重组IBV多抗原肽疫苗和鸡卵黄抗体的制备及效应分析

Preparation and effectiveness assessment of a recombinant IBV multiple antigenic peptide vaccine and laying hen yolk antibodies

摘要:

目的 设计并表达融合传染性支气管炎病毒(infectious bronchitis virus, IBV)已证实抗原表位的重组蛋白rMKIBV，作为疫苗以期提供全方位保护，同时探讨经rMKIBV疫苗免疫蛋鸡收获的多克隆抗体卵黄抗体(IgY)在IBV防治中的潜力。方法 通过网站获取的IBV已证实抗原表位序列与GenBank数据库收录的具有代表性的IBV毒株序列进行比对，设计柔性肽连接所有抗原肽。构成的氨基酸序列经过分析处理后，逆翻译并进行密码子优化，随后插入pET-28a(+)克隆载体，导入大肠杆菌进行表达。纯化脱盐、去内毒素处理后的rMKIBV蛋白作为疫苗免疫动物，探究其免疫原性及是否可以刺激蛋鸡产生特异性IgY。结果 检索的IBV抗原表位序列与代表性22株IBV毒株的公开N蛋白和S蛋白序列在相应区域均高度相似。rMKIBV蛋白预测的等电点和分子量分别为10.25和63.39 kDa；二级结构显示无规则卷曲占比最高，表明抗原性较强。成功构建重组质粒pET-28a-mkibv，并在大肠杆菌中表达出高纯度的rMKIBV蛋白。该蛋白能与抗His-tag抗体、N蛋白抗体、S蛋白抗体特异性结合。免疫小鼠后，脾脏指数增大(P<0.05)，血清特异性IgG抗体水平显著升高(P<0.01)，IFN-γ水平升高(P<0.05)，IL-2水平无明显变化。免疫蛋鸡后，成功提取蛋黄中IgY，其特异性IgY抗体水平均显著升高，且IgY抗体滴度在免疫后逐步增大，至50 d左右达到最高，而后缓慢下降，达到稳定水平。结论 本研究成功构建并表达了融合IBV已证实抗原表位的重组蛋白rMKIBV，该蛋白具有较好的免疫原性，能够刺激小鼠和蛋鸡产生特异性抗体。特别是从免疫蛋鸡的蛋黄中获取的IgY，为IBV的防治提供了新的思路，对开发针对IBV的疫苗具有重要的科学意义和应用价值。

Abstract:

Objective To design and express a recombinant protein rMKIBV incorporating confirmed antigenic epitopes of infectious bronchitis virus (IBV) as a vaccine to provide comprehensive protection. Additionally, it explores the potential of polyclonal yolk antibodies (IgY) harvested from laying hens immunized with the rMKIBV vaccine in the prevention and control of IBV.Methods The antigenic epitope sequences of IBV, obtained from online databases, were compared with sequences of representative IBV strains from GenBank. Flexible peptides were designed to link all antigenic peptides. The constructed amino acid sequence was analyzed, reverse-translated, codon-optimized, and then inserted into the pET-28a(+) cloning vector. The recombinant vector was introduced into Escherichia coli for expression. The purified, desalted, and endotoxin-removed rMKIBV protein was used as a vaccine to immunize animals for investigation of its immunogenicity and ability to stimulate specific IgY production in laying hens.Results The retrieved IBV antigenic epitope sequences showed high similarity with the published N and S protein sequences of 22 representative IBV strains. The predicted isoelectric point and molecular weight of rMKIBV were 10.25 and 63.39 kDa, respectively. The secondary structure of rMKIBV included a high proportion of random coils, which suggested strong antigenicity. High-purity rMKIBV was obtained from E. coli transformed with the recombinant plasmid pET-28a-mkibv. This protein specifically bound to anti-His-tag antibodies, N protein antibodies, and S protein antibodies. The mice immunized with this protein showed increases in the spleen index (P<0.05), elevations in the levels of serum-specific IgG antibodies (P<0.01) and IFN-γ (P<0.05), and no significant change in the IL-2 level. Immunized laying hens successfully produced IgY in egg yolks, with specific IgY antibody levels significantly increasing. Moreover, the IgY antibody titer gradually rose after immunization, reaching the peak after about 50 days and then gradually declining to reach a stable level.Conclusion We successfully constructed and expressed the recombinant protein rMKIBV. The protein demonstrated good immunogenicity, stimulating specific antibody production in both mice and laying hens. Notably, the IgY extracted from the yolks of immunized laying hens offers a novel approach to IBV prevention and control. These findings hold significant scientific and practical value for the development of vaccines against IBV.