含禽流感病毒M2e 氨基端抗原表位的重组传染性法氏囊病毒VP2 蛋白的免疫原性鉴定

【目的】构建传染性法氏囊病毒VP2蛋白展示禽流感M2e抗原表位的重组蛋白,研发预防H5或H9亚型禽流感和传染性法氏囊的基因工程疫苗。【方法】根据现有禽流感疫苗株M2e的氨基端12个氨基酸多肽序列(nM2e)序列,结合GenBank中H5和H9亚型禽流感病毒nM2e的比对结果,确定nM2e序列。用融合PCR分别将1拷贝H5或H9的nM2e序列插入IBD B87株VP2基因的PBC区,获得VP2BCnM2e重组基因。将重组基因克隆至杆状病毒表达系统,转染Sf9细胞进行表达。经间接免疫荧光和Western blotting检测Sf9细胞表达重组基因后,扩繁重组病毒,制备疫苗,间隔4周对非免鸡作2次重复免疫,用间接ELISA和鸡胚成纤维细胞中的病毒血清中和试验检测血清中VP2和nM2e的抗体效价。【结果】成功构建含H5或H9 nM2e的VP2BCnM2e重组基因,该重组基因在Sf9细胞中得到表达。经免疫鸡,两重组蛋白均能激发针对VP2和nM2e的抗体,VP2BCnM2eH5组抗体效价高于VP2BCnM2eH9组。【结论】两重组蛋白均具有免疫原性,VP2BCnM2eH5免疫原性更佳。

Characterization the immunogenicity of recombinant VP2 of infectious bursal disease virus containing N-terminal M2e of avian influenza virus

Objective]We developed subunit vaccines against H5 or H9 subtype avian influenza viruses(AIV) and infectious bursal disease viruses(IBDV).Viral protein 2(VP2) of IBDV was used as cargo protein to display a 12-amino-acid(aa) immunodominant epitope derived from N-terminal M2 extracelluar domain(nM2e) of H5 or H9 subtype AIV.Methods] The aa and nucleotide sequence of nM2e was determined by comparing the available avian influenza vaccine strains and alignment the AIV sequence available in GenBank.One copy of H5 or H9 nM2e was inserted into PBC region of VP2 origin from IBDV B87 vaccine strain by fusion polymerase chain reaction.The VP2BCnM2e recombinants were cloned into Bac-to-Bac expression system and transfected to Sf9 cell.The expressed chimeric protein was characterized by indirect immunofluorescence assay and Western blotting,and subsequently was used as antigen to develop vaccine.The non-immunized chicken was given two injections with the vaccine at a 4-week interval.Serum against VP2 and nM2e was tested by indirect ELISA and virus neutralization in chick embryo fibroblast.Results] Both VP2BCnM2e recombinants were successfully constructed and expressed in Sf9 cell.Both chimeric proteins elicited antibody against VP2 and nM2e.The antibody level elicited by VP2BCnM2eH5 vaccine was higher than that of VP2BCnM2eH9.Conclusion] Both chimeric proteins were immunigenic,and the efficacy of VP2BCnM2eH5 was higher than VP2BCnM2eH9 in chicken.