Human Granulocyte-Macrophage Colony Stimulating Factor (hGM-CSF) Gene Expression In Anabaena Sp.PCC7120 And The Reconstruction Of Its Expression System

Human Granulocyte-Macrophage Colony Stimulating Factor (hGM-CSF) is the first haematopoietic cytokines that has been cloned and been utilized by recombinant DNA technology. It has potent effects in stimulating proliferation, maturation and function of haematopoietic cell. It occupied large percent of recombinantion medicine market. In the meantime, commercial produced hGM-CSF usually use E. coli to be the host. For its disadvantages of overexpression and endotoxin, people try to express hGM-CSF in many other hosts just as yeast, mammal, hexapod, etc. But the results have not been optimized. Cyanobactreria as the potential candidate host to produce genetic engineering medicine have simple structure and makes them be a potentially powerful model system of modern biotechnology, and they provide advantageous hosts to produce organic substances. Based on these advantages, we attempt to express hGM-CSF gene in cyanobacterial cell.In this article, base on successfully expressed hGM-CSF in Anabaena sp. PCC 7120, we modified hGM-CSF gene by add shine-dalgarno(SD) sequence in N-terminal and adjusted the distance between it and ATG which affects the efficiency of translation. Then inserted it into downstream of the promoter PpsbA, ligated it with pDC-08 to contruct the shuttle expression vector, pDC-GMs. Then transformed it into Anabaena sp. PCC 7120 by the triparental conjugation transfer method. Gained transgenic sp. PCC 7120 harboring pDC-GMs by screening with neomycine. Then optimized the culture conditions to found the restrict curver factors. And for the reason of the prophase shuttle vectors which we constructed have the unstable disadvantage, we cloned glnA gene which encoding glutamine synthetase by TD PCR in Anabeaba sp. PCC 7120 as a integrative platform. Then, an integrative expression vector pRG-GM which contained hGM-CSF gene and endogenous promoter PpsbA was constructed. It provides reference to improve the expression level and the stability of foreign hGM-CSF gene in Anabeaba sp. PCC 7120.The contents and results of this experiment were as follows:1. We add -GGAGAG- SD sequence in hgm-CSF N-terminal and adjusted the sequence between SD and ATG into 6bp by PCR. Then inserted it into downstream of the strong promoter, PpsbA of shuttle vector pRL-439, then ligated it with pDC-08 to contruct the shuttle expression vector, pDC-GMs. Then transformed it into Anabaena sp. PCC 7120, by the triparental conjugation transfer method. Screening of positive clones were performed on BG-11(-N) agar plates supplemented with neomycine. PCR amplification of...