中文摘要：

为探索猪瘟病毒（CSFV）的复制机理,本实验在前期CSFV流行株GD53全基因组测序的基础上,利用新霉素抗性基因（Neor）替换病毒结构蛋白基因以构建CSFV GD53株的亚基因组复制子（GD53-SGR）。首先构建含有T7-5＇UTR-N^pro-Neor-EMCV/IRES-NS2-NS3-NS4A-NS4B-NS5A-NS5B-3＇UTR的重组质粒pc DNA-GD53-SGR,将其利用T7 RNA聚合酶经体外转录制备的RNA转录本转染猪睾丸细胞系（ST）后,利用G418筛选含有GD53-SGR的ST细胞,构建ST-GD53-SGR细胞系。酶切鉴定及测序结果表明,重组质粒pc DNA-GD53-SGR构建正确,利用T7 RNA聚合酶体外转录后获得了与预期大小一致的GD53-SGR RNA。经RT-PCR和间接免疫荧光鉴定结果表明,获得了含有GD53-SGR的ST细胞系。本研究构建的CSFV ST-GD53-SGR细胞系有助于进一步了解CSFV非结构蛋白的功能,同时为CSFV的复制机制研究及抗病毒药物的研发奠定基础。

英文摘要：

In order to explore the mechanism of classical swine fever virus（CSFV） replication, RNA replicon of CSFV GD53 strain was constructed through replacing its structural protein genes with neomycin resistance gene. Recombinant plasmid pc DNA-GD53-SGR containing T7-5＇UTR-N~（pro）-Neor-EMCV/IRES-NS2-NS3-NS4A-NS4B-NS5A-NS5B-3＇UTR was constructed through homologous recombination of synthetic T7-NS3 fragment and amplified NS3-3＇UTR, which was confirmed by restriction enzyme digestion and DNA insert sequencing. The obtained recombinant plasmid was used as the template for in vitro transcription of GD53-SGR, which was analyzed by agarose gel electrophoresis and RT-PCR and subsequently transfected into swine testicle cells（ST）. ST cell clones containing GD53-SGR were screened with culture medium containing G418 and confirmed by amplification and sequencing of full-length GD53-SGR and indirect fluorescent antibody test（IFA）. The RNA subgenomic replicon of CSFV GD53 and GD53-SGR-expressing ST cells were established and will be used as an efficient tool for studies on viral replication and infection of CSFV.