Studies On The Expression Of HGM-CSF In Anabaena Sp.PCC 7120 And The Effects On Its Efficiency By The Gene Modification

It is well known that since the first recombination medicine, human insulin, had beenappeared in America in 1982, more and more recombination medicines were come intomarket. Further investigations are found that recombination medicines will be a hotspot inthe medicine fields either in nowadays or in the future.Human granulocyte-macrophage colony stimulating factor (hGM-CSF) is one of afamily glycoprotein cytokines that have the potent effects in stimulating proliferation,maturation and function of hematopoietic cell. In 1977, GM-CSF was purified from lungtissues of mice that had been pre-injected with endotoxin; however, due to its strongbiological activity at minute amounts, starting material from 100,000 mice was required.Thus, it suggested that a potential autocrine role for hGM-CSFwould overexpress theprotein in host cell. Experimental overexpression of the hGM-CSF protein has beenachieved using two strategies: establishment of transgenic mice, and insertion of retroviralvectors expressing GM-CSF into the bone marrow of mice, but both of them are notsuccessful.Cyanobacteria are autotrophic prokaryotes with perform oxygenic photosynthesissimilar to that of higher plants, and they provide advantageous hosts to produce organicsubstances, since cyanobacteria use solar energy, CO2, H2O, and inorganic substances veryefficiently. Consequently, we can obtain useful organic products with little expenditure ofenergy and resources. It is quite possible that recombinant protein products accumulated incyanobacteria, in some instance, may not require further processing and purification,offering the option of oral delivery. Based on these advantages, we attempt to expresshGM-CSF gene in cyanobacterial cell.Although many expression vectors of cyanobacteria have been reported, expressionlevels of the foreign genes have not been optimized. The upstream sequence of hGM-CSFgene includes a GC-rich region and some codons that are discriminated in prokaryoticorganism. A pair of oligo-nucleotide primers was design to modify at the matureN-terminal cDNA sequence of hGM-CSF according to the degeneracy codon rules, selectfor prokaryotic usage codons, as well as add cyanobacterial SD sequence to promotehigh-level expression by PCR. The DNA fragment enoding mature hGM-CSF was insertedinto downstream of the strong promoter, PpsbA of shuttle vector pRL-439, then ligatedwith pDC-08 to produce the shuttle expression vector, pDC-GM₁. At the same time, theshuttle expression vector, pDC-GM₀ was constructed according to pDC-GM₁ method as anexperimental control without any modifications. Then pDC-GM₀ and pDC-GM₁ were...