An Expression System Of Recombinant IFN-β In E.coli

OBJECTIVE: To construct the E.coli expression system, in orderto get IFN-βexpression in the colon bacillus.Method: IFN-βgene was amplified by PCR, inserted intoPGEX4T-1 plasmid cutted by EcoRI and BamHI by T4DNA linkerenzyme, structure recombinant plasmid. The recombinant plasmid wastransinfected into Escherichia coli (E. coli) DH5a, and plasmid DNAwas extracted , identified with restriction endonucleases EcoRI andBamHI. Following transformation PGEX4T-1(+)-IFN-βinto DH21,the isopropyl-beta-D-thiogalactopyranoside (IPTG) induced cultureswith the expression of the expected protein were analyzed by sodiumdodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)RESULTS: A segment about 500bp is detected by agarose gelelectrophoresis, prove amplifying of IFN-βgene is successful. The sizeof empty plasmid pGEX4T-1 reduces to a big segment about 4Kb by thecutting of EcoRI and BamHI compared with it's original size of 4.3Kb.Escherichia coli transinfected by recombinant plasmid product moreplasmid after cultivation,which can be cut to 2 segments,one is 4Kb aboutanother 500bp. Amplifying this plasmid generates a segment about 500bp.All the three results prove success of recombinating. E.coli BL21expresses protein after cultivation.A production about 46KD is detected by SDS-PAGE which not detected in nomal E.coli BL21, prove successof expressing.Conclusion: Succeed in construct PGEX4T-1 (+)-IFN-β,then canexpress IFN-βfusion protein through E.coli BL21. This experimentsucceeds in structuring the basic system of recombinating the colonbacillus to express IFN-βand relevant detection methods.