鲤春病毒血症病毒糖蛋白的截短表达及其免疫原性分析

摘要

To reveal the main immunogenic domain of the glycoprotein of spring viremia of carp virus (SVCV), the glycoprotein encoding gene was truncated and expressed, and the immunogenicity of the truncated glycoprotein was analyzed using a rabbit polyclonal antibody produced in response to the recombination protein. The transmembrane, antigenic and hydrophilic domains of SVCV were analyzed by SOSUI and DNAStar 6.0 software. The predicted main immunogenic domain was amplified by reverse transcription polymerase chain reaction and cloned into a prokaryotic expression plasmid. The truncated glycoprotein was expressed in BL21 cells. After purification and refolding, the pro-tein was used to produce antiserum in rabbits. The antiserum titer was tested by an enzyme-linked immunosorbent assay and the immunogenicity of the protein was tested by immunoblotting and indirect immunofluorescence assay (IFA). The results showed that the truncated glycoprotein gene was 1339 bp encoding a 439 aa peptide (from 29 to 467aa of the full-length glycoprotein) with a predicted molecular weight of 49.6 kD. The antiserum titer against the recombinant glycoprotein was 1 : 64000. Immunoblotting and IFA results demonstrated that the antiserum reacted immunologically with the natural glycoprotein on the surface of SVCV-HN. There was no difference in immunogenicity between the truncated glycoprotein produced in this study and the natural glycoprotein of SVCV. Therefore, the truncated glycopro-tein of SVCV had good immunogenicity and could be used in immunological diagnosis and genetic engineering vaccine development of SVCV.%为了揭示鲤春病毒血症病毒(spring viremia of carp virus, SVCV)糖蛋白编码基因的主要免疫原性决定区域,本研究对 SVCV 的糖蛋白基因进行截短表达,并用纯化的重组蛋白作为免疫原制备了兔抗血清以分析其免疫原性。通过SOSUI以及DNAStar 6.0软件对SVCV糖蛋白基因的跨膜区、亲水性以及抗原表位进行分析后,采用RT-PCR 对该基因主要抗原决定区域编码片段进行扩增,并构建重组表达质粒 pGEX-Gtr,对其进行诱导表达后获得截短的 SVCV 糖蛋白的重组蛋白。将表达的重组蛋白进行纯化复性后,作为免疫原制备兔抗血清,采用 ELISA法检测其效价,采用免疫印迹以及间接免疫荧光技术检测该重组蛋白的免疫原性。研究结果显示,截短表达的糖蛋白编码基因长1317 bp,编码439个氨基酸(29~467),推测分子量为49.6 kD。利用该重组蛋白制备的兔抗血清,其与重组蛋白的反应效价为1∶64000,。免疫印迹及间接免疫荧光结果显示,该兔抗血清与SVCV-HN株能发生特异性反应,表明该重组蛋白与天然的病毒表面糖蛋白的免疫原性无差异。上述研究结果表明,截短表达的重组蛋白具有很好的免疫原性,可应用于免疫诊断技术研发与基因工程疫苗的研制。