新城疫病毒Italien株辅助质粒的构建及功能鉴定

摘要

目的 构建基于T7启动子的含新城疫病毒(NDV)Italien株核衣壳蛋白(NP)、磷酸化蛋白(P)和蛋白(L)基因的表达质粒,作为构建重组NDV所需的辅助质粒.方法 将NDV的NP、P和L基因酶切后置于T7启动子和核糖体进入位点序列的下游,分别构建NP、P和L基因的表达质粒,转染BSR-T7/5细胞,采用间接免疫荧光法(1FA)检测病毒蛋白的表达,利用微型基因组(MG-L)质粒检测辅助质粒组装核糖核酸蛋白质复合物(RNP)的功能.结果 测序证实辅助质粒构建正确.IFA检测可观察到NP和P基因的表达.与MG-L单转染对照组和空白对照组相比,辅助质粒和MG-L共转染后,报告基因萤火虫荧光素酶得到高效表达(P＜0.001).结论 成功构建了基于T7启动子的NDV辅助质粒,为进一步构建重组溶瘤NDV Italien株奠定了基础.%Objective Newcastle disease virus (NDV) is anaturally oncolytie virus that has been shown to be safe and effective for cancer therapy. NDV virions possess a non-segmented negative-sense single-stranded RNA genome which contains six genes encoding the nucleocapsid protein (NP), phosphoprotein (P), large polymerase protein (L), matrix protein, fusion protein, and hemagglutinin-neuraminidase. The ribonucleoprotein (RNP) complex consisting of the genomic RNA and the three proteins NP, P, and L are the active template for transcription and replication of the viral genome. The purpose of this study was to construct the expression plasmids of NP, P and L genes of NDV Italien strain in which phage T7 promoter was a transcription promoter for the aim of generation of recombinant NDV. Methods NP, P and L genes were cloned from the genome RNA of NDV Italien followed by introduction into the downstream of T7 promoter and internal ribosome entry sites to construct the expression plasmids of NP, P and L, respectively. Expression of exogenous gene in BSR-T7/S cells which constitutively express phage T7 RNA polymerase and transfected with plasmids of NP and P was detected by indirect immunofluorescence assay. The function of NP, P and L proteins expressed by constructed plasmids to facilitate the genomic RNA to form RNP complex was tested using minigenome of NDV ItaLien carrying firefly Luciterase as a reporter gene. Results The expression plasmids of NP, P and L genes were confirmed by DNA sequencing. Using the indirect iromunofhiorescence assay, we detected the expression of viral NP and P proteins in BSR-T7/5 cells. When the helper plasmids were co-trsnsfected with NDV minigenome plasmid, the expression of firefly luciferase was more significant compared with the control group (P<0.001). Conclusion The helper plasmids of NDV Italien strain using T7 promoter as a transcription promoter has been constructed successfully, and it provides a basis for the construction of recombinant NDV.