Expression of β-galactosidase by recombinant respiratory syncytial viruses for microneutralization assay
Expression of β-galactosidase by recombinant respiratory syncytial viruses for microneutralization assay
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DOI：
10.1016/s0166-0934(02)00122-2
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发表时间：
2002-09-01
影响因子：
3.1
通讯作者：
Jin, H
中科院分区：
文献类型：
--
作者：
Cheng, X;Munoz, MG;Jin, H
将β-半乳糖苷酶基因（lacZ）插入A亚群呼吸道合胞病毒（RSV）A2株（命名为A-lacZ）和嵌合RSV（命名为B-lacZ）中，所述嵌合RSV具有由B RSV 9320株的G和F表面糖蛋白替代的A2的G和F表面糖蛋白。两种重组RSV，A-lacZ和B-lacZ，在组织培养中生长良好，并表达高水平的β-半乳糖苷酶。使用这两种表达β-半乳糖苷酶的重组RSV，开发了一种新的微量中和试验来测量来自亚组A或亚组B RSV感染的血清抗RSV中和抗体。该试验在96孔板中进行，在将感染的细胞裂解物与酶底物氯酚红β-D-吡喃半乳糖苷（CPRG）孵育后，通过分光光度法测量β-半乳糖苷酶促反应定量未中和的病毒。通过与空斑减少中和试验相比的微量中和试验，检查了通过蛋白质印迹分析显示的抗RSV抗体阳性的成人血清和亚组A或亚组B RSV感染的猴血清的抗RSV中和抗体水平。当用同源RSV进行中和试验时检测到的抗体滴度高于用异源RSV进行的抗体滴度，表明中和试验可以区分两种RSV亚组之间的抗原差异。微量中和试验的灵敏度与空斑减少中和试验相当，但它快速、省力，适合筛选大量样品。(C)2002 Elsevier Science B. V.保留所有权利。
The P-galactosidase gene (lacZ) was inserted into a recombinant respiratory syncytial virus (RSV) A2 strain of subgroup A RSV (designated as A-lacZ) and a chimeric RSV that had the G and F surface glycoproteins of A2 replaced by those of the subgroup B RSV 9320 strain (designated as B-lacZ). Both recombinant RSVs, A-lacZ and B-lacZ, grew well in tissue culture and expressed high levels of beta-galactosidase. Using these two beta-galactosidase-expressing recombinant RSVs, a novel microneutralization assay was developed to measure serum anti-RSV neutralizing antibody from subgroup A or subgroup B RSV infection. The assay was carried out in 96-well plates and the unneutralized virus was quantitated by spectrophotometric measurement of the beta-galactosidase enzymatic reaction following incubation of the infected cell lysate with the enzyme substrate, chlorophenol red beta-D-galactopyranoside (CPRG). Adult human sera positive for anti-RSV antibody as shown by Western blot analysis and subgroup A or subgroup B RSV infected monkey sera were examined for the levels of anti-RSV neutralizing antibodies by the microneutralization assay in comparison with the plaque reduction neutralization assay. A higher antibody titer was detected when the neutralization assay was performed with the homologous RSV than the heterologous RSV, indicating that neutralization assay could distinguish antigenic differences between the two RSV subgroups. The microneutralization assay is comparable to the plaque reduction neutralization assay in sensitivity, but it is rapid, less laborious and suitable for screening a large number of samples. (C) 2002 Elsevier Science B.V. All rights reserved.