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【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 investigate the immunogenicity of recombinant protein coexpressed with P30 and gp90 in retina of Reticuloendotheliosis Virus (REV) group, and provide the basis for developing a novel REV antibody diagnostic kit. 【Method】 Two pairs of primers were designed and synthesized based on the proviral genomic cDNA sequence of spleen necrosis virus (SNV). The pPB101 plasmid was used as template to amplify the fragments of REV p30 gene and gp90 gene, respectively. The PCR products were sequentially cloned into the expression vector pET-28a (+). The recombinant plasmid pET-p30-gp90 was screened by restriction enzyme analysis and sequencing analysis. The recombinant strain was induced by isopropylthiogalactopyranoside (IPTG) and analyzed by SDS-PAGE. Western blot was used to detect the reactivity between the expressed protein and the specific serum. The antisera of the expressed protein was prepared, and the immunofluorescence of the expressed protein was verified by indirect immunofluorescence assay (IFA) using the antiserum with REV-infected chick embryo fibroblasts (CEFs). 【Results】 The expected size of the band was observed after SDS-PAGE electrophoresis. Western blot results showed that the recombinant protein reacted with REV antiserum. The expressed product was purified and immunized Balb / c mice to prepare p30-gp90 antiserum, the antiserum and REV infected CEF showed specific fluorescence reaction in IFA. 【Conclusion】 The recombinant protein of REV p30-gp90 was expressed in tandem in vitro and the expressed protein was of good immunogenicity.