【摘要】 为研制预防新城疫病毒(NDV)和传染性法氏囊病毒(IBDV)两种病毒病的疫苗,本研究通过基因工程抗体和病毒反向遗传操作技术,以NDV LaSota株为载体,在病毒基因组的不同部位,分别或同时插入具有中和活性的鸡源IBDV抗体重链(H)或轻链(L)片段,构建并拯救出重组病毒,分别命名为rNDV-IRES-H-L(P/M)、rNDV-H(NP/P)-L(P/M)、rNDV-L(NP/P)-H(P/M)、rNDV-H(P/M)、rNDV-H(NP/P),并对其进行HA、TCID50、EID50、MDT、ICPI等试验以检测上述重组病毒的生物活性。结果显示,重组病毒不仅保持了其亲本病毒(NDV LaSota株)的感染能力而且还保持了其弱毒株的生物学特性。生长曲线结果显示,重组病毒与亲本NDV生长特性基本一致,表明外源基因的插入对病毒的复制能力无影响。ELISA和western blot结果显示rNDV-H(NP/P)、rNDV-H(P/M)中的H基因,rNDV-IRES-H-L(P/M)、rNDV-H(NP/P)-L(P/M)、rNDV-L(NP/P)-H(P/M)中的H和L基因均获得了表达,且rNDV-H(P/M)中的H基因和rNDV-L(NP/P)-H(P/M)中的H和L基因表达量高。用表达量高的重组病毒接种14日龄SPF鸡,分别检测其诱导SPF鸡NDV和IBDV抗体的产生情况。结果显示,在NDV抗体水平方面:HI实验结果表明重组病毒抗体效价均能在14 d达到保护临界值(4 log2);在IBDV抗体水平方面:ELISA结果表明重组病毒均能产生IBDV抗体。利用上述表达量高的重组病毒对人工感染IBDV(经典株BC6/85)的SPF鸡进行免疫保护试验,结果表明rNDV-H(P/M)的免疫保护效果要好于卵黄抗体和rNDV-L(NP/P)-H(P/M)。本研究获得了表达IBDV抗体的重组NDV即rNDV-H(P/M),为利用NDV载体表达本动物源抗体提供了实验依据。更多还原

【Abstract】 In order to develop vaccine preventing Newcastle disease（ND） and infectious bursal disease（IBD）, the NDV LaSota strain was used as a vector to express the heavy chain（H）, light chain（L） or both chains of chicken-derived neutralizing antibody against IBDV. The techniques of genetic engineering antibody and reverse genetics in viruses were used to construct and rescue the recombinant viruses. Nucleotide sequences of the heavy chain（H）, light chain（L） or both chains of chicken-derived neutralizing antibody were inserted into different intergenic regions of the NDV vector, named as rNDV-IRES-H-L（P/M）, rNDV-H（NP/P）-L（P/M）, rNDV-L（NP/P）-H（P/M）, rNDV-H（P/M）, and r NDV-H（NP/P）, respectively. The HA, TCID₅₀, EID₅₀, MDT, ICPI, and growth curve experiments were performed to determine the biological activity of the recombinant viruses. The results showed that the insertions of above mentioned nucleotide sequences had no effect on any biological characteristic of NDV LaSota strain as an attenuated vaccine. The growth curve showed that the insertion of foreign nucleotide sequence did not influent the replication of LaSota viruses. The ELISA and western blot results showed that foreign genes were successfully expressed in all recombinant viruses, among which r NDV-H（P/M） and rNDV-L（NP/P）-H（P/M） showed the highest levels of expression of foreign genes. The production of antibody upon the inoculation of recombinant viruses was detected in 21-day-old SPF chickens. For the production of anti-NDV antibody, HI results showed that the titer of antibody induced by the inoculation of recombinant viruses reached the protection value at 14 days post inoculation. For the production of anti-IBDV antibody, ELISA results showed that all recombinant viruses could induce the production of anti-IBDV antibodies. Immunoprotection experiments were performed to analyze the protective effects of the recombinant viruses against IBDV infection. The results showed that immunoprotective effect of rNDV-H（P/M） was stronger than those of yolk antibody and r NDV-L（NP/P）-H（P/M）. In conclusion, the present study generated a novel recombinant Newcastle disease virus（rNDV-H（P/M）） expressing the heavy chain（H） of chicken-derived neutralizing antibody against IBDV, which lays a theoretical and experimental foundation for the use of Newcastle disease virus vector to express the animal-derived antibody.更多还原