重组禽呼肠孤病毒S1133 σ3基因表达及其免疫原性研究

摘要

[Objective ]Eukaryotic recombinant plasmid of avian reovirus (ARV) SI 133 σ3 gene was constructed to study its immunogenicity and provide references for developing ARV gene vaccine. [ Method ]The σ3 gene of avian reovirus was amplified with the specific primers by RT-PCR. The amplified product from RT-PCR was purified and ligated with pcDNA3.1 ( + ) vector and then transferred into E.coli DH5α. DNA of the recombinant plasmid was extracted and identified by double restriction endonuclease analysis,PCR assay,and DNA sequencing. Positive recombinant plasmid pcDNA3.1-σ3 was injected into 7-old days SPF chick as a vaccine. In contrast,the same protein from three groups viz.,inactive S1133,Σ3 protein,and saline were injected. Two weeks after the second injection,chick serum antibody was determined using Western blotting. The chick from four groups were injected with reovirus S1133 strain,and four days later ARV detection was conducted. [Result]Double digestion and PCR amplification of recombinant pcDNA3.1-Σ3 got the same result for obtaining 999 bp base bands. The positive rates of reovirus from pcDNA3.1-Σ3,inactive S1133,Σ3 protein,and saline group were 7.4,3.7,11.1,and 29.6%,respectively. The pcDNA3.1-σ3 group had significant difference (P<0.05) with saline group but no significant difference (P>0.05) with inactive S1133 andσ3 protein group. The joint had the highest positive rate followed by kidney and thymus (0%). [Conclusion]Owing to relatively good immunity protection,eukaryotic recombinant plasmid of ARV S1133 σ3 gene could be used as immunizing antigen for further vaccine neutralization experiment.%[目的]构建禽呼肠孤病毒(ARV)S1133 σ3基因的真核重组质粒,研究其表达蛋白的免疫原性,为研发ARV基因疫苗奠定基础.[方法]采用RT-PCR对ARV S1133毒株的σ3基因进行RT-PCR扩增,扩增产物与真核表达载体pcDNA3.1(+)连接后,转化大肠杆菌DH5α,然后提取重组质粒的DNA,经双酶切、PCR扩增鉴定和纯化.以阳性重组质粒pcDNA3.1-σ3免疫7日龄SPF雏鸡,同时设灭活S1133、表达σ3蛋白及生理盐水对照,二次加强免疫两周后,采用Western blotting检测鸡群血清抗体；并用S1133毒株进行攻毒,4 d后进行ARV检测.[结果]重组质粒pcDNA3.1-σ3的双酶切和RT-PCR扩增鉴定结果一致,均能扩增出约999 bp目的条带；免疫攻毒试验结果表明,pcDNA3.1-σ3处理、灭活S1133处理、表达σ3蛋白处理和生理盐水处理的病毒检出率分别为7.4％、3.7％、11.1％和29.6％,pcDNA-σ3处理与生理盐水处理的差异显著(P＜0.05),但与灭活S1133处理、表达σ3蛋白处理的差异不显著(P＞0.05)；病毒检出部位最高是关节,其次是胸腺和肾脏,胸腺检出率为零.[结论]真核表达ARV S1133 σ3蛋白具有较好的免疫保护,可作为免疫抗原进行下一步的疫苗中和试验.