利用酵母双杂交系统筛选与鉴定鱼类神经坏死病毒衣壳蛋白互作因子

Using the yeast two-hybrid system to screen and identify interacting factors with the envelope protein of fish necrosis virus

  • 摘要:
    目的 探究神经坏死病毒在鱼类感染过程中的作用机制,筛选与鉴定鱼类神经坏死病毒衣壳蛋白(CP)的互作因子。
    方法 首先提取SSN-1(striped snakehead cell,SSN-1)细胞总RNA,构建SSN-1细胞的酵母双杂交cDNA文库;利用PCR扩增神经坏死病毒衣壳蛋白基因,克隆于pBT3-STE表达载体,以神经坏死病毒衣壳蛋白作为诱饵,利用酵母双杂交技术,在SSN-1细胞cDNA文库中筛选出神经坏死病毒衣壳蛋白的互作因子。
    结果 酵母双杂文库容量为7.2×106 CFU,插入片段的平均长度在1 000 bp以上,重组率为100%。初步筛选得到24个阳性克隆,经测序比对和一对一验证,最终获得7个候选互作蛋白。功能预测结果显示,互作蛋白涉及免疫调控、转录调节、胁迫相应、信号转导等方面的功能。
    结论 本研究利用酵母双杂交技术从神经坏死病毒的易感细胞SSN-1中成功筛选出衣壳蛋白的互作因子,可为进一步探究神经坏死病毒在感染鱼类过程中的作用机制提供数据支撑。

     

    Abstract: Nervous necrosis virus (NNV) is a significant pathogen affecting a wide range of marine fish species. The capsid protein (CP) of NNV, as its major structural protein, not only participates in viral particle assembly but also plays a critical role in viral entry into host cells and the subsequent pathogenesis. However, the host protein factors that interact with CP remain largely uncharacterized. To investigate the mechanism of nervous necrosis virus (NNV) infection in fish and, identify CP-interacting factors, total RNA was extracted from SSN-1 cells to construct a yeast two-hybrid cDNA library. The NNV CP gene was PCR-amplified and cloned into the pBT3-STE bait vector. Using CP as bait, we screened the SSN-1 cDNA library via yeast two-hybrid assay. The library capacity reached 7.2×106 CFU with 100% recombination rate and average insert size >1000 bp. Initial screening yielded 24 positive clones. After sequencing, alignment, and pairwise validation, seven candidate interacting proteins were identified. Functional analysis indicated their roles in immune regulation, transcription, stress response, and signal transduction. This study successfully identified CP-interacting factors in NNV-susceptible SSN-1 cells, providing data to elucidate NNV infection mechanisms.

     

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