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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 the two major outer membrane proteins Com1 and A (adaA) of C. burnetii antigen in prokaryotic cells. The recombinant Com1 and adaA were identified by mass spectrometry and antigenicity. Methods The gene sequences coding for Com1 and adaA were synthesized and cloned into the prokaryotic expression vector pET-20b (+). The constructed vector was transformed into E. coli BL21 (DE3), isopropylthio β-D Inducible expression of recombinant Com1 and adaA by galactoside (IPTG). Recombinant Com1 and adaA were purified by nickel affinity column chromatography, and purified protein bands were cut out for mass spectrometry identification. The purified recombinant Com1 and adaA and Q hot-positive bovine serum were analyzed by Western blot to test their immune response characteristics. Results The prokaryotic expression vectors pET-20b (+) - Com1 and pET-20b (+) - adaA were constructed. The recombinant antigens Com1 and adaA were efficiently expressed and purified in soluble form. SDS-PAGE showed that the relative molecules The mass (Mr) was 27 000 and 25 000, respectively, and the mass spectrometry results confirmed that it was the C. albictii antigenic protein Com1 and adaA. Recombinant Com1 and adaA positive Q positive cattle bovine serum reaction bands, Mr band size and SDS-PAGE results. Conclusion Soluble recombinant Com1 and adaA were successfully expressed and confirmed their immunoreaction specificity.