Human Granulocyte-Macrophage Colony Stimulating Factor (hGM-CSF) Gene Expression In Anabaena Sp. PCC7120 With Promoter

Cyanobacteria are autotrophic prokaryotes performing oxygenic photosynthesis, who can use light, carbon dioxide, and other inorganic nutrients efficiently. Due to their properties, such as wide-spread, simple growth requirement, simple structure, large-scale culture and clean bioprocesses, cyanobacteria become a potentially powerful model system of modern biotechnology, as well as for the study of higher plant processes. Concerning their simple structure and ease of handling, cyanobacteria make an ideal and economical host for expression of valuable foreign proteins such as genetic engineering medicine. 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 recombination medicine market. hGM-CSF expression in cyanobacteria has been researching in our lab since 2001. However, the level of expression for the product of hGM-CSF in cyanobacteria hampers the utilization of these transgenic cyanobacteria.In order to improve the expression level of foreign gene in cyanobacteria, based on the character of Anabaena sp.PCC 7120 RNA polymerase and the structure of promoter. we select two cyanobacteria endogenesis promoters---- Pcpcβwhich the 340 bp upstream fragment of cpcβgene encodes theβ-subunit of phycocyanin and a heat shock promoter PgroESL which indwells in the groESL operon of Synechococcus sp.PCC 7942 .The two promoters were obtained by polymerase chain reaction (PCR) from genomic DNA of cyanobacterium Synechococcus sp.PCC7002 and Synechococcus sp.PCC 7942 respectively. Then integrative expression vector pDC-C-GM which contained promoter Pcpcβand hGM-CSF foreign gene, pDC-G-GM which contained PgroESL and hGM-CSF gene , were constructed and introduced into Anabaena sp.PCC 7120 via triparental conjugation transformation. Selected by neomycin,the stable transgenic cyanobacterium were obtained. PCR analysis indicated the existence of vectors pDC-C-GM,pDC-G-GM and Western blotting demonstrated the expression of hGM-CSF in Anabaena sp.PCC 7120.The contents and results of this experiment of were as follows:1.Pcpcβwas cloned by PCR from genomic DNA of marine cyanobacterium Synechococcus sp.PCC 7002. Then inserted it into upstream of the foreign gene hGM-CSF and ligated with pDC-08 to construct the shuttle expression vector pDC-C-GM , transformed it into Anabaena sp.PCC 7120 by triparental conjugation method. Selected by neomycin,the stable transgenic cyanobacterium cGM was obtained .2 . Obtained the promoter PgroESL was by PCR from genomic DNA of Synechococcus sp.PCC 7942. Then placed the foreign gene hGM-CSF into the downstream of PgroESL and ligated with pDC-08 to construct the shuttle expression vector pDC-G-GM ,induced it into Anabaena sp.PCC 7120 by triparental conjugation transformation. Selected by neomycin and obtained the stable transgenic cyanobacterium gGM .3.PCR amplification of wild type and two transgenic Anabaena sp.PCC 7120 cells(cGM and gGM strain) with specific primers revealed no band in wild type and plasmid free cells, whereas transgenic cGM cells yielded a about 750 bp band.( Pcpcβand hGM-CSF) and transgenic gGM cells yielded a about 625 bp band.( PgroESL and hGM-CSF).4.Results of the growth curve of cells over time were showed as follows: In the extension of this experiment, the optimal growth temperature and PH of cGM and gGM transgenic Anabaena strain are 30℃, 9.5 respectively and the apt light intensity is 60-90μmol m-2·s-1 .These messages will help us to optimize growth of the two transgenic cyanobacteria, further express more hGM-CSF in the transgenic cells.5.Western blotting GM1,cGM and gGM transgenic Anabaena strain analysis further confirmed that hGM-CSF is expressed in the two transgenic Anabaena sp.PCC 7120 harboring pDC-C-GM and pDC-G-GM. Moreover, the expression level of hGM-CSF in cGM strain with promoter Pcpcβis 90%. as compared to GM1 strain with PpsbA and the expression level of hGM-CSF in gGM strain with promoter PgroESL by 42℃induced 1h is 459% as compared to GM1 strain.The aim of the present study is to make a higher-level expression of hGM-CSF in cyanobacteria cell by replacing stronger promoters and optimize growth of the two transgenic cells by investigating various conditions. In a word , all the results proved that two promoters were cloned and heightened the level expression of hGM-CSF in Anabaena sp.PCC 7120 . The conclusions offer the evidences for the further produce genic engineering medicine and how to enhance the expression of foreign gene in cyanobacteria.