表达鸡传染性支气管炎病毒S1和N蛋白重组新城疫病毒的构建及免疫保护评价

Virulent strains of Newcastle disease virus (NDV) and Infectious bronchitis virus (IBV) can cause serious respiratory diseases in poultry worldwide. These viruses are highly contagious and possess serious threats to food safety, public health, and biosecurity. The IBV disease is usually accompanied by secondary bacterial infection that can increase high incidence and mortality, resulting in the significant economic losses to the poultry industry. Vaccination combined with strict biosecurity practices has been the recommendation for controlling both NDV and IBV diseases in the field. Live, attenuated IBV vaccines appear to be effective against IBV disease routinely, but the stability and safety of these live attenuated vaccines remain a concern because of the potential of virulence revertance. Moreover, the IBV genome mutationresult in protecting with fail. More researcher foucos on development novel vaccine expressing one protein as vaccine against IBV, But those vaccine were not enough to complete protection against virulent IBV strain challenge. Thus, there is a pressing need to develop a save, effective and economic bivalent vaccine that can be readily administered in the field to control these viral diseases. Spike glycoprotein (S1) and Nucleocapsid protein are the important antigen against IBV challenge, Internal ribosome entry site (IRES) is a nucleotide sequence that allows for translation initiation in the middle of a messenger RNA (mRNA) sequence as part of the greater process of protein synthesis. lentogenic NDV strains LaSota, are routinely used as live vaccines throughout the world to prevent Newcastle disease. Therefore, this research proposed to develop NDV LaSota vaccine strain-based recombinant viruses that co-express IBV Spike glycoprotein (S1) and Nucleocapsid protein protein with IRES firstly as a bivalent vaccine against these viral diseases. In addition, the recombinant NDV LaSota vaccine vector can be used to develop bivalent vaccines against other avian viral diseases and may also have potential applications in anti- other viral diseases.

新城疫和禽传染性支气管炎分别由新城疫病毒（NDV）和禽传染性支气管炎病毒（IBV）引起家禽呼吸道感染一种急性高度接触性传染病，在全世界传播，其高病死率造成养禽业重大经济损失，并严重威胁着食品、生物及公共卫生安全。现今用于预防IBV的灭活苗和弱毒苗存在缺陷，当前研究的新型疫苗多是表达单个基因，免疫保护较低，加上基因突变常导致免疫失败，其防控当今仍未解决，因此，研发安全有效的新型疫苗刻不容缓。S1和N蛋白是IBV 重要保护性抗原，IRES可独立启动mRNA 翻译，LaSota不但可直接作为疫苗用于NDV防控，又是较安全有效的活疫苗载体，本研究拟以LaSota 为载体，首次利用IRES连接IBV M41 株S1和N基因插入基因组F-HN 间，构建共表达S1和N蛋白的重组病毒，对其生物学特性及免疫保护评价，为获得可抵抗NDV和IBV安全有效的双价苗提供候选株，也为其他疫病共同预防提供新的研究思路。

新城疫和禽传染性支气管炎分别由新城疫病毒（NDV）和禽传染性支气管炎病毒（IBV）引起家禽呼吸道感染一种急性高度接触性传染病，在全世界传播，其高病死率造成养禽业重大经济损失，并严重威胁着食品、生物及公共卫生安全。现今用于预防IBV的灭活苗和弱毒苗存在缺陷，当前研究的新型疫苗多是表达单个基因，免疫保护较低，加上基因突变常导致免疫失败，其防控当今仍未解决。. 本研究以LaSota 为载体，利用反向遗传学，首次利用IRES连接IBV M41 株保护性抗原基因S1和N基因通过infusion PCR插入LaSota 基因组F-HN 间，构建含有S1和N蛋白的重组新城疫cDNA 克隆，通过转染试剂将其转染到HEP-2细胞内，拯救重组病毒，在9日龄SPF 鸡胚内增殖病毒，并检测重组新城疫病毒rLS/IBV-SI-N的生物学特性、间接免疫荧光鉴定S1-N蛋白的表达，在 SPF 雏鸡内对重组新城疫病毒进行免疫保护评价，并与PBS 对照组和亲本病毒LaSota作对照。结果表明rLS/IBV-SI-N的毒力在SPF鸡胚和1日龄雏鸡内轻微致弱，其生物学特性与LaSota 株相似。免疫保护评价结果显示，SPF雏鸡免疫接种rLS/IBV-SI-N后可完全保护NDV强毒株的攻击，首免后约70%的雏鸡可诱导产生特异性IBV 抗体，用强毒IBV M41攻击时，与对照组LaSota 相比，其临床症状表现轻微，病毒在呼吸道内的检出率仅为20%，明显提高了对IBV的保护率。. 本研究结果表明，以 LaSota为载体表达IBV –S1和N蛋白的重组NDV病毒，对NDV强毒株完全保护，对IBV 有明显保护作用，可作为抵抗NDV和IBV的候选双价疫苗，用于生产实践。同时，在本研究中所构建的重组NDV载体还可用于其他多价疫苗和基因治疗方面的研究，具有广泛的应用前景。