包虫病诊断抗原分子Eg-00512的筛选、重组表达及抗原性鉴定

Expression,purification,and analysis of the immunogenicity of Eg-00512 and screening for that antigen to detect echinococcosis

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 for the protoscolex-specific molecule Eg-00512 of Echinococcus granulosus and to express,purify,and identify the immunogenicity of that molecule in order for it to serve as a specific diagnostic antigen.Methods The expression profiles of E.granulosus in four different stages of development were obtained from the China National Center for Human Genome Research（CHGCS）,and those profiles were analyzed and compared.Eg-00512 is highly expressed only in the protoscolex phase,so the gene was screened for.Eg-00512 encodes proteins,and the software package DNAStar was used to analyze epitopes in Eg-00512.An appropriate fragment of Eg-00512 was chosen to serve as a target fragment.The Eg-00512 gene in a pET-24 avector was used to genetically engineer a BL21 strain of E.coli.That strain（Eg-00512/pET-24a/BL-21）was used to express the recombinant protein rEg-00512,and that protein was purified.BALB/c mice were randomly divided into an immunization group,an adjuvant control group,and a PBS control group.The immunization group was subcutaneously injected with the recombinant protein at multiple points,the adjuvant control group was subcutaneously injected with PBS＋ Freund＇s adjuvant,and the PBS control group was subcu-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 also obtained through the eyeball 5weeks after immunization.Changes in the serum levels of specific IgG antibodies at different time points were identified using ELISA.The immunogenicity of the recombinant protein was analyzed using Western blotting,with mouse serum as the primary antibody. Results The protoscolex-specific molecule Eg-00512 was screened for.The molecule encodes 203 amino acids,it has a molecular mass of 23 ku,and is rich in epitopes,which are mainly located at amino acids 1-15,55-93,111-119,126-149,166-194,and 199-202 or in the vicinity.The genetically engineered strain Eg-00512/pET-24a/BL-21 was successfully constructed.Expression of the protein Eg-00512 was induced and the protein was purified to obtain the recombinant protein rEg-00512.Mice in the immunization group produced specific anti-IgG antibodies.The serum level of specific anti-IgG antibodies peaked at 5weeks,with an optical density of1.807±0.767 at A450.This optical density was significantly higher than that in the adjuvant group（0.111±0.014）or in the PBS control group（0.132±0.0246）（P0.01）.Western blotting indicated that the recombinant protein rEg-00512 was recognized by serum from immunized mice and serum from mice with a secondary infection,but it was not recognized by serum from mice in the PBS control group. Conclusion The protein Eg-00512 was successfully screened for and the recombinant protein rEg-00512 was obtained.This protein is antigenic and can potentially be used as an immunodiagnostic antigen.