NDV F48E9株与La Sota株F蛋白B细胞表位差异分析区段的鉴定和免疫原性比较

本文对预测的NDV F48E9强毒株和La Sota 疫苗株F蛋白优势表位进行比较分析。根据预测结果设计克隆了两个毒株F蛋白各4段表位区，分别将每个毒株的4段表位区连接到原核表达载体上进行了体外表达，鉴定得到4段多肽的大小分别是P1：8.0KD、P2:10.8KD、P5:13.5KD、P6:10.5KD。应用F48E9和LaSota特异性抗血清对表达各肽段进行了抗原性鉴定，P1、P2、P5、P6段均呈阳性反应，表明其中含有线性B细胞表位。其中F48E9 P5 (FP5)和La Sota P5(LP5)与血清反应时有明显差异。FP5和LP5与鸡IL-18成熟肽（m ChIL-18）蛋白以不同组合免疫SPF鸡，ELISA检测各组的免疫活性，结果发现FP5与m ChIL-18蛋白联合免疫组的抗体水平高于LP5与m ChIL-18蛋白联合免疫组、FP5、LP5单独免疫组；用NDV F48E9株攻毒结果显示FP5具有20%的保护率，说明P5段与F蛋白免疫原性相关，而LaSota P5不能抵抗强毒的攻击，证明该表位区域在两个毒株之间存在着免疫原性差异。

F protein B Cell Epitopes between Comparison of Immunogenicity of NDV F48E9 and La Sota strains

To identify and compare predicted the B cell-epitopes between NDV F_ 48 E_9 and La Sota strain, The predicted 4 poly-epitopes genes segments of each strain weve cloned, then were constructed into Prokaryotic vector separately. The expressed 8 recombinant proteins were analysed by SDS-PAGE, molecular weight of four peptides segments P1, P2, P5 and P6 are 8.0kDa, 10.8kDa, 13.5kDa, 10.5kDa, respectively.The Antigenicity of expressed peptides was identified with anti-F48E9 and anti-La Sota strains serum. The result showed that peptides P1, P2, P5 and P6 of NDV F_ 48 E_9 and La Sota could reacted with both anti-serum. However, the Western blot of peptide P5 against anti-F48E9 and anti-La Sota serum showed a strain specific reaction. Then P5 ploypeptide and mChIL-18 protein were purified by Ni-NTA Resin. Using mChIL-18 as adjuvant, immunized SPF chickens with P5 peptide of F48E9 or La Sota,serum were collected and NDV specific antibody titers were detected with ELISA. The results indicate that F48E9 P5 peptide (FP5) in combination with mChIL-18 protein has the highest antibody titer among all immunized groups, and it is the only group that could protect some of immunized chicken from death (20%). Finding of immunogenicity difference between FP5 peptide and LP5 peptide, suggested that there are strain specific epitopes exist in F48E9 and La Sota F protein.