鸭IFN-α真核表达质粒基因枪免疫对鸭瘟弱毒疫苗免疫鸭细胞免疫调节作用初探

Preliminary Study on Effect of Cell Mediated Immunity Regulation of Duck Enhanced by Duck IFN-α Eukaryon Expression Plasmid Inoculated with Gene-gun when DPV Attenuated Vaccine Fellow Vaccination

为探索鸭α干扰素(IFN-α)真核表达质粒(pcDNA-SDIFN-α)对鸭瘟弱毒疫苗免疫鸭的细胞免疫调节作用,本研究将pcDNA-SDIFN-α以1、3和6μg/只3个剂量用基因枪轰击法分别免疫28日龄鸭,以PBS和空载体质粒pcDNA3.1(+)为对照,所有鸭15 d后接种鸭瘟(DP)弱毒疫苗。接种后第3、7、14、21、28、35、49、63、84天采血用淋巴细胞增殖试验(MTT法)测定鸭外周血中T淋巴细胞转化效果;第7、14、21、28、35、49天采血用流式细胞仪(FACS)测定CD3+T淋巴细胞数量的动态变化。结果发现:①T淋巴细胞对ConA的反应能力(OD值),不同剂量pcDNA-SDIFN-α免疫组鸭外周血T淋巴细胞转化功能于第3-84天均高于PBS和空载体pcDNA对照组,其中第3-84天1μg/只组极显著(P≤0.01)高于PBS和pcDNA对照组,3μg/只组极显著(P≤0.01)或显著(P≤0.05)高于PBS和pcDNA对照组,6μg/只组于第7-49天极显著(P≤0.01)或显著(P≤0.05)高于PBS和pcDNA对照组;1μg/只组第3-35天显著(P≤0.05)高于3、6μg/只组;3μg/只组于第14-35天高于6μg/只组,但差异不显著(P≥0.05);pcDNA对照组略高于PBS对照组,但差异不显著(P≥0.05);②CD3+T淋巴细胞数量变化,不同剂量pcDNA-SDIFN-α免疫组鸭于第7-49天均高于PBS和pcDNA对照组,其中1μg/只组于第14-49天极显著(P≤0.01)高于PBS和pcDNA对照组,3μg/只组于第21-49天极显著(P≤0.01)高于PBS对照组和显著(P≤0.05)高于pcDNA对照组,6μg/只组于第7-49天显著(P≤0.05)或极显著(P≤0.01)高于PBS和pcDNA对照组;1、3和6μg/只组之间差异不显著(P≥0.05);pcDNA组于第14-49天高于PBS组,但差异不显著(P≥0.05)。研究表明,pcDNA-SDIFN-α提前15 d免疫能显著增强DP弱毒疫苗诱导的鸭细胞免疫力,以基因枪免疫1μg/只的效果最佳,它是一种良好的增强DP弱毒疫苗细胞免疫的分子佐剂。

In order to study the effect of cell mediated immunity regulation of duck IFN-α eukaryon expression plasmid （pcDNA-SDIFN-α） on DPV attenuated vaccine in ducks , pcDNA-SDIFN-α was administrated to 28-day old ducks at the dose of 1, 3, 6 μg/duck, respectively, by genegun. PBS and empty vector pcDNA were used as control. 15 days later, all ducks were injected with DPV attenuated vaccine, and blood samples were collected at 3, 7, 14, 21, 28, 35, 49, 63, 84 days after injection. T lymphocyte proliferation tests （MTT） were used to detect the T-lymphocyte proliferation in peripheral blood（PBL） of ducks. Blood samples collected at 7, 14, 21, 28, 35, 49 days were detected by fluorescence activated cell sorter （FACS） for recording the number of CD3^＋ T lymphocytes of ducks. Results were as follows： ① Reaction of T lymphocytes in PBL to ConA （OD value）of ducks treated with pcDNA-SDIFN-α were higher than that of PBS and pcDNA control groups in 3-84 days. There were highly significant difference between 1 μg/duck group and two control groups in 3-84 days （P≤0. 01）, between 3 μg/duck group and two control groups in 3-84 days （P≤0.01,P≤0.05）, and between 6 μg/duck group and two control groups in 7--49 days （P≤0.01 ,P≤0. 05）. The significant difference was also present between 1 μg/duck and 3,6 μg/duck groups in 3-35 days（P≤0.05）. Moreover, there was no significant difference between 3 μg/duck group and 6 μg/duck group （P≥0.05）. pcDNA control group was higher than PBS control group, but no difference was found （P≥0. 05）.② Change of the number of CD3^＋ T-lymphocyte of ducks administrated with different doses of pcDNA-SDIFN- α were higher than that of PBS and pcDNA control groups in 7-49 days. 1 μg/duck group was significantly higher than PBS and pcDNA control groups in 14-49 days （P≤0.01）, there were significant difference between 3 μg/duck group and two control groups in 21-49 days （P≤0.01, P≤0. 05）,significant difference between 6 μg/duck group and two control groups in 7-49 days （P≤0.01,P≤0. 05）. No significant difference was found among the groups of 1, 3, 6 μg/duck （P≥0.05）. There was no significantly difference between two control groups（P≥0.05）. The results indicate that pcDNA-SDIFN-α administrated 15 days before injection of DPV attenuated vaccine could significantly enhance cellular immunity induced by DPV attenuated vaccine, pcDNA- SDIFN-α is an excellent DPV attenuated vaccine molecular adjuvant, best result could be obtained with the dose of 1 μg/duck of pcDNA-SDIFN-α inoculated by gene-gun.