血清1型Ⅰ群禽腺病毒Fiber-2蛋白的表达及其中和作用研究

Expression and Neutralization of Fiber-2 Protein of Group Ⅰ Fowl Adenovirus Subtype 1

为研究血清1型Ⅰ群禽腺病毒(FAdV-1)的Fiber-2蛋白对FAdV-1在细胞水平上的中和作用,研究根据FAdV-1亚型Fiber-2基因序列设计引物,进行Fiber-2全基因序列PCR扩增并构建重组质粒,转入BL21(DE3)感受态细胞,经IPTG诱导表达,表达产物纯化后免疫SPF鸡制备抗血清,通过鸡LMH细胞研究Fiber-2蛋白抗血清对FAdV-1感染的中和作用。结果显示,FAdV-1 Fiber-2基因扩增产物大小为1 233 bp,构建了pCold-FAdV-1-Fiber-2重组质粒,表达的FAdV-1 Fiber-2蛋白分子量大小为105 ku;FAdV-1 Fiber-2蛋白的抗血清孵育LMH细胞后,与阳性对照组相同第4天细胞出现病变,而阴性对照组细胞均无病变。结果表明,FAdV-1 Fiber-2蛋白抗血清在体外试验中不能中和本血清型病毒的感染,FAdV-1 Fiber-2蛋白作为亚单位疫苗的研究存在风险。

To study the neutralization of Fiber-2 protein of fowl adenovirus group Ⅰ subtype 1(FAdV-1) at the cellular level, primer was designed according to Fiber-2 gene sequences of FAdV-1, the whole gene sequence of Fiber-2 gene was amplified by PCR and the recombinant plasmid was constructed. Plasmid was transferred into BL21(DE3) cells and expressed induced by IPTG. Antiserum against Fiber-2 protein was prepared by immunization with expressed protein in SPF chickens. Neutralization of Fiber-2 protein antiserum on FAdV-1 was studied in LMH cells. The results showed that the amplified FAdV-1 Fiber-2 gene was 1 233 bp. The recombinant plasmid pCold-FAdV-1-Fiber-2 was successfully constructed, and the molecular weight of expressed Fiber-2 protein of FAdV-1 was 105 ku. After incubating LMH cells with antiserum of FAdV-1 Fiber-2 protein, characteristic lesions appeared on the forth day in the same way as in the positive control group, while the cells in the negative control group had no lesions. It′s suggested that the FAdV-1 Fiber-2 protein antiserum could not neutralize the infection of this serotype virus in vitro, and the use of FAdV-1 Fiber-2 protein as a subunit vaccine was risky.