传染性造血器官坏死病病毒CJ-13株糖蛋白的原核表达及免疫原性

Prokaryotic expression and immunogenicity of glycoprotein gene of infectious hematopoietic necrosis virus(IHNV)CJ-13 strain

【目的】克隆传染性造血器官坏死病病毒(IHNV)CJ-13株糖蛋白(G蛋白)基因,构建原核表达载体,并检测其在大肠杆菌BL21中的表达情况,为IHNV诊断试剂盒和疫苗的研制奠定基础。【方法】设计1对G基因特异引物对G基因进行RT-PCR扩增,将扩增产物克隆到pGEM-T Easy载体上,构建重组质粒pGEM-T Easy-G,经双酶切鉴定、核苷酸序列分析后,将G基因亚克隆到pET-32a(+)原核表达载体上,构建传染性造血器官坏死病病毒G基因原核表达质粒pET-32a-G,转化至宿主菌BL21中,诱导表达目的蛋白,采用SDS-PAGE、Western blot和ELISA等方法对表达产物进行分析。【结果】成功克隆了IHNV G蛋白基因,该基因长度为1 527bp。成功构建了原核表达质粒pET-32a-G,诱导表达出约76ku的产物,与预期分子质量大小相符。Western blot分析结果显示,所表达的G蛋白能够被小鼠抗IHNV血清识别。间接ELISA结果显示,小鼠抗G蛋白血清能够识别IHNV全病毒。【结论】成功构建了IHNV G蛋白原核表达系统,重组G蛋白具有良好的反应原性和免疫原性。

[Objective] The study cloned IHNV CJ-13 strain glycoprotein gene and constructed prokaryotic expression vector for observation of expression in host Escherichia coli strain BL21 to provide basis for further research and development of IHNV diagnostic kit and DNA vaccine. [Method] The surface glycoprotein of CJ-13 isolated from rainbow trout in Changchun was amplified and cloned into pGEM-T Easy vector to construct recombinants pGEM-T Easy-G and sub cloned into prokaryotic expressing vector pET-32a（＋）. The pET-32a-G was transformed into the E. coli strain BL21 and the expression of G protein was induced. G protein was analyzed by SDS-PAGE, Western blot and ELISA. [Result] G gene with the length of 1 527 bp was cloned and the expressed product was about 76 ku. The G protein was identified specifically with the mouse anti-IHNV serum by Western blot analysis. ELISA results showed that IHNV could react specifically with the serum of G protein. [Conclusion] The prokaryotic expression of IHNV G protein was constructed successfully and the expressed G protein had immunogenic and antigenic characters as native IHNV G protein.