| IL-1 is a kind of important cytokine which displays extensive biological activities. Its effects are exerted via IL-1RI. IL-1 takes part in many pathological processes. It is a therapy to decrease IL-1 activity. Soluble forms of IL-1RI(sEL-1RI) are produced from proteolytic cleavage of the extacellular portion of the receptors on cell surface. It is still able to bind with IL-1 and can be used to treat some diseases as an antagonist of IL-1RI or screen new drugs as a target. In this study, we focused on the cloning and expression of human sIL-1RI in Streptomyces lividans TK54.A new plasmid pSGL1 was isolated from S.globisporus in our laboratory. To develope it as a useful vector, the nucleotide sequence of the minimal replicon of plasmid pSGL1 was confirmed .Compared with the database of Genebank, it is a new sequence and belongs to the rolling-circle replication plasmid . With the total DNA of S.globisporus as template, the DNA fragment involving C1027 apoprotein signal peptide encoding sequence (gpp) was amplified by PCR. After this fragment was inserted into the derivative plasmid of pSGL1, one new Streptomyces -E.coli shuttle plasmid was obtained namely as pSGLgpp.With the total RNA of HepG2 cell as template , the cDNA of human sIL-1RI was amplified by RT-PCR technique. The cDNA of hsIL-1RI was fused with melC1 signal peptide encoding sequence downstream in correct reading frame. The fusion gene was cloned into pIJ702. At meantime, the cDNA of hsIL-1RI was fused with gpp downstream in pSGLgpp. The results of Southern blot showed that both of the two recombinant plasmids contain the hsIL-1RI cDNA.To investigate the secrected expression of hsBL-1RI controlled by gpp and melC1 in streptomyces, the two recombinant strains were cultured with shaking in a medium containing enzymatic hydrolysate casein up to 72hr respectively. SDS-PAGE and... |
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