杆状病毒口服感染因子模块化的研究

Oral infection, mediated by 10 oral infection factors (PIF), is primary step of baculovirus infection cycle. Due to the large size of viral genome and dispersed distribution of pif genes on the genome, the oral infection mechanism and host range study are greatly restricted. This project intends to apply synthetic biology technology to solve this problem through the modularization of pif gene. On the previous basis of artificially synthesizing a baculovirus, firstly, the tests of 10 pif genes are carried out through single gene deletion to determine the optimal deletion strategy, and then a baculovirus genome with all pif genes deleted is constructed; secondly, a pifs gene module is constructed and repaired to the genome with all pif genes deleted, and a baculovirus similar to parental strain is obtained through optimization and screening and a platform for pif genes modularization is established; at last, the replacements of different baculovirus PIFs are carried out based on the platform and the application efficacy of the platform of pif genes modularization is detected and evaluated. The successful establishment of the platform of baculovirus pif genes modularization will provide a key technology platform for oral infection mechanism and host range research, and also provide theoretical support for modularization of other functional genes.

口服感染是杆状病毒感染宿主的首要环节，也是影响病毒宿主域的重要因素，其由10个口服感染因子（PIF）介导。由于pif基因在基因组上呈散在性分布，且杆状病毒基因组较大，极大地限制了其口服感染机制及宿主域研究。本项目拟应用合成生物学技术，通过pif基因模块化研究解决这一难题。在前期已经人工合成了一株杆状病毒的基础上，首先对10个pif基因进行单基因缺失验证，确定最佳缺失策略，构建pif基因全缺失的病毒基因组；其次构建pif基因模块，将其回复到pif基因全缺失的基因组上，通过优化筛选，获得一株与亲本株特性相似的病毒，建立pif基因模块化平台；最后利用该平台实现不同病毒PIFs的替换操作，检测评估pif基因模块化平台的应用效果。杆状病毒pif基因模块化平台的成功构建，将为其口服感染机制及宿主域研究提供了关键技术平台，也为其他功能基因的模块化提供了理论支撑。

口服感染是感染的起始阶段，也是影响宿主域的重要因素。影响口服感染的10个基因（pif0-pif9）散在分布在基因组上，协同发挥作用。为了能够方便后期对10个pif基因的整体功能研究以及其对宿主域范围影响的机制研究，本项目对开展了10个pif基因的模块化研究。以苜蓿银纹夜蛾核型多角体病毒（AcMNPV）为研究对象，利用合成生物学技术， 通过合成pif基因单缺失的病毒确立了10个pif基因缺失方案。然后构建了10个pif基因同时缺失的病毒，然而此病毒的复制效率受到了极大影响。将AcMNPV的10个pif基因构建成一个模块并组装到病毒的基因组上，重组病毒的复制效率有一定程度的提升。口服感染结果显示，pif模块化的病毒具有口服感染甜菜夜蛾幼虫的能力。用其他杆状病毒的pif模块替换AcMNPV的pif模块，可以获得不同口服感染能力重组病毒。以上结果表明，虽然pif基因的模块化会在一定程度上影响病毒的复制，但pif基因模块化的病毒可以发挥口服感染能力，因此pif基因的模块化可以用于后续PIF复合物功能研究和影响病毒宿主域机制研究。