表达可溶性猪唾液酸黏附素重组腺病毒的构建和鉴定

Construction and identification of recombinant adenovirus expressing the soluble form of porcine sialoadhesin

猪唾液酸黏附素(pSn)是猪繁殖与呼吸综合征病毒(PRRSV)感染宿主的重要受体。采用RT-PCR和基因合成方法构建能够表达猪Sn蛋白前4个Ig样结构域(Sn4D)和IgG1Fc片段融合蛋白的穿梭载体pShuttle-Sn4D-Fc,利用AdEasyTMAdenoviral Vector System制备重组腺病毒。透射电子显微镜观察表明,构建重组病毒rAd-Fc和rAd-Sn4DFc具有典型腺病毒形态;在重组腺病毒感染的细胞中,RT-PCR能检测到预期的Fc和Sn4D-Fc转录子;在重组腺病毒转导细胞裂解物及培养上清中,Western-blot能检测到预期的28ku Fc或72ku Sn4D-Fc蛋白;从重组病毒转导细胞培养上清中纯化的Fc和Sn4D-Fc蛋白为单一的条带,能被抗猪IgG或Sn抗体识别。结果表明:成功构建了分泌表达猪Fc片段和Sn4D-Fc融合蛋白的重组腺病毒,为用Sn可溶性受体阻断抗PRRSV感染研究打下基础。

Sialoadhesin is the main receptor that mediates the PRRSV entry. In this study, the full lenth gene of pig IgG1 Fc portion and the first four IgV domains （Sn4D） of porcine Sn gene were fused and cloned into adenoviral vector pShuttle-CMV, designated pShuttle-Sn4D-Fc. The two recombinant adenoviruses were generated using AdEasyTM Adeno- viral Vector System. The recombinant virus had a typical morphology of adenoviruses. Due to the presence of the Fc or Sn4D expression cassette in the viral genome was confirmed, and an expected 28 ku Fc protein or 72 ku Sn4D-Fc fusion protein was detected in the lysates of cell culture and supermatabt. Furthermore, the purified Fc and Sn4D-Fc protein were recognized by anti-pig IgG and/or anti-Sn serum. The recombinant adenovirus secretively expressing porcine Sn4D- Fc protein was successfully constructed, which paved a way to explore the feasibility of blocking PRRSV infection in vivo using the soluble Sn.