利用本研究小组克隆的猪IgGⅡ类Fc受体cDNA(swFcγRⅡ)基因序列(DQ026064)设计引物,应用RT-PCR技术,从猪外周血白细胞cDNA中扩增了完整的960 bp swFcγRⅡ ORF序列,利用基因重组技术将swFcγRⅡ ORF基因亚克隆到真核表达载体pcDNA3,经PCR及双酶切鉴定:扩增出901bp的PCR产物;Kpn I/Eco R I双酶切,切出5376和901bpDNA片段,表明重组质粒pcDNA/swFcγRⅡ构建成功。然后脂质体法转染COS-7细胞,用玫瑰花环试验鉴定了swFcγRⅡ配体亲和特性。
The Fc receptor cDNA sequence of porcine IgG Ⅱ cloned by us was used to design primers. Using RT- PCR technology, a 960 bp of swFcγRⅡ sequence covering the whole coding region was amplified from the peripheral blood leucocyte cDNA, and the was sub-cloned into the expression vector pcDNA3. Through double digestion by Kpn I/EcoR I and PCR identification, a 5 375 bp and a 901 bp of DNA fragments were obtained, indicating that the recombinant plasmid pcDNA/swFcγRⅡ was successfully constructed. The 901 bp fragment was then transfected into COS-7 cells by the lipofection method, and the ligand affinity of swFcγRⅡ was identified by rosetting procedures.