作　　者： ; ; ; ;

机构地区： 广州医学院第二附属医院

出　　处： 《山东大学耳鼻喉眼学报》 2006年第2期105-107,123,共4页

摘　　要： 目的:克隆人粒细胞巨噬细胞集落刺激因子全长cDNA以构建相应腺病毒表达载体。方法:根据人粒细胞巨噬细胞集落刺激因子基因序列设计合成可扩增人粒细胞巨噬细胞集落刺激因子cD-NA的特异性引物,用RT-PCR法从骨髓细胞总RNA中扩增人粒细胞巨噬细胞集落刺激因子cDNA,并克隆至pGEM-T载体中,经酶切鉴定后再行序列分析。结果:逆转录多聚酶链反应扩增产物长度与预期的456 bp一致;用M13正、反向引物行荧光测序,证实克隆出的序列与GenBank的人粒细胞巨噬细胞集落刺激因子cDNA序列完全一致;人粒细胞巨噬细胞集落刺激因子全长cDNA被成功地插入到质粒pGEM-T中。结论:克隆人粒细胞巨噬细胞集落刺激因子全长cDNA为构建相应腺病毒表达载体及应用B7-1行肿瘤免疫基因治疗提供了可能性。 Objective： To construct recombinant granulocyte-macrophage colony-stimulating factor （GM- CSF） retrovirus expressing vector by cloning of GM-CSF genes from human blood and construct the recombinant plasmids encoding for GM-CSF. Methods： Primers for GM-CSF were designed and synthesized according to the sequences of human GM-CSF genes derived from GenBank. The full length cDNA of GM-CSF was cloned by RT-PCR techniques. The recombinant plasmids pGEM-T-GM-CSF were constructed by recombinant gene techniques. Results： The length of RT-PCR product coincided with that of authors＇ anticipation（456bp）,and the recombinant plasmid was confirmed by Xba I /Not I restriction enzyme digesting. The sequencing result of the cDNA was identical to the sequence of GM-CSF cDNA in GenBank, and the full length cDNA of human GM-CSF was successfully inserted into the vector of pGEM-T. Conclusion： The successful cloning of human GM-CSF cDNA, as well as construction of its retrovirus expressing vector enables us to further investigate the role of GM-CSF in tumor immunogene therapy.

关 键 词： 粒细胞巨噬细胞集落刺激因子 基因 克隆 分子

领　　域： [生物学]