【摘要】 利用多聚酶链反应(PCR)方法,从活化的人外周血单个核细胞中克隆了人粒-巨噬系集落刺激因子(GM-CSF)的cDNA。DNA序列测定证实此片段为完整的GM-CSF cDNA。应用DNA重组技术,将此cDNA重组于逆转录病毒载体pDORneo上,以Lipofectin介导转染病毒包装细胞PA317,用NIH3T3细胞测定病毒滴度。选取高滴度病毒上清感染中国仓鼠卵巢(CHO)细胞,经G418筛选获抗性克隆,PCR方法鉴定重组载体整合于CHO细胞的基因组DNA中。用CFU-GM集落形成实验检测GM-CSF活性,证实转染后的CHO细胞有GM-CSF的稳定、高效表达。更多还原

【Abstract】 A DNA seguence encoding human granulocyte-macrophage colony-stimulating factor (GM-CSF) has been cloned by reverse transcription and polymerase chain reaction (RT-PCR) from mRNA derived from activated human peripheral blood Ivmphocvtes. The recombined retroviral vector pDORGM was constructed by cloning the GM-CSF cDNA into the replication defective retroviral vector pDORneo, and transferred into packaging cell line PA317 by Lipofectin method. The colonies were setected in G418 and determined the virai titer with NIH3T3 cell. The clone supernatant with higher titer was used to infect the Chinese hamster ovary (CHO) cells. The drug-resistant cells were cloned and ex-panded in G418 medium. PCR was used to identify the integration of the fusion neo gene in genomic DNA of G418-resistant colonies. The GM-CSF activity assay in the supernatant demonstrated that transferred CHO celis stably secreting human GM-CSF. These results provided a basis for study of GM-CSF in hematopoiesis regulation and use in cancer gene therapy.更多还原