鸭α-干扰素真核表达质粒的构建及其对DVH弱毒苗免疫鸭细胞免疫调节作用研究

为检测鸭α-干扰素对鸭细胞免疫的调节作用,本文按常规方法构建了鸭α-干扰素真核表达质粒(pcDNA-DuIFN-α),并以50、100、200μg分别肌注28日龄樱桃谷鸭,15 d后免疫鸭病毒性肝炎(DVH)弱毒疫苗,设空载体+DVH弱毒疫苗、生理盐水、DVH弱毒疫苗以及pcDNA-DuIFN-α对照组,采用流式细胞仪(FACS)和淋巴细胞增殖试验(MTT法)分别对免疫鸭外周血的CD3+淋巴细胞总数和T淋巴细胞转化能力进行动态检测。结果:①CD3+淋巴细胞数量:7~56 d实验组极显著(P≤0.01)高于生理盐水和显著高于(P≤0.05)DVH弱毒苗、空载体+DVH弱毒疫苗和pcDNA-DuIFN-α对照组,14~28 d 100μg组显著(P≤0.05)高于50和200μg组;②T淋巴细胞对ConA的反应能力(OD值):21~56 d实验组极显著(P≤0.01)高于生理盐水和显著高于(P≤0.05)DVH弱毒苗、空载体+DVH弱毒疫苗和pcDNA-DuIFN-α对照组,50和100μg组差异不显著,28~56 d均略高于200μg组。研究表明pcDNA-DuIFN-α是一种优秀的鸭细胞免疫的分子增强剂和DVH弱毒疫苗的分子佐剂,肌注100μg/只的效果最佳。

Construction of Duck Interferon-α Eukaryotic Expression Plasmid and Studies on the Cellular Immunological Regulation to Ducks Immunized with DVH Attenuated Vaccine

In order to determine cellular immunological regulation of duck interferon-α,duck interferon-α eukaryotic expression plasmid(pcDNA-DuIFN-α) had been constructed and administrated to 28-day old cherry valley ducks at the dose of 50,100,200 μg per duck respectively by intramuscular injection and 15 days later duck viral hepatitis(DVH) attenuated vaccine was administrated to these ducks,with empty vector+DVH attenuated vaccine,psychological saline,DVH attenuated vaccine and pcDNA-DuIFN-α as controls.Flow cytometry method was employed to study the number of CD3+ lymphocytes in peripheral blood and MTT method was used to study the transformation ability of T lymphocyte respectively.Results: ①Numbers of CD3+ lymphocyte of 7～56 days experimental groups were extremely significantly higher than(P≤0.01) that of the psychological saline group and 14～28 days significantly higher(P≤0.05) than that of DVH attenuated vaccine,empty plasmid+DVH attenuated vaccine and pcDNA-DuIFN-α control group.Number of CD3+ lymphocyte of 21～56 days 100 μg group was significantly higher(P≤0.05) than that of 50 and 200 μg group.②The reaction of T lymphocyts to ConA(OD values) of 21～56 days experimental groups were extremely significantly higher than(P≤0.01) that of psychological saline group and significantly higher(P≤0.05) than that of DVH attenuated vaccine,empty plasmid+DVH attenuated vaccine and pcDNA-DuIFN-α control group.There was no significant difference between 50 and 100 μg group,which was little higher than 200 μg group 28～56 days.The results demonstrated that pcDNA-DuIFN-α was an excellent molecular immunological promoter of ducks and DVH attenuated vaccine molecular adjuvant.Best result could be obtained at the dose of 100 μg pcDNA-DuIFN-α per duck by intramuscular injection.