禽网状内皮组织增生症病毒p30-gp90串联表达蛋白及其免疫原性

Immunogenicity of co-expressed p30-gp90 against Reticuloendotheliosis virus

【目的】研究禽网状内皮组织增生症病毒(Reticuloendotheliosis Virus,REV)群特异性抗原P30与囊膜糖蛋白gp90体外共表达蛋白的免疫原性,为研发新型REV抗体诊断试剂盒提供基础。【方法】根据REV脾脏坏死病毒(spleen necrosis virus,SNV)株的前病毒基因组cDNA序列,设计合成2对引物,以pPB101质粒为模板,分别扩增REV p30基因和gp90基因片段。将PCR产物依次克隆入表达载体pET-28a(+)中,通过酶切鉴定和测序分析,筛选阳性重组克隆pET-p30-gp90。重组菌经异丙基硫代D-半乳糖苷(IPTG)诱导后,通过SDS-PAGE电泳分析表达情况,Western blot检测表达蛋白与特异性血清之间的反应性。制备表达蛋白的抗血清,以该抗血清与REV感染的鸡胚成纤维细胞(CEF)进行间接免疫荧光实验(IFA),验证表达蛋白的免疫原性。【结果】经SDS-PAGE电泳后能观察到预期大小的表达条带,Western blot结果显示,重组蛋白能与REV抗血清反应。将表达产物纯化后免疫Balb/c小鼠,制备p30-gp90抗血清,该抗血清与REV感染CEF在IFA中呈现特异性荧光反应。【结论】体外串联表达REV p30-gp90蛋白,表达蛋白具有良好的免疫原性。

［Objective］ To study the immunogenicity of co-expression of p30，one of a group specific antigens，and glycoprotein gp90 genes of Reticuloendotheliosis virus （REV）．［Methods］p30 and gp90 genes were amplified with the template of plasmid pPB101 containing the whole sequence of spleen necrosis virus（SNV），and cloned into pET-28a（＋） vector．The positive clone pET-p30-gp90 was identified by enzyme analysis and sequencing．The co-expressed protein p30-gp90 was confirmed by SDS-PAGE and Western blot analysis． After quantitation，the purified protein p30-gp90 was immunized to Balb/c mice three times to prepare the p30-gp90 specific anti-serum．Afterwards，we detected the REV infected Chick Embryo Fibroblasts （CEF） by Immunofluorescence assay （IFA） with the prepared anti-serum．［Results］The p30-gp90 was co-expressed efficiently by SDS-PAGE and Western blot analysis．The anti-serum of p30-gp90 reacts with REV infected CEF in IFA．［Conclusion］The expressed protein of p30-gp90 keeps good immunogenicity．