Study On The Gene Synthesis, Prokaryotic Expression And Activity Of Human Granulocyte Colony-stimulating Factor

Hematopoietic cell growth factor is an important component of cell factors, which plays a significant role in the mechanism of blood cells generation and regulation. Human granulocyte colony-stimulating factor (hG-CSF) is a kind of Hematopoietic cell growth factors. Since the Granulocyte colony-stimulating factor (G-CSF) was approved as a drug by the US FDA in 1991.18 pharmaceutical enterprises have been approved to produce hG-CSF in china, including 69 registered number of approval. This dissertation contains gene systhesis. clone, prokaryotic express and bio-activation examination. Through full coden optimization of G-CSF gene. the expression level will be enhanced in order to further reduce the production cost.Mature G-CSF protein contains 174 amino acid residues. According to the codon preference of E.coli. the hG-CSF gene is optimized to 596bp in length. which contains the 5' element restriction endonuclease site, initiation codon. His-tag. protease site, the 3'element termination codon and restriction endonuclease site. The whole sequence is splitted to 16 oligonucleotides by GeneDesigen 3.0 and was synthesized by two-step total gene synthesis method.At the same time, hG-CSF gene is obtained and amplified by another method—RT-PCR technology. Then it is cloned to pET23a vector by the same method as coden optimized gene to construct the expression vector pET-sCSF. The expression vectors which are construsted respectively by coden optimized gene and reverse transcriptase gene transfect to the E.coli 21(DE3) and construct E.coli sCSF. and E.coli hCSF expression strain respectively. Further studies of start time of IPTG induction. duration of induction. induced concentration. and incubation temperature are conducted to test the influence on the expression of G-CSF and its proportion of the total protein in the cell influence. The optimum conditions of expression of foreign genes are as follows:incubation at 37℃6h after inoculation. adding a final concentration of 40μmol/ml of IPTG. and induction 6h. Then harvest cell. and conduct the correlation detections after sonication.The codon-optimized rhG-CSF gene significantly increased protein expression levels in all cells tested. A strong expression vector was constructed. and a simple purification procedure was established. The final expression level of hG-CSF was increased 21.9% compared to the the result of RT-PCR gene. The proportion of hG-CSF in the cell increased 17.2%. And the expressed protein exists mainly in soluble form (90.9%). which accounted for 35.2% of total protein content in the supernatant.