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为制备一种安全、有效的人偏肺病毒（Human metapneumovirus,HMPV）假病毒阳性标准品，并用于核酸检测的质量控制，本研究对335条HMPV全基因序列比对分析，选择HMPV基因表达量高且进化较为保守的N基因构建重组假病毒颗粒。合成全长1 185 bp的N基因序列并与CD513B载体进行重组连接，采用第三代慢病毒包装体系构建HMPV假病毒颗粒，超速离心浓缩后感染HEK293T细胞，RT-qPCR核酸检测N基因和透射电镜观察鉴定重组假病毒颗粒，确定假病毒阳性标准品使用浓度，进一步评价阳性标准品的热稳定性和均一性。研究结果显示，包装获得的HMPV假病毒颗粒能够感染HEK293T细胞，假病毒颗粒为圆形或椭圆形病毒样结构，直径在150～200 nm之间，并带有G蛋白刺突。HMPV假病毒稀释103倍后Ct值最适于进行核酸检测，并且均一性和稳定性可达到阳性标准品要求。本研究成功构建了含有HMPV N基因全长的假病毒颗粒，可作为核酸检测方法的阳性标准品，实现HMPV核酸提取和核酸检测过程的全程质量控制。
Abstract：To develop safe and effective positive controls for human metapneumovirus(HMPV) pseudovirus that can be used in the quality control of nucleic acid detection, this study compared and analyzed 335 full-length HMPV gene sequences. The N gene, known for its high expression levels and evolutionary conservation, was selected to construct recombinant pseudovirus particles. The full-length 1185 bp N gene sequence was synthesized and recombined with the CD513B vector. Using a third-generation lentiviral packaging system, HMPV pseudovirus particles were constructed, concentrated by ultracentrifugation, and subsequently used to infect HEK293T cells. The recombinant pseudovirus particles were identified via RT-qPCR targeting the N gene and observed through transmission electron microscopy. The optimal concentration for the positive control was determined, and the thermal stability and homogeneity of the controls were further evaluated. Results indicated that the packaged HMPV pseudovirus particles successfully infected HEK293T cells, exhibiting spherical or elliptical virus-like structures with diameters ranging from 150 to 200 nm and displaying spike proteins. Following a 103-fold dilution, the HMPV pseudovirus showed Ct values that were ideal for nucleic acid detection, with satisfactory homogeneity and stability to meet the requirements for a positive control. This study successfully developed pseudovirus particles containing the full-length of the HMPV N gene, which can serve as a positive control for nucleic acid detection, enabling comprehensive quality control for HMPV nucleic acid extraction and detection processes.
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基本信息:
DOI：10.13242/j.cnki.bingduxuebao.004615
中图分类号:R373.1
引用信息:
[1]郑诗源,黄益曼,孟小草等.人偏肺病毒假病毒阳性标准品的制备与鉴定[J].病毒学报,2024,40(06):1263-1273.DOI:10.13242/j.cnki.bingduxuebao.004615.
基金信息:
中国疾病预防控制中心病毒病预防控制所青年科学基金（项目号：IVDC-202304）,题目：基于铁蛋白的呼吸道合胞病毒和人偏肺病毒二联纳米颗粒疫苗研究~~