论文部分内容阅读
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(P<0.01)。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。
Objective To screen Eg-00512 antigen of Echinococcus granulosus of Echinococcus granulosus and identify, purify and characterize its immunogenicity in order to make it a specific antigen for diagnosis of hydatid disease. Methods The expression profiles of Echinococcus granulosus in four different developmental stages published by the National Human Genome Southern Research Center (CHGCS) were compared and analyzed. The Eg-00512 gene, which was specifically expressed only at the stage of the progenitor cells, was screened out. The DNAStar software package The protein fragment of Eg-00512 was analyzed by antigenic epitope analysis. The suitable fragment was selected as the target fragment to construct recombinant plasmid Eg-00512 / pET-24a / BL-21. The recombinant protein rEg-00512 was obtained by inducible expression. The BALB / c mice were randomly divided into immune group, adjuvant control group and PBS control group. The BALB / c mice were injected subcutaneously with recombinant protein, PBS + Freund’s adjuvant and PBS respectively. Blood was collected from tail vein of mice in each group before immunization and at 2 and 4 weeks after the first immunization. Blood samples were collected by eyeball method five weeks after immunization. The serum levels of specific IgG antibodies in each group were determined by ELISA. Primary antibody, Western blot analysis of recombinant protein immunoreactivity. Results The Eg-00512 gene was highly expressed only in the stage of metaplasia of Echinococcus granulosus. The gene encodes a protein of 203 amino acids with a molecular mass of 23 ku and its antigenic epitope is mainly located in and around the amino acid residues 1-15, 55-93, 111-119, 126-149, 166-194 and 199-202. The genetically engineered strain Eg-00512 / pET-24a / BL-21 was successfully constructed and the recombinant protein rEg-00512 was induced by IPTG. The purified recombinant protein rEg-00512 immunized mice induced the production of specific IgG antibodies, wherein the highest level of antibody 5 weeks after immunization A450 value of 1.807 ± 0.767, compared with the adjuvant control group 0.111 ± 0.014 and PBS control group 0.132 ± 0.0246 difference There was statistical significance (P <0.01). Western blot showed that the recombinant antigen was immunized with mouse antisera and Echinococcus granulosus infection in serum of mice, but not with normal mouse serum. Conclusion The recombinant protein rEg-00512 was successfully screened and obtained, which has good antigenicity and can be used as a candidate antigen for immune diagnosis of hydatid disease.