摘要
目的分析肝片形吸虫(Fasciola hepatica)重组组织蛋白酶L(CatL)的免疫反应性,以及对SD大鼠的免疫原性。方法诱导含重组原核表达质粒pET30a-FhCatL的大肠埃希菌BL21(DE3)宿主菌表达,SDS-PAGE分析表达产物,以感染肝片形吸虫山羊血清为一抗,蛋白质印迹(Western blotting)分析其免疫反应性。20只SD大鼠随机均分为重组蛋白免疫组和佐剂对照组,免疫组大鼠皮下注射纯化的重组FhCatL,200μg/(只·次),共免疫3次,每次间隔3周;对照组用等量PBS与佐剂混合后注射。于第2次和末次免疫前,以及末次免疫后3、6和9周尾静脉采血,分离血清。利用间接ELISA法检测免疫大鼠血清IgG抗体水平,噻唑蓝比色法(MTT法)检测脾淋巴细胞增殖情况。结果经纯化获得重组FhCatL蛋白,相对分子质量(Mr)约42 000。Western blotting分析结果表明,纯化重组蛋白能被感染肝片形吸虫的山羊血清识别。重组FhCatL蛋白免疫SD大鼠后可诱导产生特异性IgG抗体,随着免疫时间的延长抗体水平逐渐升高,于末次免疫后3周抗体效价达到峰值(1∶102 400),明显高于对照组(1∶1 000)。免疫组脾淋巴细胞刺激指数为2.176±0.047,显著高于对照组(1.171±0.032)(P<0.05)。结论重组FhCatL蛋白具有较好的免疫反应性和免疫原性。
Objective To analyze the immunoreactivity of recombinant cathepsin L-like proteases（CatL） protein of Fasciola hepatica and its immunogenicity in SD rats. Methods The E. coli BL21（DE3） cells harbouring recombinant plasmid pET30a-FhCatL were inoculated in LB medium, and the protein expression was induced with IPTG. The recombinant protein FhCatL was analyzed by SDS-PAGE and the immunoreactivity was identified by Western blotting with sera from Fasciola hepatica-infected goat as the primary antibody. Twenty SD rats were randomly divided into immunized group and adjuvant control group. SD rats in immunized group were injected subcutaneously with 200 μg of purified FhCatL protein. All the rats received three immunizations at 3-week intervals. The adjuvant control group with 10 SD rats received only adjuvants emulsified with the same amount of PBS. Serum samples were collected at the day before the second and final immunization, 3, 6, and 9 weeks after the final immunization. The IgG antibody of rats′ sera was examined by indirect ELISA and spleen lymphocyte proliferation（SLP） was tested by methyl thiazolyl tetrazolium（MTT）.Results The molecular weight of purified FhCatL was about Mr42 000. The recombinant FhCatL was recognized by pool sera of goats naturally infected with F. hepatica. The titer of specific antibody IgG in SD rats induced by the recombinant protein against CatL protein was significantly higher than that of the control, and the antibody titer reached the peak at three weeks after the final immunization（1 ∶ 102 400）. The stimulation index of splenocytes in immunized group was 2.176 ±0.047, which was significantly higher than that of the control（1.171 ±0.032）（P 0.05）. Conclusion The recombinant FhCatL protein bears stronger immnoreactivity and immunogenicity.
出处
《中国寄生虫学与寄生虫病杂志》
CAS
CSCD
北大核心
2014年第4期289-292,共4页
Chinese Journal of Parasitology and Parasitic Diseases
基金
黑龙江省普通高校青年学术骨干支持项目(No.1251G042)
黑龙江省科技计划项目(No.GZ11B208)~~
关键词
肝片形吸虫
组织蛋白酶L
免疫原性
免疫反应性
Fasciola hepatica
Cathepsin L-like proteases（CatL）
Immunogenicity
Immunoreactivity
