通过基因组合成构建美洲型猪繁殖与呼吸综合征病毒感染性克隆

发布时间：2018-06-25 20:53

本文选题：猪繁殖与呼吸综合征病毒 + 基因合成　；参考：《中国兽医科学》2017年07期

【摘要】：根据已测定的猪繁殖与呼吸综合征病毒(PRRSV)GSWW分离株的全长基因组序列信息,采用合成生物学的方法,将全长基因组序列分为两段(7 636 bp和7 774 bp)分别连接到低拷贝载体p BR-322载体中,然后将2个半长片段相连接得到全长c DNA克隆。将质粒线性化后,体外转录获得全长RNA,经电穿孔转染BHK21细胞拯救获得了初代病毒。将初代病毒接种感染Marc-145细胞进行传代,接毒后第36小时即可观察到完全的致细胞病变效应(CPE)。通过RT-PCR、间接免疫荧光、测序验证,证明成功获得了PRRSV GSWW株的感染性克隆。通过实时荧光定量法检测拯救病毒与原田间分离毒的复制能力,没有显著差异,采用实时荧光定量法测得的拯救病毒复制的最高效价为1×1010copies/m L。本研究表明,采用长片段基因合成的方法进行PRRSV感染性克隆的构建,方法可行且速度快,为PRRSV流行毒感染致病机理、免疫机制和基于反向遗传学的新型疫苗研制提供了重要的分子工具。
[Abstract]:According to the information of the full-length genome sequence of porcine reproductive and respiratory syndrome virus (PRRSv) GSWW isolate, the synthetic biology method was used. The full length genomic sequence was divided into two segments (7 636 BP and 7 774 BP), respectively, which were ligated to the low copy vector pBR-322, and then two half long fragments were ligated to obtain the full length cDNA clone. After the plasmid was linearized, the full-length RNAs were obtained by transcription in vitro, and the primary virus was obtained by electroporation transfection into BHK21 cells. The primary virus was inoculated into Marc-145 cells for passage, and the complete cytopathogenic effect (CPE) was observed 36 hours after inoculation. The infectious clone of PRRSv GSWW strain was successfully obtained by RT-PCR, indirect immunofluorescence and sequencing. There was no significant difference between the replicating ability of the rescue virus and the original virus isolated in the field by real-time fluorescence quantitative method. The highest titer of the replicating of the rescue virus was 1 脳 1010copies/m / L measured by the real-time fluorescence quantitative method. The results showed that the construction of PRRSv infectious clone by long fragment gene synthesis was feasible and rapid, which was the pathogenic mechanism of PRRSv infection. Immune mechanisms and the development of new vaccines based on reverse genetics provide important molecular tools.
【作者单位】：中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室;
【基金】：甘肃省农业生物技术研究与应用开发项目(GNSW-2009-10) 中国农业科学院创新工程经费资助
【分类号】：S852.651

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