摘要
采用RT-PCR技术扩增禽呼肠孤病毒ARV S1133毒株和广西分离株R1的σ2基因,将σ2基因克隆至真核表达载体pcDNA3.1(+)载体上,对获得的重组质粒经PCR酶切以及序列分析鉴定。结果表明,插入的片段为σ2目的基因,插入的位置、大小和阅码框架均正确,成功构建了真核表达载体重组质粒pcDNA-S1133-σ2、pcDNA-R1-σ2;分别免疫SPF鸡2次后,用ARV S1133进行攻毒,使用RT-PCR方法进行检测,结果表明,pcDNA-S1133-σ2、pcDNA-R1-σ2 DNA疫苗免疫组检出率与对照组差异显著(p<0.05),与灭活苗免疫组及σ2蛋白免疫组差异不显著(p>0.05)。使用ARVσ2基因构建的真核表达质粒来免疫SPF鸡,鸡只获得了较好的免疫保护作用。
The σ2 gene in ARV S1133 R1 of avian reovirus,isolated from Guangxi,was amplified by using RT-PCR.The purified RT-PCR product（σ2 gene） was inserted into pcDNA3.1（＋） vector.The recombinant plasmid was identified by restriction endonuclease analysis and PCR.It was proved by DNA sequencing that the acquired recombinant plasmid contains complete σ2 gene,the inserting location,size and open reading frame（ORF） were correct,therefore the recombinant DNA vaccine plasmid of pcDNA-S1133-σ2,pcDNA-R1-σ2 was constructed.SPF chickens were immunized with plasmids pcDNA-S1133-σ2 and pcDNA-R1-σ2 twice,then they were challenged with ARV S1133,and then by using RT-PCR to detect the chickens,the results showed that the positive rate of chickens immunized with pcDNA-S1133-σ2 and pcDNA-R1-σ2 had significant difference with that of control（P0.05）,and no significant difference with chickens immunized with inactivated vaccines or σ2 protein.The result showed that the SPF chickens immunized with pcDNA-S1133-σ2 and pcDNA-R1-σ2 could obtain better immune protection.
出处
《湖南农业科学》
2011年第1期126-129,133,共5页
Hunan Agricultural Sciences
基金
广西科技攻关项目(桂科攻0428001-2)
