Kovács Beáta Marianna, Szilágyi László, Janan Janbaz, Rudas Péter
Department of Physiology and Biochemistry, Faculty of Veterinary Science, Szent István University, Budapest, Hungary.
Amyloid. 2005 Jun;12(2):109-14. doi: 10.1080/13506120500106941.
We defined the nucleotide-sequence of the full-length goose serum amyloid A and compared it to SAA sequences of the duck. The aim of this work was to clone and express recombinant goose SAA and to produce antibody against this protein: Total RNA was isolated from goose liver and used to synthesise first strand cDNA. The coding region of the goose SAA cDNA was amplified by PCR using primers corresponding to the appropriate conservative regions of duck SAA mRNA. The product was subcloned into pET-15b expression vector to result in a His*Tag fusion protein expression. The protein was purified by affinity chromatography. Rabbits were then immunized against the recombinant purified goose SAA protein. The anti-SAA serum was tested by Western blotting. Full-length goose SAA mRNA sequence has been obtained and sequenced.
我们确定了全长鹅血清淀粉样蛋白A的核苷酸序列,并将其与鸭的血清淀粉样蛋白A序列进行了比较。这项工作的目的是克隆和表达重组鹅血清淀粉样蛋白A,并制备针对该蛋白的抗体:从鹅肝脏中分离出总RNA,并用于合成第一链cDNA。使用与鸭血清淀粉样蛋白A mRNA的适当保守区域相对应的引物,通过PCR扩增鹅血清淀粉样蛋白A cDNA的编码区。将产物亚克隆到pET-15b表达载体中,以产生His*标签融合蛋白表达。通过亲和层析纯化该蛋白。然后用重组纯化的鹅血清淀粉样蛋白A蛋白免疫兔子。通过蛋白质印迹法检测抗血清淀粉样蛋白A血清。已获得并测序了全长鹅血清淀粉样蛋白A mRNA序列。