viral titer was determined by NIH3T3 cells.Results The recombinant retroviral vector,including HOXA9 genes,was determined by the restriction endonuclease digestion and sequencing.The cell clones efficiently producing virus were screened by G418 and designated as PT67/MSCVneo-HOXA9 and its titer was 5×105 CFU/ml.Conclusion We have successfully obtained the recombinant retrovirus vector containing homeobox gene HOXA9 and the packaging cell lines,which could produce viruses efficiently and correctly.This study provides a basis for the further research on the function of HOXA9 gene in the regulation of hematopoiesis.