Tavassoli Amin, Soleymani Safoura, Haghparast Alireza, Hashemi Tabar Gholamreza, Bassami Mohammad Reza, Dehghani Hesam
1Division of Biotechnology, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Azadi Square, Mashhad, Iran.
2Immunology Section, Department of Pathobiology , Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran.
Biol Proced Online. 2020 May 1;22:9. doi: 10.1186/s12575-020-00119-3. eCollection 2020.
The BioBrick construction as an approach in synthetic biology provides the ability to assemble various gene fragments. To date, different BioBrick strategies have been exploited for assembly and cloning of a variety of gene fragments. We present a new BioBrick strategy, here referred as Asis-Sal-Pac BioBrick, which we used for the assembly of NDV as a candidate for single-stranded non-segmented, negative-sense RNA genome viruses.
In the present study, we isolated three NDVs from clinical samples which were classified into the VIId genotype based on their pathogenicity and phylogenetic analyses. Then, SalI, AsisI, and PacI enzymes were used to design and develop a novel BioBrick strategy, which enabled us to assemble the NDV genome, adopting the "rule of six". In this method, in each assembly step, the restriction sites in the newly formed destination plasmid are reproduced, which will be used for the next insertion. In this study using two overlapping PCRs, the cleavage site of the F gene was also modified from RRQKRFto GRQGRL in order to generate the attenuated recombinant NDV. Finally, in order to construct the recombinant NDV viruses, the plasmids harboring the assembled full-length genome of the NDV and the helper plasmids were co-transfected into T7-BHK cells. The rescue of the recombinant NDVwas confirmed by RT-PCR and HA tests.
These findings suggest that the combination of reverse genetic technology and BioBrick assembly have the potential to be applied for the development of novel vaccine candidates. This promising strategy provides an effective and reliable approach to make genotype-matched vaccines against specific NDV strains or any other virus.
生物砖构建作为合成生物学中的一种方法，能够组装各种基因片段。迄今为止，已开发出不同的生物砖策略用于多种基因片段的组装和克隆。我们提出了一种新的生物砖策略，在此称为Asis-Sal-Pac生物砖，我们将其用于新城疫病毒（NDV）的组装，新城疫病毒是单链、不分节段的负链RNA基因组病毒的候选对象。
在本研究中，我们从临床样本中分离出三株新城疫病毒，根据其致病性和系统发育分析将它们归类为VII d基因型。然后，使用SalI、AsisI和PacI酶设计并开发了一种新型生物砖策略，该策略使我们能够采用“六规则”组装新城疫病毒基因组。在这种方法中，在每个组装步骤中，新形成的目的质粒中的限制酶切位点会被复制，用于下一次插入。在本研究中，通过两次重叠PCR，F基因的切割位点也从RRQKRF修饰为GRQGRL，以产生减毒重组新城疫病毒。最后，为了构建重组新城疫病毒，将携带组装好的新城疫病毒全长基因组的质粒和辅助质粒共转染到T7-BHK细胞中。通过RT-PCR和血凝试验证实了重组新城疫病毒的拯救。
这些发现表明，反向遗传技术和生物砖组装的结合有潜力应用于新型候选疫苗的开发。这种有前景的策略提供了一种有效且可靠的方法来制备针对特定新城疫病毒株或任何其他病毒的基因型匹配疫苗。
