Development of an Antibody‐Based Assay for Determination of Baculovirus Titers in 10 Hours
Development of an Antibody‐Based Assay for Determination of Baculovirus Titers in 10 Hours
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开发基于抗体的 10 小时内测定杆状病毒滴度的方法
DOI：
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发表时间：
2002
期刊：
影响因子：
--
通讯作者：
Enoch Y. Park
中科院分区：
文献类型：
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作者：
M. Kwon;T. Dojima;Masaru Toriyama;Enoch Y. Park
杆状病毒表达系统已被用于通过表达目的蛋白的重组杆状病毒感染昆虫细胞来产生大量的生物活性蛋白。为了有效地表达目标蛋白，有必要用足够量的病毒感染昆虫细胞。然而，目前的方法费时费力，要么存在技术困难，要么由于使用报告基因的病毒表达机制而受到限制。开发了一种新的方法，可在10h内获得病毒滴度，该方法易于使用96孔板进行操作，并且适用于任何基于家蚕核型多角体病毒(BmNPV)的重组杆状病毒(BmNPV)和任何一种银纹夜蛾核型多角体病毒(AcNPV)。这项检测使用DNA结合蛋白的抗体通过免疫染色来检测受感染的细胞。滴度是通过在荧光显微镜下对病毒感染产生的病灶进行计数来确定的。由于感染细胞在感染后4h就表达了病毒抗原，因此潜伏期大大缩短，因此10h就足以估计病毒滴度，包括病毒感染时间、昆虫细胞培养和病毒滴度的估计。用这种免疫测定法测定的效价在价值和有效性上都与用传统方法测定的效价相当，前提是原料的效价高于103pfu/mL。
The baculovirus expression system has been used to produce large amounts of biologically active proteins by infecting insect cells with a recombinant baculovirus expressing the target protein. For an efficient expression of the target protein, it is necessary to infect insect cells with an adequate amount of virus. However, current methods are time‐consuming and either have technical difficulties or are limited as a result of virus expression mechanism using a reporter gene. A novel method is developed to yield virus titers in 10 h that is easy to perform using 96‐well plates and applicable to both any Autographa californica nucleopolyhyderovirus (AcNPV) and Bombyx mori nucleopolyhedrovirus (BmNPV)‐based recombinant baculovirus. This assay uses an antibody to a DNA‐binding protein to detect the infected cells via immunostaining. The titer is determined by counting foci produced as a result of infection of the virus under a fluorescent microscope. The required incubation period was shortened considerably because infected cells expressed viral antigens at the post‐infection time of 4 h. Therefore, 10 h was enough to estimate the virus titer including virus infection time, insect cell culture, and estimation of virus titer. Titers determined using this immunological assay are comparable, both in value and validity, to those obtained using a traditional method, provided that the stocks have titers above 103 pfu/mL.
DOI：
10.1073/pnas.91.23.11212
发表时间：
1994-11-08
影响因子：
11.1
作者：
KOOL, M;AHRENS, CH;VLAK, JM
通讯作者：
VLAK, JM
用于估计表达 β-半乳糖苷酶的杆状病毒滴度的 24 小时测定。
DOI：
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发表时间：
1995
期刊：
BioTechniques.
影响因子：
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作者：
Sussman,DJ
通讯作者：
Sussman,DJ