[Purification of the recombinant Com1 and adaA of Coxiella burnetii and identification of the antigenicity].

Abstract

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 Mr 27 000 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.

• Authors: Jingxian Liu, Yihong Ji, Zhiyang Shi, Yongjun Jiao
• Journal: Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology (Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi) Vol. 33 Issue 6 Pg. 726-730 (Jun 2017) ISSN: 1007-8738 [Print] China
• PMID: 28615092 (Publication Type: Journal Article)

Chemical References

• Antigens, Bacterial
• Bacterial Outer Membrane Proteins
• Recombinant Proteins
• acute disease antigen A, Coxiella burnetii

Topics

• Animals
• Antigens, Bacterial (genetics, immunology, isolation & purification)
• Bacterial Outer Membrane Proteins (genetics, immunology, isolation & purification)
• Cattle
• Coxiella burnetii (immunology)
• Escherichia coli (genetics)
• Molecular Weight
• Recombinant Proteins (isolation & purification)