传染性法氏囊病病毒 VP2/4/3-ChIL-2融合基因DNA疫苗免疫原性研究

应用重叠延伸剪切技术(splicing by overlapping extension,SOE),经3次PCR将传染性法氏囊病病毒(infectiousbursal disease virus,IBDV)多聚蛋白(VP2/4/3)基因和鸡白细胞介素2(Chicken IL-2,ChIL-2)基因进行融合,定向插入真核表达载体pCI的CMV启动子下游,获得重组质粒pCI-VP2/4/3-IL-2和pCI-IL-2-VP2/4/3。将其制备成DNA疫苗,肌肉注射14日龄非免疫鸡,2周后加强免疫,定期测定鸡抗IBDV血清ELISA抗体效价及病毒中和抗体效价。加强免疫后3周用IBDV标准强毒株攻击,连续观察3天后全部扑杀,计算保护率及囊体比,并进行组织病理学检查。结果表明:1)融合基因重组质粒pCI-VP2/4/3-IL-2、pCI-IL-2-VP2/4/3免疫后能明显增强IBDVDNA疫苗对强毒的攻击保护(保护率分别为83.3%、91.6%),显著高于pCI-VP2/4/3单独免疫对照组(58.3%);2)诱导产生的抗IBDV血清ELISA抗体效价明显增高(P<0.05),同时能提高DNA疫苗诱导产生的中和抗体效价(P<0.05);3)能显著促进鸡外周血液T淋巴细胞增殖反应。上述结果提示:IBDV VP2/4/3与ChIL-2基因融合后发挥了相互协同作用,ChIL-2产生了分子免疫佐剂效应;融合基因DNA疫苗能增强IBDV DNA疫苗的免疫原性,促进了机体特异性免疫应答。

Enhanced Immunogenicity of DNA Vaccine Encoding Infectious Bursal Disease Virus VP2/4/3 and ChIL-2 Fusion Gene

A fusion gene encoding infectious bursal disease virus(IBDV) polyprotein(VP2/4/3) and chicken interleukin 2(ChIL-2) was achieved by using the technique of splicing by overlapping extension(SOEing).This fusion gene was cloned into pCI,an eukaryotic expression vector,to obtain recombinant expression plasmids pCI-VP2/4/3-IL-2 and pCI-IL-2-VP2/4/3.Fourteen-day-old non-immunized chickens were vaccinated intramuscularly with different plasmid-combined DNA vaccines, and boosted two weeks later.All test groups were challenged with reference virulent IBDV strain BC6/85 three weeks after boosting.The results showed that protective efficacy could be significantly enhanced after injection with recombinant plasmids pCI-VP2/4/3-IL-2 and pCI-IL-2-VP2/4/3,and that anti-IBDV ELISA antibody level and neutralization antibody level were significantly increased,the peripheral blood T lymphocyte proliferation response was also significantly enhanced.These data indicated that chicken IL-2,acting as a molecular immune adjuvant when interacted with IBDV ployprotein VP2/4/3 in vivo,could enhance the immunogenicity of IBDV DNA vaccine and promote the specific immune response of the organism.