番木瓜环斑病毒西瓜株系衣壳蛋白的原核表达及抗血清制备

Prokaryotic Expression and Antiserum Preparation of Papaya Ringspot Virus-Watermelon Strain Coat Protein

以感染番木瓜环斑病毒西瓜株系(papaya ringspot virus-watermelon strain,PRSV-W)的西葫芦叶片为供试材料,通过RT-PCR克隆PRSV-W衣壳蛋白(coat protein,CP)基因,并将其连接到原核表达载体pEHISTEV上得到重组质粒pEHISTEV-PRSV-W-CP。将其转化大肠杆菌Rosetta,经IPTG诱导后可表达分子量约为37 kD的融合蛋白。将融合蛋白从凝胶中切下,乳化后免疫新西兰长耳兔,制备PRSV-W CP的抗血清。间接ELISA测定该血清效价为1∶8192。Western blot检测结果表明,该抗血清只与感染PRSV的西葫芦样品有特异性反应,而与健康西葫芦、感染西瓜花叶病毒的西葫芦及感染马铃薯Y病毒的普通烟样品均无反应。本研究为PRSV的快速检测以及CP蛋白功能的研究奠定了基础。

The coat protein( CP) gene of papaya ringspot virus-watermelon strain( PRSV-W) was amplified by RT-PCR from infected zucchini leaf samples and cloned into the prokaryotic expression vector pEHISTEV to produce recombinant plasmid pEHISTEV-PRSV-W-CP. The recombinant plasmid was transformed into Escherichia coli strain Rosetta and expressed a fusion protein with molecular weight of 37 kD after induction with IPTG. The fusion protein was cut from the gel,emulsified and used to immunize the New Zealand rabbits to produce polyclonal antiserum against the CP of PRSV-W. The titer of the resultant antiserum was 1∶ 8192 in ELISA. In Western blot analysis,the antiserum showed specific positive reaction only with the zucchini plants infected with PRSV,but not with healthy zucchini plants or those infected with Watermelon mosaic virus,or Nicotiana tabacum plants infected with Potato virus Y. This research laid foundations for the detection of PRSV and functional studies of PRSV CP.