贝纳柯克斯体重组抗原Com1和急性Q热抗原A(adaA)的纯化和鉴定  

Purification of the recombinant Com1 and adaA of Coxiella burnetii and identification of the antigenicity

作　　者：刘静娴[1] 姬艺洪 史智扬[1] 焦永军[1] 

机构地区：[1]江苏省疾病预防控制中心,江苏南京210009 [2]南京农业大学兽医学院,江苏南京210095

出　　处：《细胞与分子免疫学杂志》2017年第6期726-730,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(31570926;81601732);江苏省医学重点人才课题(ZDRCA2016031)

摘　　要：目的原核表达并纯化2种贝纳柯克斯体(C.burnetii)抗原主要外膜蛋白质Com1和急性Q热抗原A(adaA),并对重组Com1和adaA进行质谱鉴定以及抗原性鉴定。方法全基因合成编码Com1和adaA的基因序列,并将其分别构建到原核表达载体pET-20b(+),将构建好的载体分别转化大肠杆菌BL21(DE3),异丙基硫代β-D-半乳糖苷(IPTG)诱导表达重组Com1和adaA。通过镍亲和柱层析纯化重组Com1和adaA,切取纯化的蛋白质条带进行质谱鉴定。将纯化的重组Com1和adaA与Q热阳性牛血清进行Western blot分析,检测其免疫反应特性。结果构建了原核表达载体pET-20b(+)-Com1以及pET-20b(+)-adaA,重组抗原Com1和adaA均以可溶形式得到了高效地表达和纯化,SDS-PAGE结果显示其相对分子质量(Mr)分别为27 000与25 000,质谱鉴定结果确证其为C.burnetii的抗原蛋白Com1和adaA。重组Com1和adaA与Q热阳性牛血清均有阳性反应条带,条带Mr大小与SDS-PAGE结果一致。结论成功高效表达了可溶重组Com1和adaA并证实其免疫反应特异性。Objective To express and purify two kinds of antigens of Coxiella burnetii（ C. burnetii）,the main outer membrane protein Com1 and the acute disease antigen A（ adaA）,in prokaryotic expression system and to validate the two recombinant antigens by mass spectrometry and identify their antigenicity. Methods The gene sequences encoding Com1 and adaA were separately synthesized and constructed into the prokaryotic expression vector pET-20b（ ＋）. The constructed vectors were transformed into E. coli BL21（ DE3）,and the recombinant proteins were induced by IPTG. The recombinant Com1 and adaA were purified by His affinity chromatography and identified by mass spectrometry. The immunoreactivity of the two antigens was identified by Western blot analysis using Q fever positive bovine serum. Results The expression vectors pET-20b（ ＋）-Com1 and pET-20b（ ＋）-adaA were constructed and the recombinant Com1 and adaA were expressed and purified in a soluble form. High-purity recombinant Com1 and adaA were obtained after purification,and the SDS-PAGE showed that their relative molecular masses were Mr27 000 and 25 000,respectively. The mass spectrometry confirmed the recombinant proteins were Com1 and adaA of C. burnetii. Both of the recombinant Com1 and adaA were able to react with the Q fever positive bovine serum in Western blotting,and the corresponding bands were in accordance with the SDS-PAGE.Conclusion We obtained high-purity Com1 and adaA in a soluble form and confirmed their immunoreactivity.

关 键 词：贝纳柯克斯体(C.burnetii) 重组抗原Com1和adaA 质谱鉴定 抗原性鉴定 

分 类 号：R392.11[医药卫生—免疫学] R392-33[医药卫生—基础医学]