Cloning And Expression Of Human Soluble Interleukin 6 Receptor In Insect Cells And Developing Its Antagonists Screening Model

Interleukin 6 (IL-6) , which displays a broad range biological activities on many kinds of cells, such as lymphocytes, hematopoietic stem cells, liver cells and neuronal cells, is a kind of important cytokine. Additionally, IL-6 is involved in many pathology processes of disease such as inflammation, rheumatoid arthritis, tumor and autoimmune diseases.IL-6 exhibits its role on target cells through a double-stranded receptor complex consisting of IL-6R and a signal transduction subunit (gpl30).IL-6R is one of transmembrane glycoprotein which has two types of natural receptors: gp130 binding with membrane and soluble IL-6R.Both of them play essential role during the process of IL-6 signal transduction.A novel antagonist screening model has been developed with recombinant hsIL-6R from Streptomyces lividans as target based on the competitive receptor-ligand interactions in this study. Using this model, we have screened 500 microorganisms metabolic products and found 10 positive strains. The positive ratio is 2%.And, the hsIL-6R gene was cloned and expressed in insect cells. The cDNA of hsIL-6R was obtained by RT-PCR technique with total RNA from HepG2 cells as template and then cloned into pUC19 for DNA sequencing. The result showed that it is same as the sIL-6R cDNA sequence reported before. Then sIL-6R cDNA was cloned into transfer vector pAcGP67B. The recombinant transfer vector was cotransfected with wild type AcMNPV DNA into insect cells with modified calcium phosphate precipitation method. The recombinant baculovirus was produced by homologous recombination in vivo. Followed this, we purified the recombinant baculovirus by plaque assay and end-point...