▽5-人粒系集落刺激因子(▽5-hG-CSF)的cDNA克隆、序列测定与表达

cDNA Cloning, Sequencing and Expression of ▽5-Human Granulocyte Colony-Stimulating Factor (V5-hG-CSF)

从LPS刺激的正常中国人外周血单核细胞中提取mRNA,经反转录(RT)-PCR扩增出不含信号肽和成熟型N端5氨基酸(V5-hG-CSF)的cDNA片段,酶切后组入大肠杆菌表达载体pJLA602中。序列测定表明,克隆片段与国外报道的高活性hG-CSF cDNA序列一致。重组子经诱导表达、小鼠骨髓细胞体外CFU-G测试表明,表达产物具明显的粒细胞集落刺激活性。

Macrophages/monocytes were isolated from freshly prepared peripheral blood of healthy Chinese human adult volunteers. After culture for 1,5 hour the non- adherent cells were removed. The resulting adherent cells were incubated with E. coli LPS, then mRNA was isolated and used to reverse-transcription(RT)-PCR. The amplified product of human granulocyte colony-stimulating factor deleted N-terminal 5 amino acids of mature form (▽5-hG-CSF) cDNA was digested with Nco I,Bam HVI, cloned into the bacteria expressing vector pJLA602, and screened by in situ hybridization with the specific probe for high active form of hG-CSF cDNA. The recombinants were further identified by DNA sequencing which showed that the cloned ▽5-hG-CSF cDNA encode for the high active form of hG-CSF with no substitution among the sequenced region(390bp), and induced to express ▽5-hG-CSF in E.coli. The lysates of expressing bacteria show obvious granulocyte colony-stimulating activity as assay in vitro.