包虫病诊断抗原分子Eg-00512的筛

发布时间：2018-01-02 22:13

本文关键词：包虫病诊断抗原分子Eg-00512的筛选、重组表达及抗原性鉴定　出处：《中国病原生物学杂志》2017年01期 　论文类型：期刊论文

【摘要】：目的筛选细粒棘球绦虫原头蚴抗原分子Eg-00512,并进行重组表达、纯化及免疫特性鉴定,以期作为棘球蚴病特异性诊断抗原。方法对国家人类基因组南方研究中心(CHGCS)发表的细粒棘球绦虫4个不同发育阶段的表达谱数据进行差异比较分析,筛选仅在原头蚴阶段特异表达的基因Eg-00512;利用DNAStar软件包对其编码蛋白Eg-00512进行抗原表位分析;选取合适的序列片段作为目的片段,构建基因工程菌株Eg-00512/pET-24a/BL-21,经诱导表达、纯化获得重组蛋白rEg-00512。将BALB/c小鼠随机分为免疫组、佐剂对照组、PBS对照组,分别经皮下多点注射重组蛋白、PBS+弗氏佐剂和PBS,2周后加强免疫一次。分别于免疫前,第一次免疫后2、4周每组小鼠尾静脉采血,免疫后5周摘眼球法采血,采用ELISA检测各时间点血清中特异性IgG抗体水平变化;以该血清为一抗,采用Western blot分析重组蛋白的免疫反应性。结果筛选出仅在细粒棘球蚴原头蚴阶段高表达的Eg-00512基因。该基因编码203个氨基酸,分子质量单位为23ku,其抗原表位主要位于1-15、55-93、111-119、126-149、166-194和199-202氨基酸残基及其附近。成功构建基因工程菌株Eg-00512/pET-24a/BL-21,经IPTG诱导表达重组蛋白rEg-00512。重组蛋白rEg-00512纯化后免疫小鼠诱导产生特异性IgG抗体,其中免疫5周时抗体达最高水平A450值为1.807±0.767,与佐剂对照组0.111±0.014和PBS对照组0.132±0.0246比较差异有统计学意义(P0.01)。Western blot显示,该重组抗原可被免疫鼠抗血清及细粒棘球蚴原头蚴感染鼠血清识别,而不与正常小鼠血清反应。结论成功筛选并获得重组蛋白rEg-00512,该蛋白具有较好的抗原性,可作为棘球蚴病免疫诊断候选抗原。
[Abstract]:The purpose of screening of Echinococcus granulosus antigen Eg-00512 and recombinant expression, purification and immunological characterization, in order for hydatid disease diagnostic antigen. Methods the National Human Genome Research Center (CHGCS) released from Echinococcus granulosus 4 expression with developmental stages of the spectral data are compared analysis of Eg-00512 gene screening was expressed only in protoscolex stage specific; package for the encoding protein Eg-00512 antigen epitope analysis by using DNAStar software; selecting appropriate sequence fragments as target fragment, gene engineering strain Eg-00512/pET-24a/BL-21 induced by expression of recombinant protein rEg-00512. BALB/c mice were randomly divided into group of immune adjuvant purification. The control group, PBS control group, were treated with subcutaneous injection of recombinant protein PBS+, Freund's adjuvant and PBS, 2 weeks after the booster immunization respectively before immunization. After the first immunization, 2,4 weeks of mice tail vein blood, blood eye method pick 5 weeks after immunization, the levels of specific IgG antibody in the serum of each time point ELISA detection; the anti serum, immune response by Western blot analysis of the recombinant protein. Results showed that Eg-00512 gene was highly expressed in protoscolex stage. The gene encoding 203 amino acids, the molecular weight of 23ku, the epitope is located in 1-15,55-93111-119126-149166-194 and 199-202 amino acid residues and its vicinity. The successful construction of the genetic engineering strain Eg-00512/pET-24a/BL-21, the expression of recombinant protein rEg-00512. recombinant protein after purification of rEg-00512 mice induced by specific IgG antibody induced by IPTG. The immune antibody reached the highest level at 5 weeks, the value of A450 was 1.807 + 0.767, 0.111 + 0.014 and adjuvant control group and PBS control group 0.132 + 0. There are significant differences between the 0246 (P0.01).Western blot showed that the recombinant antigen can be immune mouse antiserum and protoscolex infected mice sera, but not with the reaction of normal mouse serum. Conclusion successfully screened and recombinant protein rEg-00512, the protein has good antigenicity, can be used as a candidate for immunological diagnosis of hydatid disease antigen.

【作者单位】：宁夏医科大学临床医学院检验系;宁夏医科大学包虫病实验室;宁夏医科大学科技中心;宁夏医科大学基础医学院;宁夏医科大学基础医学院病原与免疫教研室;
【基金】：国家自然科学基金项目(No.81360249)
【分类号】：R532.32;R446.6
【正文快照】： ***taneously injected with PBS.The adjuvant control group and the PBS control group were given a booster after 2weeks.Blood was obtained via the caudal vein before immunization and 2and 4weeks after the first immunization.Blood was alsoobtained through t

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