Study On Construction Of Transgenic Anabaena With The Gene Coding RhG-CSF And Its Its Characteristics

Granulocyte colony-stimulating factor (G-CSF) is an important hematopoietic cytokine which stimulates the basal stress-induced production of neutrophils and enhances some of anti-microbial process. Production of G-CSF in large quantities as a recombinant protein in Escherichia coli has allowed its clinical use as a drug to treat neutropenia due to a variety of causes. There are some problems in the process of producing G-CSF from Escherichia coli .For example, the price is high for the complex process purifying G-CSF; the purified protein is easy to decompose. Cyanobacteria are remarkable group of simple photosynthetic micro-organisms which belong to prokaryotic organisms. For their extensive distribution, strong adoptability, and rapid growth, their cultural costs are low. So they have been regarded as a model organism in gene engineering. With the development of the cyanobacteria gene engineering, cyanobacteria are paid more attention as a host of gene engineering. The collectivity of our laboratory has attempted to produce G-CSF with cyanobacteria gene engineering. However, we didn't acquire transgenic cyanobacteria which express protein effectively by now. This work is to construct shuttle expression vector with the gene G-CSF whose 5'was modified and express G-CSF successfully in filamentous cyanobacterium Anabaena sp.PCC7120. In order to increase the expression effectively, the codons of the first four amino acids of G-CSF were altered by redesigning PCR primers on the principle of codon degeneracy. The middle vector pUC-G-CSF was gained by cloning the correct gene into vector pUC-19 between EcoRI and BamHI sites ;And then, the middle vector pUC-G-CSF was inserted between two BamHI sites in pRL-489, after the promoter PpsbA to construct shuttle expression vector pRL-G-CSF. Correct connection was certified by restriction analysis and the gene sequence is valid by sequencing. The vector pRL-G-CSF was introduced into Anabaena sp. PCC 7120 by triparental conjugative transfer. The transformation was confirmed by PCR amplification and restriction analysis. After transferring into, the expression efficiency is 0.0409% which was measured using ELISA. Assaying photosynthetic light curve proved that expression of the G-CSF gene enhanced respiration of host, which aggravates the metabolic burden. Compared with the wide type, the true photosynthetic of transgenic Anabaena enhanced 15.5%.Assaying the growth curve of transgenic and wild type Anabaena sp. PCC 7120,the cell numbers of transgenic Anabaena enhanced 89% when culturing 20 days in liquid medium, which are helpful to culture transgenic Anabaena in scale. No report has been seen in this field up to now. In this work, the expression of G-CSF gene in cyanobacteria may be first reported although the expression efficiency was rather low. This work will be continued until recombinant G-CSF from cyanobacteria will be production in scale.