基于双质粒拯救系统的新城疫病毒LaSota株的拯救

A two-plasmid rescue system for rescue of recombinant LaSota strain of newcastle disease virus

摘要:

传统新城疫病毒(newcastle disease virus, NDV)的拯救系统包括一个cDNA克隆质粒和分别表达NDV的核衣壳蛋白(NP)、磷蛋白(P)、聚合酶蛋白(L)的3个辅助质粒，且必须满足4个质粒同时转染进入同一个宿主细胞才能完成病毒的组装，效率相对低下。【目的】提高NDV的拯救效率，并建立双质粒高效拯救系统。【方法】将NP、P、L基因表达盒串联克隆至真核表达载体pCI中，构建为可同时表达NP、P、L蛋白的单辅助质粒PCI-NPL；同时，采用分段克隆再拼接的方式，将NDV LaSota株基因组cDNA克隆于真核表达质粒pCI的CMV启动子下游，并分别在P和M基因中插入报告基因增强型绿色荧光蛋白(enhanced green fluorescent protein, EGFP)、5'端引入锤头状核酶序列、3'端引入丁型肝炎病毒核酶序列，构成全基因组转录质粒pCI-LaSota-EGFP；以pCI-LaSota-EGFP和pCI-NPL组成病毒拯救系统共转染至BHK-21细胞，拯救获得重组子代病毒rLaSota-EGFP，并进行系列生物学特性鉴定。【结果】经RT-PCR、荧光显微镜观察、Western blotting、生长特性测定等系列鉴定，证明rLaSota-EGFP构建正确，成功拯救获得了重组病毒rLaSota-EGFP，且与野生型(wild-type, WT) LaSota具有相似的生物学特性。【结论】基于CMV启动子的NDV双质粒新型拯救系统构建成功，为重组NDV及其他副黏病毒的高效拯救奠定了基础。

关键词:反向遗传学技术;新城疫病毒;病毒拯救;双质粒拯救系统

Abstract:

The conventional rescue system of newcastle disease virus (NDV) contains a cDNA clone plasmid and three helper plasmids expressing NDV: nucleocapsid protein (NP), phosphoprotein (P) and polymerase protein (L), respectively. These four plasmids have to be transfected into the same host cell simultaneously to complete the assembly of the virus, which is relatively inefficient. [Objective] To improve the rescue efficiency of NDV, this study aims to establish a two-plasmid rescue system. [Methods] The expression cassettes of NP, P, and L genes were constructed and sequentially cloned into the eukaryotic expression vector pCI to generate a single-helper plasmid, named pCI-NPL, capable of co-expressing NP, P, and L proteins. During this process, the genomic cDNA of the NDV LaSota strain was incorporated downstream of the CMV promoter in the pCI expression vector. Simultaneously, the reporter gene EGFP was inserted into the genome between the P and M genes, accompanied by the introduction of a hammerhead ribozyme sequence at the 5′ end and a hepatitis delta virus ribozyme sequence at the 3′ end. This culminated in the development of the full-genome transcriptional plasmid termed pCI-LaSota-EGFP. The two plasmids pCI-LaSota-EGFP and pCI-NPL were co-transfected as a two-plasmid system into BHK-21 cells to rescue the recombinant virus rLaSota-EGFP. The biological characteristics of the virus were then examined. [Results] RT-PCR, fluorescence microscopy, Western blotting, and growth characterization confirmed that rLaSota-EGFP was correctly constructed and expressed the foreign gene. The rescued recombinant virus rLaSota-EGFP had similar biological characteristics to wild-type (WT) LaSota. [Conclusion] A novel two-plasmid rescue system for NDV based on the CMV promoter was successfully established, laying a foundation for the efficient rescue of recombinant NDV and other paramyxoviruses.

Key words:reverse genetics;newcastle disease virus;virus rescue;two-plasmid rescue system