1st affiliated hospital endocrinology, Liaoning Medical University, Jinzhou, 121000 Liaoning, China.
Liaoning Medical University, Jinzhou, 121200 Liaoning, China.
J Virol Methods. 2014 Dec;209:82-5. doi: 10.1016/j.jviromet.2014.09.007. Epub 2014 Sep 17.
Human astrovirus (HAstV) are important pathogens that cause acute viral diarrhea in infants. Little is known about the mechanisms of astrovirus-induced diarrhea. Previous studies have suggested that an apoptosis inducer may be encoded in the non-structural protein (nsP1a) of astrovirus and contribute to virus-induced diarrhea. To study the biological function of nsP1a and to gain further insight into nsP1a protein-host cell interactions, good quality antibodies must be produced. The nsP1agene of HAstV-1 was cloned into a bacterial expression vector Pgex-6P-1. The recombinant plasmid Pgex-6P-nsP1a was transformed into Escherichia coli BL21 (DE3) and expressed as a fusion protein that contains N-terminal GST tags. The expressed recombinant protein was purified and used as an antigen to produce an nsP1a antiserum in rabbits. ELISA was used to detect the titer of specific antibodies. Specificity activity was detected by Western blot and immunofluorescence analysis. The titer of specific antibodies was up to 1:30,000. Western blotting and immunofluorescence analysis indicated that the polyclonal antibody could recognize specifically the HAstV-1 nsP1a protein.
人星状病毒（HAstV）是引起婴幼儿急性病毒性腹泻的重要病原体。目前对于星状病毒诱导腹泻的机制知之甚少。先前的研究表明，凋亡诱导剂可能编码在星状病毒的非结构蛋白（nsP1a）中，并有助于病毒诱导的腹泻。为了研究 nsP1a 的生物学功能，并进一步了解 nsP1a 蛋白与宿主细胞的相互作用，必须产生高质量的抗体。将 HAstV-1 的 nsP1a 基因克隆到细菌表达载体 Pgex-6P-1 中。将重组质粒 Pgex-6P-nsP1a 转化入大肠杆菌 BL21（DE3）中，并表达为含有 N 端 GST 标签的融合蛋白。表达的重组蛋白被纯化并用作抗原，在兔子中产生 nsP1a 抗血清。ELISA 用于检测特异性抗体的效价。通过 Western blot 和免疫荧光分析检测特异性活性。特异性抗体的效价高达 1:30000。Western blot 和免疫荧光分析表明，该多克隆抗体能够特异性识别 HAstV-1 的 nsP1a 蛋白。
