Simple and rapid plaque assay for recombinant baculoviruses expressing influenza hemagglutinin.
Simple and rapid plaque assay for recombinant baculoviruses expressing influenza hemagglutinin.
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DOI：
10.1177/00368504211004261
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发表时间：
2021-01
期刊：
影响因子：
2.1
通讯作者：
Quan, Fu-Shi
中科院分区：
文献类型：
--
作者：
Basak, Swarnendu;Kang, Hae-Ji;Chu, Ki-Back;Oh, Judy;Quan, Fu-Shi
重组杆状病毒已被广泛用于产生病毒样颗粒，表达抗原蛋白的杆状病毒已成为诱导保护性免疫的有效工具。然而，目前产生杆状病毒的方法成本高，效率低。因此，发展一种简单、快速、准确的杆状病毒滴定方法至关重要。我们建立了一种利用免疫染色方法进行空斑分析的方法，通过该方法可以在光学显微镜下轻松地显示Sf9细胞中的空斑。用表达H1N1(A/California/04/09)或rH5N1(A/越南/1203/04)流感血凝素表面蛋白的重组杆状病毒感染Sf9细胞。感染细胞与抗HA抗体孵育，3‘，3-二氨基联胺(DAB)显色显示斑块。感染后第1天至第6天观察到斑块，Sf9细胞种植密度的不同导致最终斑块数量的不同。接种浓度为5.5 × 10 4个/孔或7.5 × 10 4个/孔的SF9细胞感染后第3、4、5天的空斑滴度均高于感染后1、2、6天。在5.5 × 10 4个/孔或7.5 × 10 4个/孔的细胞浓度下，重组杆状病毒对RBVHA(H1N1)的滴度为6 × 10 7 pfu/ml，对RBVHA(RH5N1)的重组杆状病毒滴度为5.4 × 10 7 pfu/ml。需要将杆状病毒与特定浓度的Sf9细胞孵育三天，才能进行快速、简单和准确的空斑分析，这可能对所有杆状病毒相关研究有重大贡献。
Recombinant baculoviruses (rBVs) have been extensively used to generate virus-like particles, and baculoviruses expressing antigenic proteins have become efficient tools for inducing protective immunity. However, current methods for generating baculoviruses are costly and inefficient. Thus, the development of a simple, rapid, and accurate method of baculovirus titration is critically important. We established a method of plaque assay using an immunostaining method by which plaques can be easily visualized in Sf9 cells under a light microscope. Sf9 cells were infected with recombinant baculoviruses expressing influenza hemagglutinin surface proteins from H1N1 (A/California/04/09) or rH5N1 (A/Vietnam/1203/04). The infected cells were incubated with anti-HA antibody and the plaques were visualized using the chromogen 3′3-diaminobenzidine (DAB). Plaques were observed from days 1 to 6 post-infection, and differences in Sf9 cell seeding densities resulted in variations in the final plaque quantification. Sf9 cells seeded at a concentration of 5.5 × 104 cells/well or 7.5 × 104 cells/well showed the higher plaque titers at days 3, 4, and 5 post-infection than those found at days 1, 2, and 6 post-infection. With 5.5 × 104 cells/well or 7.5 × 104 cells/well of cell concentrations, recombinant baculovirus for rBV-HA (H1N1) showed 6 × 107 pfu/ml of titer and rBVs for rBV-HA (rH5N1) showed 5.4 × 107 pfu/ml of titer. Three days of baculovirus incubation with a certain concentration of Sf9 cells seeded are required for a rapid, simple, and accurate plaque assay, which could significantly contribute to all baculovirus-related studies.
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影响因子：
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