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

发布时间：2017-12-28 11:18

本文关键词：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　出处：《微生物学报》2014年03期 　论文类型：期刊论文

【摘要】：【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。
[Abstract]:[Objective] to study the Avian Reticuloendotheliosis Virus (Reticuloendotheliosis, Virus, REV) immunogenic protein co expression of group specific antigen P30 and glycoprotein gp90 in vitro, provide the basis for the development of new diagnostic kit for REV antibody. [Methods] 2 pairs of primers were designed and synthesized according to the cDNA sequence of REV spleen necrosis virus (SNV) strain. PPB101 REV was used as template to amplify REV P30 gene and gp90 gene fragment respectively. The PCR products were cloned into the expression vector pET-28a (+) in turn, and the positive recombinant clone pET-p30-gp90 was screened by enzyme digestion and sequencing. The recombinant bacteria were induced by isopropyl thiosulfate D- galactoside (IPTG). The expression was analyzed by SDS-PAGE electrophoresis, and Western blot was used to detect the reactivity between the expressed protein and the specific serum. The antiserum of the expressed protein was prepared, and the immunogenicity of the expressed protein was verified by indirect immunofluorescence assay (IFA) with REV infected chicken embryo fibroblast (CEF). [results] the expected size of the bands could be observed after SDS-PAGE electrophoresis, and the results of Western blot showed that the recombinant protein could react with the antiserum of REV. The expression products were purified and immunized with Balb/c mice to prepare p30-gp90 antiserum. The antiserum and REV infected CEF showed a specific fluorescence reaction in IFA. [Conclusion] REV p30-gp90 protein is expressed in series in series, and the expression protein has good immunogenicity.
【作者单位】：华南农业大学兽医学院;广东大华农动物保健品股份有限公司;中国农业科学院生物技术研究所;
【基金】：重大动物疫病新型疫苗的关键技术研究与产业化(2011A090200117)~~
【分类号】：R373
【正文快照】： 禽网状内皮组织增生症(Reticuloendotheliosis,RE)是由逆转录病毒科的禽网状内皮组织增生症病毒(REV)引起的鸡、鸭、火鸡和其他禽类的一种以淋巴-网状细胞增生为特征的肿瘤性病理综合征[1-2]。REV已经呈世界性分布,该病在澳大利亚、匈牙利、英国、德国等国均有报道,我国于1988

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