Construction And Expression Of Recombinant Human Single-chain IL-23and Identification Of Its Biologic Activity

Objective:(1) To construct human single-chain IL-23(hscIL-23) fusion gene andpMD18-T-hscIL-23recombinant plasmid.(2) To construct prokaryotic expressionplasmid pET-28a(+)-hscIL-23and eukaryotic expression plasmid pcDNA3.1(+)-hscIL-23.(3) To express hscIL-23fusion protein in E. coli.(4) To express hscIL-23fusion protein in eucaryotic cells.(5) To identify biologic activity of hscIL-23fusionprotein.Methods:(1) The recombinant gene, p40+linker+p19(hscIL-23), was constructed byconnecting p40and p19from pMD18-T-p40and pMD18-T-p19constructed in our labwith a flexible polypeptide linker (Gly4Ser)3. The hscIL-23gene was inserted intopMD18-T vector, then the genes cloned were sequenced.(2) Plasmid pMD18-T-hscIL-23DNA was extracted and incubated with enzymes HindⅢ and Nhe I,thenhscIL-23genes was purified and connected with pcDNA3.1(+) eukaryotic expressionvector, which incubated with enzymes HindⅢ and NheI also. The prokaryoticexpression vector pET-28a(+)-hscIL-23was constructed as the same way.(3) Theprokaryotic expression vector pET-28a(+)-hscIL-23was transformed into E. coli.BL21(DE3+), use IPTG to induce the recombinant PvDBPII protein fused with His tag,and purify the prot ein by His-NTA affinity chromatography, the recombinant proteinwas identified by SDS-PAGE and western blot.(4)The DNA of plasmidpcDNA3.1(+)-hscIL-23was constructed and transfected to the hela cells according todifferent ratio, at the same time groups of tranfected with DNA of plasmid pcDNA3.1(+)and untranfected were set up, then the cells and supernatants were collected aftertransfected for48h. The expression of pscIL-12fusion protein was detected by ELISA.(5) Human PBMCs were isolated, and then incubated with the supernatant of cells,which transfected for72h. The proliferation dynamics was detected by MTT.Results:(1) Using a linker, we constructed pMD18-T-hscIL-23recombinant plasmid,pcDNA3.1(+)-pscIL-12eukarya expressing plasmid and pET-28a(+)-hscIL-23prokaryo-tic expression plasmid. Sequence analysis showed no bases was differentcompared with the target genes.(2) The recombinant fusion protein can be expressed in E.coli transformed by prokaryotic expression plasmid pET-28a(+)-hscIL-23.SDS-PAGE and Western blot analysis showed that the recombinant fusion protein(hscIL-23) was about60kDa, and can be recognized by IL-23p19antibody.(3) Theexpressions of hscIL-23fusion protein in the culture supernatant were detected byELISA assay. Amount of hscIL-23fusion protein were the group untranfected gothscIL-23fusion protein9.11±11.78pg/ml, the group transfected with pcDNA3.1(+) got10.47±9.04pg/ml, whereas the group transfected with pcDNA3.1(+)-hscIL-23got254.34±2.77pg/ml, and the results have significant difference (P<0.01).(4) By MTT assay, proliferation activity of human lymphoblastoid cells that werestimulated with supernatant of cells transfected by untransfected, pcDNA3.1(+),pcDNA3.1(+)-hscIL-23and PHA were0.553±0.038,0.595±0.289,1.135±0.048,1.968±0.018, respectively. The proliferation activity of human lymphoblastoid cells thatwas stimulated with culture supernatant of hela cells, which transfected withpcDNA3.1(+)-hscIL-23is significant higher than that of others (P<0.01).Conclusion:(1)Human single-chain IL-23(hscIL-23) fusion geneandpMD18-T-hscIL-23recombinant plasmid were constructed successfully.(2)Prokaryotic expression plasmid pET-28a(+)-hscIL-23and eukaryotic expressionplasmid pcDNA3.1(+)-hscIL-23construct were constructed successfully.(3) HscIL-23fusion protein was successfully expressed in E.coli transformed by prokaryoticexpression plasmid pET-28a(+)-hscIL-23.(4) HscIL-23fusion protein was successfullyexpressed in hela cells transfected by pcDNA3.1(+)-pscIL-23recombinant plasmid.(5)Culture supernatants containing hscIL-23fusion protein can obviously enhancedproliferation of human lymphoblastoid cells.