Optimization Of Expression And Purification And Identification Of VMIP-â…¡ In E.coli Cells

Objective This study is to establish an effective method for expression and purification of vMIP-II encoded by viral gene to homologize human chemokine nMIP-1 .This study is the bases of clinical application and acquiring amounts of single strand vMIP-II which acts as an antagonist of chemokine receptors.Methods The expression of vMIP-II in transformed bacteria was induced by IPTG, fusion protein was purified by affinity chromatography of amylose resin, aimed protein was tested by SDS-PAGE and Western blot, the bioactivity of vMIP-II was identified by Transwell plate and 96 wells plate coaded by FN. The affects of vMIP-II on toxity and proliferation to PBMC were detected by MTT.Results The fusion protein of vMIP-II/MBP was highly expressed in E. coli. High expressions were obtained in TB medium with 0. 3 mmol/L IPTG at 28癈 inducing for 4h.High purification was also got at the conditions of P2 buffer with 30 % sucrose, 50 ml MgS04 with the concentration of 5 mmol/L MgS04 per gram wet bacteria. Using these methods we reached high active vMIP-II which inhibits the chemotaxis, adherence of PBMC by 50 %. The cellular toxity was low and had no inhibitory effect to PBMC' s proliferation stimulated by PHA was shown.conclusions 1. The temperature of induction and types of medium could influence the expression of vMIP-II/pMAL-P in transformed E. coli. The concentration of IPTG didn' t affect the expression of fusion protein.2. The fusion protein fused at C - end with MBP detected by Western blot proved that pMAL-P vector expressed the MBP fusion protein successfully and had no error stop of transcript.3. Affinity chromatography of MBP fusion protein relied on thebinding of ligand and ligand body, which were not affected by the concentration of MgSO4. Many other proteins in the supernatant could interfere the binding of destined protein. 4. By affinity chroraatography of vMIP-II/MBP fusion protein, destined proteins were demonstrated with high purity and high activity. vMIP-II/MBP could block chemokine receptors of PBMC and inhibit chemotaxis and adherence of PBMC.