Patients’ sera were provided by Dr. Ronaldo F. Dias of the Bahia Farma Diagnostics (Simoes Filha, Bahia, Brazil) which were collected according to the “Bioethics and Security” and only unnamed samples described as numbers. The plasmid containing cDNA of E2 was provided by Genbody Inc. (Cheonan, Chungnam, Korea). Intrinsically unstructured region of E2 (GenBank no. KF925313.1) protein was predicted by IUPred server (http://iupred.enzim.hu/). The target fragment was amplified by PCR with designed primer sets as follows: for pMALc-5X sense 5ʹ-CCC CAT ATG TCC TTA ACA TGT TCT CCC CAC CGT CAG-3ʹ and antisense 5ʹ-CGG GAT CCG TCT GTA GTT ATT AGT CCT TCA TTT GAG CC-3ʹ, and for pGEX4T-1 sense 5ʹ-CGG GAT CCC CGT CCT TAA CAT GTT CTC CCC-3ʹ and antisense 5’-CGC TCG AGG TCT GTA GTT ATT AGT CCT TCA TTT GAG CC-3ʹ. Digested fragments were ligated into pGEX-4T-1 and pMAL-c5X vectors, respectively. The recombinant plasmids, pGEX-4T-1/E2 and pMAL-c5X/E2, were transformed into DH5α Escherichia coli. After confirmation of plasmids, the current plasmids were transformed into BL21(DE3)pLysS E. coli. Expression of glutathione S-transferase (GST)-E2 fusion proteins and maltose-binding protein (MBP)-E2 fusion proteins were induced at 0.5 mM IPTG. Total lysates of E. coli were dissolved by SDS-PAGE and transferred onto nitrocellulose membranes (Whatman GmbH, Dassel, Germany). The membranes were blocked with 5% skim milk (Difco Laboratories, Detroit, Michigan, USA) in PBS with 0.5% Tween-20 (PBST). After washing with PBST, the membrane was incubated with 1:100 diluted sera and then with HRP-conjugated anti-human IgG antibody (Sigma Aldrich, St. Louis, Missouri, USA). Chemiluminescence signals were detected with ECL Western blotting kit (Millipore Co., Billerica, Massachusetts, USA).

Both GST-E2 and MBP-E2 fusion proteins were well expressed with molecular weights of 54 kDa and 74 kDa, respectively, by rabbit monoclonal GST antibody (Santa Cruz Biotechnology, Santa Cruz, California, USA) and mouse monoclonal MBP antibody (Cell Signaling Technology, Danvers, Colorado, USA). The second antibodies were horseradish peroxidase (HRP)-conjugated anti-rabbit antibody and HRP-conjugated anti-mouse antibody (Sigma Aldrich) (Fig. 1A). Our data identified a high sensitivity and high specificity of recombinant E2 fusion antigens. The sensitivity and specificity of 2 recombinant antigens were shown in Fig. 1B and C. In western blot, 30 sera of CHIKV patients and 30 sera of normal humans were reacted. Among 30 patients’ sera, both GST-E2 and MBP-E2 were reacted with 25 sera based on the signals in western blots, which showed a high sensitivity of 83.3% with both recombinant fusion antigens. GST-E2 and MBP-E2 were reacted with 1 human normal serum among 30 normal sera, which demonstrated a high specificity (96.7%) (Table 1).

In conclusion, the seroprevalence of anti-IUD of E2 IgG antibodies in patients infected with CHIKV was analyzed by western blot. Two kinds of tag fusion proteins were confirmed to find out the best recombinant antigen with high sensitivity, specificity, and solubility. These recombinant antigens can be used for further applications such as developing an RDT kit. Fortunately, GST-E2 expressed in E. coli is soluble, and MBP-E2 is very soluble to operate easily as promising recombinant antigens for the diagnosis of CHIKV infection.