Construction,Expression Of Chicken Recombinant IL-2 And IL-4 Gene Fusd Expression Vector,and Its Bioactivities In Vitro

In order to improve the immune effect,strengthen the immune protection,reduce the side effect,and shorten the producing immunity time of coccidiosis live vaccine for chicken,chIL-4 and chIL-2 were regarded as objects of the research.They(chIL-4 and chIL-2)were used for construction of a novel cytokine immuno adjuvant chIL-4 and chIL-2 fusion gene expression vector,so that the adjuvant effect of fusion gene can be learned.The test results showed that:(1)Recombinant eukaryotic expression vectors(pCI-chIL-4-EGFP,pCI-chIL-2-EGFP,and pCI-chIL-4-chIL-2-EGFP)which carry chIL-4,chIL-2,and both two fusion genes identificated by sequencing technology were constructed successfully through gene synthesis and double enzyme cutting connection technique.(2)Transfection of mouse myoblast C2C12 was done by HilyMax liposome mediated method.C2C12 was observed using the fluorescence microscope after the transfection conditions were optimized.And the cumulative optical density was analyzed by IPP software.The results showed that the recombinant vector pCI-chIL-2-EGFP could be correctly expressed in murine myoblasts,and transfection efficiency can be the highest when the rate of DNA and Hilymax is 1:3(29.65%±2.58%).The expression of protein was highest after transfection of 72h.(3)Chicken embryo cecum epithelial cells and chicken embryo fibroblasts were transfected by three recombinant plasmids(pCI-chIL-4-EGFP,pCl-chIL-2-EGFP,and pCI-chIL-4-chIL-2-EGFP)in 24h-96h.The results showed that,the expression of mRNA increased first and then decreased,while expression on 48h was obviously higher than others(P<0.01).The cumulative optical density increased first and then decreased,and the cumulative optical density on 48h reached the peak value,which was much higher than that on 24h(P<0.05)and that on 48h(P<0.01)while the cumulative optical density on 96h cannot be detected.Fluorescence intensity of cell supernatant increased first and then decreased with the peak value on 72h which was much higher than other periods(P<0.01).Mouse myoblast was transfected by three recombinant plasmids(pCI-chIL-4-EGFP,pCI-chIL-2-EGFP,and pCI-chIL-4-chIL-2-EGFP)in 24h-96h.The expression of mRNA and protein increased first and then decreased with the peak value on 72h which was much higher than other periods.Fluorescence intensity of cell supernatant increased first and then decreased with the peak value on 72h which was much higher than other periods(P<0.01).These results indicate that all three recombinant plasmids can be expressed in three kinds of cells for more than 72h.Three kinds of recombinant proteins can be secreted to the extracellular domain and reach the peak value at 72h.(4)Chicken embryo cecum epithelial cells,chicken embryo fibroblasts and mouse myoblast were were transfected by three recombinant plasmids(pCI-chIL-4-EGFP,pCI-chIL-2-EGFP,and pCI-chIL-4-chIL-2-EGFP)in 24h-96h.Supernatant of three kinds of cells can promote the proliferation of spleen lymphocyte in chicken(P<0.01),while the stimulation index increased first and then decreased with the peak value on 72h.The results showed that all the three recombinant proteins obtain the bioactivity.The proliferation efficiency of spleen lymphocyte in chicken by cell supernatant after being transfected by pCI-chIL-4-chIL-2-EGFP was much higher(P<0.01)than that by two other recombinant plasmids.The results showed that the bioactivity of chIL-4-chIL-2 fusion protein was significantly higher than that of chIL-4 or chIL-2.