Molecular Cloning Of China White Rabbit IL-6 CDNA And Immune Response Of RHDV VP60 Gene DNA Vaccine Enhanced By PcDNA-IL6 As Molecule Adjuvant

Many of scholars pay close attention to cytokine and DNA vaccine, but little isreported about rabbit cytokine and RHD DNA vaccine. In this research, IL-6 genecDNA was cloned and sequenced, and the recombinant eukaryotic expression plasmidof rabbit IL-6 was constructed, enhanced immune responses of IL-6 was initiallystudied. At the same time, Viro cell line and rabbit as model were used to study theexpression in Vero cell line and the immune response of eukaryotic expressionplasmid pcDNA-VP60.Results as follows:Rabbit Interleukin 6 gene cDNA was acquired from the total RNA of rabbitperipheral lymphocyte which had been stimulated and induced by Con A, and it wascloned into pMD18-T vector to sequence and was insected into eukaryotic expressionvector pcDNA3.1(+) to constructe a eukaryotic expression plasmid pcDNA-IL6. Thesequencing result indicated that the completed gene of China White rabbit was 723bp. Analysing the sequence of IL-6 showed that the homology of IL-6 gene is 100%between Oryctolagus cuniculus and China rabbit. But compared acquired IL-6 geneof rabbit to the published,shared 84.9%-91.1% nucleotide identities, genetic treeanalysis showed that both Oryctolagus cuniculus and china rabbit are closely relative.At domestic, this is also the first report about rabbit IL-6 gene cloning, and it providea condition for research immunology effect of rabbit IL-6.RHDV WHNRH isoform have been sequenced and the recombinant eukaryoticexpression plasmid of RHDV VP60 (DNA vaccine) have been constructed in ourlab. However, there are no reearch about immunogenicity of pcDNA-VP60, to date.In order to study its immunogenicity, the pcDNA-VP60 was expressed in the Vero cellline by cationic liposome transfection,and immunofluorescence was used to test theVP60 expression. In conclusion,the result demonstrate that the pcDNA-VP60 in Veto cell line can expressed immunogenic VP60 protein.Immunology experiment: In order to study the immunogenicity of pcDNA-VP60and that IL-6 as molecule adjuvants, whether can enhance immune responses andprotective efficacy of pcDNA-VP60 and RHD tissue inactivity, the plasmid ofpcDNA-VP60 and pcDNA-IL6 were applied to Immunology experiment. In thisresearch, the two plasmids were maxi-prepared by alkaline method and purifid byPEG8000 precipitation. Moreover, the plasmid both pcDNA-VP60 and pcDNA-IL6were made into pcDNA-VP60/liposome and pcDNA-IL6/liposome complexes, and twocomplexes were used to Immunology experiment.For vaccination, 25 rabbits, 20 days old and 600-700g weight, were randomlydivided into 5 groups, 5 animals per group (Group one: pcDNA3.1(+), Group two:RHD inactivity vaccine, group three: RHD inactivity vaccine andpcDNA-IL6/liposome, group four: pcDNA-VP60/liposome,group five:pcDNA-VP60/liposome and pcDNA-IL6/liposome complexes). Prevaccation, theposition of vaccination would be injected procaine. The methods of four pointintramuscularly injecting in legs and twice vaccination were be adopted, and theinternal of two times vaccination are 20 days. Blood was drew and serum wascollected at 0 day, 7 days, 14 days, 21 days, 28 days, 35 days and 70 days aftervaccination. Hemagglutination test was used to detect antibodies against RHDV andSPSS was used to statistics and analyze the data of experimental.Results indicated that: Both pcDNA-VP60/liposome complexes andpcDNA-VP60/pcDNA-IL6/liposome complexes can induce immune responses, andcompared to the pcDNA3.1(+)/liposome, the antibodies induced against RHDVshowed extremely significant difference (p＜0.01). But compared to the inactivity ofRHD, it exhibited no significant difference. The top of antibodies induced bypcDNA-VP60 against RHDV was 35 days after vaccination. Moreover, both RHDDNA vaccine and inactivity inducing immune response can be enhanced bypcDNA-IL6, and immune responses reduced by pcDNA-VP60+pcDNA-IL6 andinactivity+pcDNA-IL6 significant stronger than by pcDNA-VP60 and inactivity(p＜0.05 or p＜0.01). At present, this is the first report showing the expression of pcDNA-VP60 in Virocell line and inducing the immune response on organism. This research providedscientific datas for further investigation of RHD DNA vaccine. At domestic, there isno report about rabbit cytokine, and this research was the earliest report aboutrabbit IL-6 gene cloning, sequencing and its molecular adjuvant effect. This studyprovided reliable dates for the resesrch and application of rabbit IL-6, moreover, itlay the foundation for further research for the immunology effacy of rabbit IL-6.