Prokaryotic Expression And Purification Of Murine IFN-β

Interferon is one kind of soluble glycoprotein produced by a variety of cells in a wide range of anti-virus, anti-tumor and immunomodulatory. IFN can be divided into typeâ… and typeâ…¡. To date, typeâ… IFN, also known as anti-viral IFN, has been found IFN-α, IFN-β, IFN-ω, IFN-κ, IFN-Ï„, and IFN-δ, while typeâ…¡IFN, until now, as immune IFN, only found IFN-γ. Scientists discover that the function of anti-viral and immune regulation for IFN has been reduced significantly because a number of viruses interfere with the induction of IFN, IFN signal transduction, and effect of protein by expression of antagonist protein. Clinical trials showed that injection of interferon products has the curable and defensive effect for some diseases treatments. For more than half a century, there has been a lot of studies for IFN, and appears a lot of IFN products that mainly concentrated on IFN-αand IFN-γ. IFN-βis a single gene product, Its role is similar to IFN-α. In recent years, scientists have gradually discovered that the effect of IFN-βis better than IFN-αfor some diseases treatments.In this study, a pair of specific primers was designed according to IFN-βsequence of mouse on the Genebank(NM₀10510) , we amplified successfully the IFN-βmature protein gene with signal peptide sequence removed by PCR technology from the liver DNA of mice. The amplified fragment was cloned into pMD18-T vector and transformed into competent cells. Then the JM109 were cultured on the solid LB medium with the resistance of benzyl ammonia. We cultured the monoclonal colony that grew better after screening. After the identification of plasmid restriction enzyme digestion, the double digested fragment was connected to the PET-28a expression plasmid and transformed into BL21 cells, then cultured on the solid LB medium with the resistance kanamycin, monoclonal colony that grow better was picked. After restriction enzyme digestion, the positive bacteria were sequenced in the Shanghai Public Health and biotechnology companies. Sequencing results are consistent with the expected results. The mature protein gene (IFN-β'genes) of recombinant mouse with signal peptide sequence removed were expressed in inclusion bodie by IPTG-induced expression. We designed the experiments of protein purification by inclusion body lysis using urea lysis and activity tests for next step. a variety of conditions were ajusted,â‘ Lysis environment, taking into account the effect of lysis, we would extend the lysis time from 4h to overnight or even 48h, and lift lysis temperature from 4℃to room temperature or 37℃;â‘¡Lysis buffer adjustments, such as the preparation of lysis buffer several times to re-regulate the PH value of lysis buffer. We also added DTT and sodium acetate in the lysis buffer considering that the protein disulfide bonds may affect lysis results.â‘¢The recombinant bacteria allowed the expression under the conditions of IPTG-induced culture of 12h at 18℃in order to produce soluble protein;â‘£Inclusion body washing, the clean inclusion body underwent urea lysis until all the impurities were removed from inclusion body by washing;⑤Taking into account the induction time and induction of IPTG concentration might affect the inclusion body formation, as well as its structure, we applied can be observed that the expression of the protein has the shortest induction time with the most at the end of the IPTG concentration for induction;â‘¥We adjusted the urea concentration from 8mol / L to 10mol / L.Experiments results show that inclusion bodies lysis of the recombinant mouse IFN-β'mature protein is difficult.It is found that 30% inclusion body of the mouse recombinant IFN-β' was pyrolysis under the ratio of lysis buffer and dry bacteria increased to 20ml: 1g.