Expression,Purification And Assays Of Recombinant Human Interleukin-6 In E.Coli

Interleukin-6(IL-6)is produced by T cells,lymphoid cells,horn cells,endothelial cells,fibroblastsand monocytes in the activated state,which is a multieffect cytokine.In 1986,the genetic structure of IL-6 was cleared and definited,and was officially named IL-6 at the Sixth Sessions Meeting of Immunology.IL-6 basically has effect on the organism through autocrine or paracrine modes.It can regulate the effection of other cytokines,thus play a major role in immunologic response,acute phase reaction and regulation of hematopoiesis.IL-6 can active target gene,no only acts as the differentiation factor and growth factor of endothelial cells,B cell,T cell,hematopoietic cell and osteoclast,etc,but also plays an important role in peripheral nervous cell and axoneure growth,differentiation,regeneration and degradation.IL-6 can recover and improve hematopoietic function,stimulate produce of platelets.On the other hand,some disease aways has a relation to IL-6 overexpression.In conclusion,IL-6 is used in thrombocytopenia due to radiotherapy and chemotherapy,the study and treatment in tumour,liver injury and so on has a broad application prospect.In this paper,using genetic engineering technology,build efficient expression of recombinant expression of IL-6 strain,pilot scale,fermentation and purification,get high active and high purity reorganization of IL-6,and its physical and chemical properties were analyzed.According to IL-6 amino acid sequence,using E.coli preference codon design nucleic acid sequence and primer,through the primer PCR technology to paragraphs joining together,get the full sequence cDNA of IL-6.And chose pCold ? build Expression strains,then the exogenous gene inserted into expression vector pCold ?,We chose the E.coli BL21 as the host.and the high expression strain was screened.The engineering strain was cultivated in 200 L fermenter at optimized fermentation conditions.The inducer was IPTG.Bacteria was collected by Centrifuge and was broke to get inclusion body,after dissolution,centrifugal,affinity chromatography capture,obtain the fusion protein.Fusion protein was desalted,and then be digested by EK enzyme,the affinity chromatography,size exclusion chromatography,such as purification steps,to obtain high active and high purity rhIL-6.In order to verify and control the quality of rhIL-6,the rhIL-6 acti-vity was measured by cell B9-11 proliferation assay.The purity of the final products was analyzed by electrophoresis and HPLC.The molecular weight was analyzed by the method of mass spectrometry.The N-terminus of IL-6 was analyzed by method of Edman degradation.The results showed that the HPLC-SEC purity of rhIL-6 was 87%and the electrophoresis purity was more than 95%.The results of mass spectrometry indicated that the molecular weigh of glycosylated rhIL-6 was 20894 Dalton.As the same as the theoretic value,The N-terminus is correct.In conclusion,the high expression of rhIL-6 in E.coli was realized by genetic engineering technology.The methods of fermentation and purification rhIL-6 were established.The physicochemical property of rhIL-6 was analysed.The results in the paper would be helpful to pilot-scale study.