乌鳢水泡病毒L蛋白的原核表达及多克隆抗体制备
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国家自然科学基金面上项目(31972832)


Prokaryotic expression of L protein of snakehead vesiculovirus and preparation of its polyclonal antibody
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    摘要:

    为研究L蛋白在乌鳢水泡病毒(snakehead vesiculovirus,SHVV)增殖过程中发挥的作用,扩增SHVV L基因的前900个碱基,将其克隆到载体pET-32a(+)中,构建原核表达质粒pET32a-L,并转化至大肠杆菌BL21(DE3)感受态细胞。设置不同温度、IPTG浓度及诱导时间,选择最佳的表达条件。通过NI-NTA亲和层析柱纯化蛋白,并利用纯化的蛋白免疫新西兰大白兔,制备多克隆抗体。用Western blot鉴定抗体特异性,间接免疫荧光观察L蛋白在SHVV感染斑点叉尾鮰卵巢细胞(channel catfish ovary,CCO)中的定位情况。结果显示,纯化的L蛋白分子质量约42 ku,与预期大小相符。制备的L蛋白多克隆抗体可以与 L蛋白发生特异性免疫反应,且L蛋白主要定位在细胞质,表明L蛋白多克隆抗体制备成功。

    Abstract:

    To study the role of L protein in the snakehead vesiculovirus (SHVV) proliferation,the first 900 bases of the SHVV L gene was amplified and inserted into pET-32a (+) to construct pET32a-L prokaryotic expression plasmid,and transformed into E. coli BL21 (DE3) competent cells. Different temperature,IPTG concentration and induction time were designed and the best expression conditions were selected. The purified protein was purified by NI-NTA affinity chromatography column,and was used to immunize New Zealand white rabbits to prepare polyclonal antibodies. Western blot was used to identify the specificity of the antibody.Indirect immunofluorescence was used to study the location of L protein in channel catfish ovary (CCO) cells after SHVV infection. The results showed that the purified L protein was about 42 ku. The L protein polyclonal antibody can react specifically with the L protein,and the L protein is mainly localized in cytoplasm,indicating that the L protein polyclonal antibody has been successfully prepared.

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张艳维,张永安,涂加钢.乌鳢水泡病毒L蛋白的原核表达及多克隆抗体制备[J].华中农业大学学报,2021,40(4):177-181

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  • 收稿日期:2021-02-05
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  • 在线发布日期: 2021-07-22
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