Nogueira-Dantas Eliana Ottati, Ferreira Antonio J Piantino, Astolfi-Ferreira Claudete Serrano, Brentano Liana
Department of Pathology, College of Veterinary Medicine, University of São Paulo, Av. Prof. Dr. Orlando Marques de Paiva 87, São Paulo, SP, 05508-000, Brazil.
Comp Immunol Microbiol Infect Dis. 2007 May;30(3):133-42. doi: 10.1016/j.cimid.2006.11.003. Epub 2007 Jan 26.
Purification of chicken anemia virus (CAV) VP3 protein, expressed in a prokaryotic expression system as histidine-tagged fusion protein is demonstrated in the present study. CAV particle was obtained from infected liver of chicken and DNA was extracted. The VP3 protein gene was amplified from the extracted DNA by polymerase chain reaction (PCR) and cloned. The recombinant expression construct (pTrc-VP3) was identified by PCR and sequencing analysis. Expression of VP3 protein with a molecular mass of approximately 21kDa was confirmed by Western blotting analysis with CAV-specific antibodies. The in vitro expressed VP3 protein was purified to near homogeneity by elution from the gel, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The purified VP3 protein was recognized by CAV antibodies in a Western blotting assay. This finding indicates that recombinant VP3 expressed in the pTrcHis2 vector system can be used as antigen to detect anti-CAV antibodies.
本研究展示了在原核表达系统中作为组氨酸标签融合蛋白表达的鸡贫血病毒（CAV）VP3蛋白的纯化过程。从感染的鸡肝脏中获得CAV颗粒并提取DNA。通过聚合酶链反应（PCR）从提取的DNA中扩增VP3蛋白基因并进行克隆。通过PCR和测序分析鉴定重组表达构建体（pTrc-VP3）。用CAV特异性抗体进行的蛋白质免疫印迹分析证实了分子量约为21kDa的VP3蛋白的表达。根据十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析判断，体外表达的VP3蛋白通过从凝胶上洗脱而纯化至接近均一。在蛋白质免疫印迹试验中，纯化的VP3蛋白可被CAV抗体识别。这一发现表明，在pTrcHis2载体系统中表达的重组VP3可作为抗原用于检测抗CAV抗体。
