Secretion Expression Of Recombinate Human Interferon α-2b By Escherichia Coli

Objective:According to gene BANK sequence of human interferon a-2b gene,we cloned human interferonα-2b gene fragment and constructed prokaryotic secretion expression vectors of human huIFNa-2b.Then tranformed these vectors into competent cells from E.coli BL21 and E.coli W3110 respectively.These vectors in different hosts express the target protein is not the same in amount and secretion efficiency.We get the pPSIFN-4L/E.coli W3110 system as the best choice through the identify methods of SDS-PAGE and Western-Blot. Methods:Mutated the 5'end gene sequence of huIFN-α2b code gene by site-directed mutagensis and amplified it with PCR according to the frequency table gene codon in E.coli with out change its amino acid compsition,and then clone the gene into sequencing vector pGEM-7zf(-), and the vector with mutated huIFN-α2b genes was transformed into competent cell of E.coli DH5a.The positive clones were identified by blue-white screen and DNA sequencing;Design primers cloned the secretion signal coding sequence of E.coli heat-stable enterotoxinⅡ(STⅡ) from E.coli genome,fused the STⅡsignal gene sequence to the mutated gene of huIFNa-2b, introducing restriction sites BamHⅠ/SalⅠ, NdeⅠ/HindⅢand NsiⅠ/Xho I to the fusion gene fragments and ligase fragments into exprssion vectors pCSE,pET-22b and pPAK4L. Transformed these recombinant vectors into E.coli BL21 and E.coli W3110 competent cells respectively.Screened some positive clones and expressed target protein under inducement, and investigated the bacteria by SDS-PAGE,Western Blot and WISH-VSV to analysis the antivirus vitalism. Results:With fusion gene, pCSE expressed huIFNa-2b protein in low yield in different hosts,nearly 50% of the protein can be secreted.Induced by IPTG, pET-22b can get high level expression of huIFNa-2b in E.coli BL21, but the STⅡsignal can not be cutted efficiently. In low-phosphate growth media, E.coli W3110 with vector pPAK4L which contains the target gene synthesized approximately 20μg huIFNa-2b/ml A550 unit of cells,and about 30% of it can be secreted into periplasimic space.It showed that the expression productions have the same immunogenecity to positive control and centrifuge to verified the specific activitity of supernate was able to obtain 1.425×105IU/ml,in the same condition inclusion body expression system only can get 1.259x103IU/ml. Conclusions:The recombinant pPSIFN-4L vector was constructed successfully and transfected into competent cells from E.coli W3110, it get high level expression of huIFNa-2b in E.coli W3110, and STⅡsignal can be cutted efficiently after inducement.