Khodabandehloo Mazaher, Shamsi Shahrabadi Mahmoud, Keyvani Hossein, Bambai Bijan
Dept. of Virology, Iran University of Medical Sciences, Rassul Akram Hospital, Tehran, Iran.
National Institute for Genetic Engineering and Biotechnology, Pazhoohesh Boulevard, Kilometrs17, Karaj Highway, P.O. Box 14455-6343, Tehran, Iran.
Iran Biomed J. 2009 Jan;13(1):9-18.
VP4 protein is as spikes on rotavirus outer capsid shell which is responsible for virus attachment to the host. VP4 induces production of neutralizing antibodies which could be used for serotyping of different isolates.
Simian rotavirus SA11 gene 4 cDNA was cloned into a cloning plasmid pDONRTM by recombination reaction using clonase II enzyme mix. The resulting clone was called VP4-entry clone. In the second recombination reaction, cloned gene was inserted into the linear DNA of the Baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) downstream of the strong polyhedrin promoter. The recombinant AcNPV-VP4 DNA was transfected by lipofection into the insect cell line, Spodoptera frugiperda (Sf9) cells. Expression of VP4 in the Sf9 cells was confirmed by the immunofluorescence test using rabbit polyclonal anti-rotavirus and anti-rabbit FTIC-conjugated antibodies by Western immuno-blotting technique. The antigenicity of the expressed protein was determined by immunizing rabbits and testing the sera by Western-blotting and neutralization method.
The cloned VP4 gene was obtained and expressed in baculovirus system. The specificity of the expressed protein was confirmed by its reactivity with anti-rotavirus antibody. Antibody produced against the expressed protein showed neutralizing activity for rotavirus indicating that the protein was biologically active and could induce natural antibody response.
The expressed protein from rotavirus VP4 gene has a potential for development of rotavirus vaccine.
VP4蛋白是轮状病毒外衣壳上的刺突，负责病毒与宿主的附着。VP4可诱导中和抗体的产生，这些抗体可用于不同分离株的血清分型。
使用II型酶混合物通过重组反应将猿猴轮状病毒SA11基因4 cDNA克隆到克隆质粒pDONR™ 中。所得克隆称为VP4进入克隆。在第二次重组反应中，将克隆的基因插入杆状病毒苜蓿银纹夜蛾核型多角体病毒（AcNPV）的线性DNA中，位于强多角体蛋白启动子的下游。通过脂质转染将重组AcNPV-VP4 DNA转染到昆虫细胞系草地贪夜蛾（Sf9）细胞中。使用兔多克隆抗轮状病毒抗体和抗兔FTIC偶联抗体，通过Western免疫印迹技术，通过免疫荧光试验确认Sf9细胞中VP4的表达。通过免疫兔子并通过Western印迹和中和方法检测血清来确定表达蛋白的抗原性。
获得了克隆的VP4基因并在杆状病毒系统中表达。表达蛋白与抗轮状病毒抗体的反应性证实了其特异性。针对表达蛋白产生的抗体对轮状病毒显示出中和活性，表明该蛋白具有生物活性并可诱导天然抗体反应。
轮状病毒VP4基因表达的蛋白具有开发轮状病毒疫苗的潜力。
