A 27?kDa immunodominant antigen Com1 was identified by all different methods mentioned above. Iraq. Less than 5% (5 out of 156) of the sera from patients with other febrile diseases reacted with the Com1. These results suggest that the modified ELISA using Com1 may have the potential to improve the detection of Q fever specific antibodies. 1. Introduction Q fever is a worldwide zoonotic disease caused by infection withCoxiella burnetiiC. burnetiiC burnetiiDNA can be detected occasionally in patient serum of acute phase Q fever with Polymerase Chain Reaction (PCR) [8C10], the current diagnosis of Q fever relies mainly on serological methods [11]. These methods include the indirect RL immunofluorescent antibody assay (IFA) [12, 13], the complement fixation assay (CFA) [14, 15], and the enzyme-linked immunosorbent assay (ELISA) [16, 17]. Among these tests, IFA is considered to be the reference test during an endemic situation [8, 18]. Because the screening of serum samples by IFA was laborious, ELISA was Wnt-C59 used during an epidemic situation because it can be automated and is easier to perform [19]. The antigen used in ELISA is mostly whole cell preparation of phase I or phase IIC. burnetii[16, 17, 19, 20]. Due to the hazard and difficulty of culturing and purifyingC. burnetiiin a biosafety level (BSL)-3 laboratory, the antigens are not available in most clinical laboratories. Although there are commercially available IFA Wnt-C59 and ELISA tests for Q fever, the serological test results vary considerably among different laboratories using the same kit. This may be due to the residual egg yolk or tissue culture proteins in the whole cell antigen preparation [2, 21]. Earlier studies focused on the identification of immunogenic antigens ofC. burnetiidiscovered protein immunogens of molecular weights from 13 to 92?kDa [22, 23]. Among them, Hsp60, Com1, Cbmip, P1, and AdaA have been cloned and characterized previously [23C27]. More recently, several proteomic studies have identified additional immunogenic proteins by 2D-gel immunoblotting [28, 29] and protein microarray approaches [30, 31]. A 27?kDa immunodominant antigen Com1 was identified by all different methods mentioned above. In this study, we cloned and purified the Com1 antigen. 33 Q fever patient sera, 10 normal human sera, and 156 other febrile patient sera were used to evaluate the usage of recombinant Com1 antigen for the detection of Q fever specific antibodies in ELISA. The results demonstrated the amplification of ELISA signal may have the potential to improve the serological assay. 2. Materials and Methods 2.1. Bacterial Strains and Vectors Top10 (Invitrogen, CA) was used for general cloning. The cloned genes were inserted into pET24a (Novagen, CA) for the expression of Com1 protein.E. coliBL21 (DE3) (Invitrogen, CA) was used for expression of proteins under the control of phage T7lacpromoter [32]. 2.2. Cloning of the Gene Coding for the Com1 Protein into Wnt-C59 the Expression Vector pET24a A primer pair Wnt-C59 [com1f (5-CGGGATCCGCCCCCTCTCAATTCAGTTTTT-3) and com1r (5-AAGAATGCGGCCGCCTTTTCTACCCGGTCGATTTCT-3)] was designed by using the nucleotide sequence of the open reading frame for the Com1 from strain RSA 493 (GenBank accession quantity NC002971.1). The coding series for proteins 22 to 252 from the Com1 proteins was amplified by PCR using genomic DNA isolated fromC. burnettiRSA 493 stress as the template. The PCR product was digested with NotI and BamHI and ligated in to the expression vector pET24a. Best10 skilled cells had been transformed using the ligation blend, and colonies had been screened for the current presence of inserts with the proper size. The ultimate sequences had been verified by DNA sequencing from the ensuing plasmid. 2.3. Manifestation and Purification from the Recombinant Com1 Proteins (rCom1) BL21 (DE3) was changed with plasmids holding the Com1 put in. The recombinantE. colicolony with high manifestation degree of the Com1 proteins was grown over night in Over night Express moderate TB (EMD Biosciences, CA) in the current presence of kanamycin at 37C with shaking at 200?rpm. Cell pellets from 500?ml cultures were resuspended in 20?ml of buffer A (20?mM Tris-HCl, pH 8.0, 1?mM EDTA, and 1?mM DTT) containing 2% deoxycholic acidity (DOC). Cells extracted from sonication (Ultrasonic Water Processor chip model VirSonic 475, VIRTIS Business,.