Construction And Expression Activity Of Novel Fusion Gene Of Procine Interleukin-6 And Interleukin-4

In order to construct a fusion cytokine protein which could have more and stronger bioactivities to enhance the immnuity than the original cytokines, we use the PCR technique to reconstruct the porcine IL-6 and IL-4 gene. The termination code of the IL-6 gene was deleted and the enzyme sites of BamHI and Sail were introduced to the two ends, named as FPIL-6 cDNA; the starting code of the IL-4 gene was cut and the enzyme sites of Sail and Xhol were added to the two flanks, designed as FPIL-4 cDNA. The reconstructed IL-6 and IL-4 gene were connected together via a linker sequence encoding Gly-Ser-Ser-Thr amino acid residues. The PCR amplified products of the IL-6 and IL-4 gene was digested respectively with BamHI and Sail, Sail and Xhol endonucleases, and then cloned into the pGEX-4T-l expression plasmid which was also digested with the sarne endonucleases. The recombinant plasmid was digested with BamH I, Sail and Xhol to identify whether the FPIL-6 and FPIL-4 cDNA fragments were inserted into the plasmid in correct orientation. .The BL21 (DE3) competent cells were transformed with the recombinant pGFPIL-6/IL-4, incubated with 2xYTA culture and induced to express the fusion protein by IPTG. The mRNA and protein expression of the recombinant plasmids were assayed by RT-PCR and SDS-PAGE. The results were found that the mRNA of the fusion gene could be correctly transcribed, and the fusion protein was expressed in the form of inclusion body with 64KD molecular weight revealed by SDS-PAGE analysis. The bioactivities of refolded fusion protein were detected by MTT assay to stimulate the proliferation of ConA blast cells of porcine. The results showed that through the treatment of denature, dilution and dialysis, the inclusion body of fusion protein restored the bioactivity and had marked bioactivity to promote the proliferation of porcine ConA blasts. These suggest that the fusion protein of porcine IL-6 and IL-4 could be employed to develop a novel effective immunoadjuvant to enhance the immunity of animals against infectious diseases.