基于内部核糖体进入位点提高重组NDV表达IBDV VP2基因的研究

An enhanced expression of vvIBDV VP2 gene in recombinant NDV by using IRES sequence

为提高重组新城疫病毒(NDV)外源基因表达量,本研究采用内部核糖体进入位点序列(IRES)构建表达鸡传染性法氏囊病病毒超强毒(vvIBDV)H LJ07株V P2双拷贝基因的重组NDV(rClone30-V P2-IRES-V P2),同时构建表达单拷贝V P2基因的重组NDV(rClone30-V P2)作为对照。间接免疫荧光试验表明V P2蛋白在感染两种重组病毒的鸡胚成纤维细胞(DF-1)中得到表达。Real-time PCR检测结果表明rClone30-VP2-IRES-V P2中VP2 mRNA转录水平明显高于rClone30-VP2。生长曲线分析表明,两株重组病毒株与亲本NDV LaSota(Clone30)株在细胞培养中可以同样稳定复制。重组病毒株在鸡胚中多次传代之后仍稳定表达VP2蛋白。本研究通过建立NDV的反向遗传操作系统构建了独立表达IBD V双拷贝V P2基因的重组NDV,并证明双拷贝基因的表达水平优于单拷贝基因,为提高重组NDV外源基因的表达量提供了新的思路。

To enhance the expression of exogenous gene in recombinant Newcastle disease virus （NDV）, we constructed the recombinant NDV （rClone30-VP2-IRES-VP2） containing double VP2 genes connected with the internal ribosome entry sites （IRES） sequence and the recombinant virus （rClone30-VP2） containing single VP2 gene as control. The expressions of VP2 protein in infected DF-1 ceils were confirmed by indirect immunofluorescence assay and mRNA transcript levels of VP2 gene were examined by real-time RT-PCR. The mRNA transcript level in rClone30-VP2-IRES-VP2 showed significantly higher than that of rClone30-VP2. The recombinant viruses had similar replication characteristic as parental NDV in DF-lcells and were genetically stable in chicken embryos. In this study, we constructed the recombinant NDV expressing double genes by using IRES, and proved the double genes had a higher expression than single gene. The study provides a new approach for improving the expression of exogenous genes in recombinant NDV.