中科院武汉病毒所人工合成杆状病毒获得成功
合成生物学技术作为21世纪的一门新兴生物学技术，推动了生命科学乃至整个自然科学领域的发展。病毒的人工合成为深入揭示病毒的本质和功能，以及病毒的遗传改造提供了强有力的工具。以往，对病毒人工合成的探索主要集中在RNA病毒上，而目前已知最大的RNA病毒基因组也仅有~30 kb。迄今为止，所报道成功合成的DNA病毒最大不超过6 kb。杆状病毒是一类大的双链DNA病毒（基因组大小80-180 kb），在生物农药、真核表达系统等领域具有重要的应用价值。近期，中国科学院武汉病毒研究所胡志红研究员课题组联合运用PCR及酵母转化相关的同源重组（Transformation Associated Recombination, TAR）技术，首次合成了杆状病毒模式种AcMNPV的全基因组，并通过转染细胞成功拯救出了有感染性的人工合成病毒。研究人员首先利用PCR扩增覆盖AcMNPV全基因的~45个片段，每个片段约3kb，相邻片段之间有大于60 bp的重叠序列。然后利用TAR技术，在酵母细胞内进行了三次重组，依次获得了9个~15 kb的片段、3个~45 kb的片段和全基因组（145,299 bp）。将合成的病毒基因组进行了454测序验证，通过转染昆虫细胞成功获得了有感染性的人工合成病毒AcMNPV-WIV-Syn1（图A）。电镜、一步生长曲线和生物测定等结果表明，合成病毒与亲本病毒具有相似的生物学特性（图B）。
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该技术的建立，不仅为杆状病毒的基础研究提供了有力工具，还可以用于改良杆状病毒的表达系统和杀虫性能。该研究是大DNA病毒研究领域的重要突破，被审稿人认为“this is a highly significant paper that will serve as a landmark in synthetic biology”。 这一研究成果已经在ACS Synthetic biology杂志上在线发表（DOI: 10.1021/acssynbio.7b00028），中科院武汉病毒所的博士生商雨为该论文的第一作者，邓菲研究员和胡志红研究员为该论文的共同通讯作者。该研究得到了中国科学院战略性先导科技专项（XDB11030400）、国家自然科学基金创新群体项目（31621061）和重点项目（31130058）、中国科学院前沿科学重点研究项目（QYZDJ-SSW-SMC021）、中科院知识创新工程项目（KSCX2-EW-Z-3）、病毒学国家重点实验室病毒学前沿科学重点研究项目（klv-2016-03）、973项目（2012CB721102）的资助。
题目：Construction and Rescue of a Functional Synthetic Baculovirus
Abstract
Synthetic viruses provide a powerful platform to delve deeper into the nature and function of viruses as well as to engineer viruses with novel properties. So far, most synthetic viruses have been RNA viruses (<30 kb) and small DNA viruses, such as bacteriophage phiX174. Baculoviruses contain a large circular dsDNA genome of 80–180 kb and have been used as biocontrol agents and protein expression vectors. Here, we report on the first synthesis of a baculovirus based on the type species Autographa californica nucleopolyhedrovirus, AcMNPV, by a combination of PCR and transformation-associated recombination in yeast. The synthetic genome, designated AcMNPV-WIV-Syn1, is 145 299 bp comprising the complete genome of AcMNPV except for the hr4a locus that was replaced with an ∼11.5 kb cassette of bacterial and yeast artificial chromosomal elements and an egfp gene. Sf9 insect cells were transfected with AcMNPV-WIV-Syn1 DNA and progeny virus was examined by electron microscopy, and assayed in one-step growth curves and oral infectivity. The results conclusively showed that the rescued virus AcMNPV-WIV-Syn1 had structural and biological properties comparable to the parental virus. We validated a proof of concept that a bona fide baculovirus can be synthesized. The new platform allows manipulation at any or multiple loci and will facilitate future studies such as identifying the minimal baculovirus genome and construction of better expression vectors. This is the largest DNA virus synthesized so far, and its success is likely to be the impetus to stimulate the fields of other large DNA viruses such as herpesviruses and poxviruses.
本文来源：中国科学院武汉病毒研究所
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