Cloning, Expression And Preliminary Pilot-scale Production Study Of RhIL-6

Interleukin-6 (IL-6) is a multifunctional cytokine, which is produced by a variety of cells and exerts a wide spectrum of biological function on a broad range of target cells. It plays not only a pivotal role in both immune and inflammatory responses, but also a primary role in regulating hematopoiesis, enhancing platelet production and reconstructing bone marrow. Now recombinant human IL-6 has been on phase II trial in abroad . In China the research and development of IL-6 has been paid more attention.Human IL-6 is a 21 — 28 kd glycoprotein comprising 184 amino acid residues. It contains two disulfide bonds which are functionally important.We cloned human IL-6 cDNA by RT-PCR with the RNA extracted from PHA-treated human PBMC. The human IL-6 cDNA we cloned was the same as that reported except the 288th base T was replaced by C. By mutation PCR, this site restored. After we had got the right human IL-6 cDNA, we inserted the IL-6 cDNA into the expression vector pBV220. Furthermore, we optimized the distance between SD sequence and the initiating code ATG. We find when the distance between SD and ATG was 6bp or lObp, there was no obvious band on SDS-PAGE. When the distance were 5bp, 7bp, 8bp, 9bp, the expression level was high.The highest expression level of IL-6 accounts 26% percent of the total cell protein. The expression product existed in the form of inclusion body. The inclusion body was extracted, solubilied, refolded, and purified by anion exchange (Q Sepharose HP). The purity of the product was about 95%. The purity of IL-6 was higher than 98% when it was purified again by gel filtration (Superdex 75). The activity of the final product was 1.1×10~8U/mg by MTT assay method. With some methods, such as western blotting, IEF and N-terminal sequencing, we proved the product was what we expected. We have a good beginning of pilot-scale production.