Expression, Seperation And Purification Of Recombinant Fusion Protein Of IFNα_2b-THYα₁

Interferonα_2b(IFNα_2b)is a drug of antivirus and anti-tumor which can regulate immunity.Thymosinα₁(THYα₁)can enhance immunity. Recombinant cell gene is a very useful drug for treatment of human cancer and some virus infection diseases as well as immunity improving as a immunity regulator.Due to the effect of cellulous genes is more powerful than the individual cell gene,we considered to construct a fusion protein molecule containing both IFNα_2band THYα₁ by molecule biology technology which can optimize drug's quality,heighten drug's value and get flourishing medical treatment foreground.In order to prepare recombinant fusion protein with the transgenic E.coli,the optimal process for the target protein expression and purification was studied in detail.The optimal experiment conditions for the genetic engineered E.coli were as follows:37℃,Kna 30mg·L^-1,The induction was started at its logarithm growth phase and lasted for 4 h with IPTG 0.5 mmol·L^-1.The heterognous protein expressed in E.coli was usually accumulated as inclusion bodies in the bacterium.Refolding correctly at high concentration with high yield has been researched.After milling 48h,the inclusion bodies denaturaing by 7mol·L^-1guanidine hydrochloride containing 10mmol·L^-1DTT.After that,the denatured liquid was added to the renaturation solution 1mol·L^-1Urea, 0.2mmol·L^-1GSSG-2mmol·L^-1GSH by a pulse way,according to 1mL every other 2h.After denaturaing and renaturating,three different chromatographic columns,namely Ni-chelating-Sepharose, SPFF-Sepharose and Sephadex G-50,were operated in series.The purity of the resulting fusion proteins was more than 90%,as shown by RP-HPLC analysis.The yield of recombinant fusion proteins can be 68mg·L^-1fermenter.The experiment results proved that the design of the molecular structure of the fusion protein was rational.A whole technics route was got through including expression,separation and purification.It provided a good reference for large expression and purification of fusion protein with bacteria.