Cloning Of Chicken Interleukin-6 And Study On The Immunoenhancement On Newcastle Disease LaSota Vaccine

Two primers were designed and synthesized according to the chicken interleukin-6 sequence published in GenBank (AJ309540). The Gallus interleukin-6 was amplified by reverse transcription-polymerase chain reaction (RT-PCR) from splenocytes lymphocytes stimulated with ConA. The gene was inserted into pMD18-T vector and sequenced. The result showed that the length of this cDNA was 603bp, encoding 195 amino acids, and sharing 100% homology with the only one ChIL-6 sequnce (AJ309540) in GenBank. Bioinformatics analysis of this sequence shows that the protein of ChIL-6 was hydrophilic and highly antigenic, including three potential protein kinase C phosphor -ylation site, six potential Casein kinaseâ…¡phosphorylation site. The cDNA was digested with Xho /and Xbaâ… and inserted into pCI-neo vector which was also digested with Xho /and Xba /to construct the eukaryotic expression vector pCI-IL-6. Then an immune experiment was designed to evaluate the immune adjuvant effect of pCI-IL-6.One hundred chickens were randomly assigned to five groups of twenty animals per treatment, including LaSota vaccine group, pCI-IL-6+LaSota vaccine group, pCI-IL-6 group, pCI-neo group and PBS control group. The immune adjuvant effect of the pCI-IL-6 to LaSota had been analyzed using the Hammaeglutination test (HI) antibody and the percentage of the CD4~+, CD8~+ and CD3~+T lymphocyte.The results shows that HI antibody of pCI-IL-6+LaSota vaccine group shows notable difference in 14,21, 35day (P<0.05) with LaSota vaccine group, shows extremely notable difference(P<0.01) in 28 days, HI antibody of pCI-IL-6+LaSota vaccine group shows extremely notable difference (P<0.01)with other treatments. The percentage of CD4~+, CD8~+ and CD3~+T lymphocyte was tested by flow cytometer technique, the results shows that the percentage of CD4~+T lymphocyte of pCI-IL-6 +LaSota vaccine group shows shows notable difference and extremely notable difference in 14 day (P<0.05) and 21, 28day (P<0.01), pCI-IL-6+LaSota vaccine group shows extremely notable difference in all days (P<0.01) with other groups. The percentage of CD8~+T lymphocyte of pCI-IL-6+LaSota vaccine group shows extremely notable difference in the 14 day (P<0.01) with LaSota vaccine group, shows notable difference in the 28 day (P<0.05). The percentage of CD3~+T lymphocyte of pCI-IL-6+LaSota vaccine group shows extremely notable difference in the 14, 21day (P<0.01) with LaSota vaccine group, shows notable difference in the 7, 28, 35day (P<0.05). Challenge test on 35th day after immunization shows that the protection ratio of co-immunization group of pCI-IL-6 and LaSota was 89.5%, while LaSota vaccine group was only 76.5%.The results indicated that pCI-IL-6 can improve the NDV immunity level of the humoral and cellualrimmunity as well as the challenge protection rate, showing immune adjuvant function. As we know, the immune adjuvant effect of eukaryotic plasmid of the ChIL-6 to LaSota vaccine hasn't been reported now, this study provides referrence to the united application of pCI-IL-6 and NDV vaccine.