新城疫病毒耐热关键因子的挖掘与利用

Newcastle disease (ND) is a severe contagious avian disease, which is a heavy threat to poultry industry worldwide. The live vaccine is important for ND control. However, most of the ND live vaccines are thermolabile, and dependent strictly on the cold chain. During the application of the live vaccine, the partial or complete failure of immunization is commonly existed. Therefore, it is very important to develop the thermostable vaccines against ND. Previously, we have obtained a thermostable vaccine strain of Newcastle disease virus (NDV), named TS09-C strain, and established the reverse genetic system of this strain. To find the key thermostable determinant of NDV TS09-C strain, we will construct a series of chimeric viruses stemmed from the thermostable TS09-C stain with the replacement of each gene by the non-thermostble Lasota strain in turn, and stemmed from Lasota strain with the replacement of gene by TS09-C strain. By analyzing the thermostable characterization of these chimeric viruses, the major gene that determines the thermosatbility of NDV will be identified. Next a series of chimeric virues with the replacement of gene fragment or site-directed mutagenesis in the thermostable gene will be constructed and analyzed, the key thermostable determinant of NDV will be identified. By utilizing the bio-informatic technology, we will discuss the effect of the key gene fragment or amino acids on the NDV thermostability. Finally, the thermostable mechanism of NDV will be clarified. By replacing the corresponding gene fragment of NDV Mukteswar strain with thermostable key determinant of TS09-C strain, a new thermostable vaccine strain will be constructed and tested for its thermostability, growth kinetics, pathogenicity and immunogenicity, the potential value of this recombinant NDV strain as thermostable vaccine candidate will be evaluated.This project will lay foundation for the development of new thermostable vaccines that can be stored and transported at room temperature.

新城疫是OIE规定的仅有的两种A类禽病之一，其爆发流行给养禽业造成了巨大的经济损失。活疫苗是防控新城疫的重要工具，其中耐热活疫苗在我国气温较高的南方地区和偏远的农村地区具有巨大优势和应用前景。但具有优良耐热特性的疫苗株在自然界比较稀有。本项目前期已选育得到新城疫耐热弱毒TS09-C株，并构建了其反向遗传操作系统。在此基础上，拟开展新城疫病毒耐热关键因子的挖掘与利用研究。应用反向遗传操作技术，以耐热TS09-C株和非耐热LaSota株基因片段互换（或点突变）为策略，寻找其关键耐热因子。应用生物信息学技术，分析关键因子对病毒耐热的影响，阐明其耐热的分子机理。利用优化的耐热关键因子对新城疫病毒Mukteswar疫苗株进行耐热改造，分析耐热改造后重组病毒的耐热、免疫原性等特性，评估其作为新耐热活疫苗的潜在价值。将为人为地赋予优秀的工程毒株耐热特性和研发可常温保存的新型基因工程疫苗提供科学依据。

新城疫是OIE规定的仅有的两种A类禽病之一，其爆发流行给养禽业造成了巨大的经济损失。活疫苗是防控新城疫的重要工具，其中耐热活疫苗在我国气温较高的南方地区和偏远的农村地区具有巨大的优势和应用前景。但具有优良耐热特性的疫苗株在自然界比较稀有。本项目前期已选育得到新城疫病毒（NDV）耐热弱毒TS09-C株，并构建了其反向遗传操作系统。在此基础上，开展了NDV耐热关键因子的挖掘与利用研究：① 利用反向遗传操作技术，通过耐热TS09-C株和非耐热LaSota株基因片段互换（或点突变）为策略，证实了NDV的HN基因为其耐热关键基因，在HN基因内部找出了可显著影响NDV热稳定性的若干氨基酸突变组合；② 此类氨基酸突变组合首先影响到了HN蛋白自身的热稳定性，随后影响到了NDV与宿主细胞结合的热稳定性，最后影响到了NDV的感染、复制能力；③ 利用此类氨基酸突变组合，对TS09-C株和LaSota株进行耐热改造（点突变策略），两个毒株的热稳定性均得到了显著提升。本项目为人为地赋予优秀的工程毒株耐热特性和研发可常温保存的新型基因工程疫苗提供了科学依据。