[Cloning and secretory expression of soluble interleukin-6 receptor gene in Streptomyces lividans].
With the total RNA extracted from HeLa cells as templates of retrotranscription, the 1,017 bp cDNA fragment of human soluble Interleukin 6 receptor (sIL-6R) was amplified by RT-PCR technique. The amplified cDNA fragment was cloned into plasmid pUC19 for DNA sequence analysis. The result showed that the cDNA fragment is identical to natural sIL-6R encoding sequence reported before. The sIL-6R cDNA was fused with melC1 signal peptide encoding sequence. The fusion gene (mel/sIL-6R) was inserted into Streptomyces plasmid pIJ459. Both Southern blot analysis and restriction enzymatic analysis proved that the sIL-6R cDNA has been inserted into pIJ459 and the recombinant plasmid was named pIJ459-mel/sIL-6R. pIJ459-mel/sIL-6R was transformed into S. lividans TK54 and got the recombinant strain S. lividans [pIJ459-mel/sIL-6R]. SDS-PAGE and receptor-ligand binding assay show that sIL-6R was expressed by this strain and the maximum level is at 72 h. It was also proved that the recombinant sIL-6R has biological activity of binding IL-6.
Wang
X Q
XQ
Institute of Medicinal Biotechnology Chinese Academy of Medical Sciences (CAMS) & Peking Union Medical College (PUMC), Beijing.
Li
Y
Y
D015139:Blotting, Southern; D003001:Cloning, Molecular; D006367:HeLa Cells; D006801:Humans; D015850:Interleukin-6 / Q000378:metabolism; D019947:Receptors, Interleukin-6 / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis; D013302:Streptomyces / Q000235:genetics*
D015850:Interleukin-6; D019947:Receptors, Interleukin-6; D011994:Recombinant Proteins