Melittin Genes Allosteric Human Interleukin -2 Fusion Genes Of Prokaryotic Plasmid Construction, Expression, Purification And Activity Assay

Objective To construct expression vector pET/Melittin-IL-2(⁸⁸Arg,¹²⁵Ala) and identify it. Screen host cells where the target gene can express stably,followed by induction, purification and determination.Then investigate activities of the recombinant protein in vitro.Methods Using pGEX-4T-2/Melittin-IL-2(⁸⁸Arg) as the template,a mutant Melittin-IL-2(⁸⁸Arg,¹²⁵Ala) was obtained by PCR point mutation.pET/M-IL-2(⁸⁸Arg, ¹²⁵Ala) was constructed by ligation pET-32a(+) and pMD18-T/M-IL-2(⁸⁸Arg,¹²⁵Ala) digested by NcoⅠand HindⅢ,which followed the T-A cloning.The construct was identified with DNA sequencing.Induction was run when the construct was transformed to BL21(DE3),and the total protein was analyzed by ELISA.The growthcurve of host cell drew during induction,meanwhile,survival host cells was determined on the media dish.The survivals were picked up and induced again.The production was monitored by SDS-PAGE electrophoresis and Western-Blotting.Purification was carried out by affinity and ion exchange chromatography.The concentration of the recombinant protein was evaluated by means of BCA method.The bacteriostasis test of the recombinant protein was performed.The proliferation assay of peripheral blood mononuclear cells(PBMC) and the growth inhibiting assay of Hela cells were run by using MTT method.All the data was evaluated with analysis of variance.Results Melittin-IL-2(⁸⁸Arg,¹²⁵Ala) was 542bp long and the mutant was identified as expected by DNA sequencing.The fused protein could be detected by ELISA.OD₆₀₀ reading of host cell decreased from 0.8 to 0.6 and survival bacteria counting of them fell from 10⁸/ml to 10⁴/ml as the induction of the recombinant was carried out.We could detect the target protein,which was about 19kDa after purification,at about 37kDa in both insoluble and soluble parts by SDS-PAGE electrophoresis and Western-Blotting.Using BCA,we determined 62μg recombinant protein could be extracted from 1 liter culture.In vitro,its bacteriostasis was concentration dependent;it could profile PBMC same as the wild hIL-2(p>0.05) and inhibit growth of the Hela but wild IL-2 cannot do(p<0.05).Conclusion The expression vector pET/M-IL-2(⁸⁸Arg,¹²⁵Ala) was constructed as expected.The investigation to the effects on its host cells suggested that the fused protein was expressed at a low level, which may be resulted from toxicity to its host cells.We obtained host cells where the target gene can express stably and the recombinant protein was purified with chromatography.It was characteristics of bacteriostasis,enhancing immune function and anti-tumor activity in vitro.