Plasmids, bacterial strains, and culture conditions
The
FLAG300 fragment (~ 300 bp) was isolated from our constructed pET17b-FLAG300 plasmid [
20], and linked downstream to the pET24a-rGroEL plasmid [
18] to generate fusion genes (~ 1900 bp). The recombinant plasmid was first harbored in
E. coli XL1-Blue for maintenance and then re-transformed into
E. coli BL21 (DE3) for chimeric protein expression. All bacteria were grown in Luria–Bertani (LB) media (Difco, Detroit, MI, USA) containing 50 μg/mL of kanamycin.
Serum samples
All human serum samples (n = 220) used in this study were collected and stored anonymized before they were used. The samples were categorized as follows: (1) culture positive melioidosis patient sera (PS) (n = 38), (2) serum from healthy normal volunteers (NS) (n = 126) obtained from endemic (n = 37) and non-endemic (n = 89) provinces of Thailand, and (iii) disease control sera (DS) (n = 56) collected from patients infected with other bacteria from endemic and non-endemic areas: E. coli (n = 10), Klebsiella pneumoniae (n = 12), Pseudomonas spp. (n = 13), gram-positive cocci (n = 7), and other glucose non-fermentative (GNF) bacteria (n = 14).
Construction of the recombinant plasmid pET24a-rGroEL-FLAG300
Initially, FLAG300 from our original clone (pET17b-FLAG300) was amplified with a set of primers at EcoRI and HindIII restriction sites for the forward (5′-GAATT CCGGCACGATCAAGGTGGCG-3′) and reverse primers (5′-CCCAAGCTTCTGGTACGC GCCCGT-3′), respectively. Primers were custom-synthesized from Bio Basic Inc., Canada. Next, polymerase chain reaction (PCR) was performed using 50 μL of the final reaction volume containing 50–100 ng of the DNA template, 0.5 μL of 20 mM of dNTPs, 20 pmol of each primer, 1X Pfu buffer with MgSO4, and 1.25 U of Pfu DNA polymerase (Fermentas, Life Sciences, Massachusetts, USA). The PCR mixture was amplified based on the following protocol: preheat at 95 °C for 5 min, followed by 30 cycles of denaturation at 94 °C for 1 min, annealing at 65 °C for 1 min, extension at 72 °C for 1 min, and a final cycle at 72 °C for 5 min. The amplicon and recombinant plasmid (pET24a-rGroEL) were completely double-digested with the EcoRI plus HindIII (Biolabs, Massachusetts, USA) and gel-purified using the GeneJET Gel Extraction Kit (Thermal Fisher Scientific). EcoRI-HindIII DNA fragments were fused with T4 DNA ligase (Biolabs) at 16 °C for 16–18 h. The recombinant plasmid containing fusion genes was designated pET24a-rGroEL-FLAG300 and transferred into competent E. coli hosts by heat-shock transformation. The carboxyl end of the chimeric protein (~ 59 kDa of rGroEL plus ~ 13 kDa of FLAG300) was fused with the hexa-histidine tag that was purified by IMAC and identified with anti-his antibody via western blotting.
Chimeric protein expression (rGroEL-FLAG300), purification, and verification
The transformant carrying pET24a-rGroEL-FLAG300 was cultured in LB-kanamycin medium at 37 °C shaken at 200 rpm for 16–18 h and sub-cultured in 800 mL of freshly prepared LB-kanamycin medium at 1% inoculation rate. With prior induction of protein expression, the bacterial culture was allowed to grow until reaching an optical density at 600 nm (OD600nm) of 0.7–0.8. Next, isopropyl-β-d-thiogalactoside (IPTG) (Fermentas, Life Sciences) was added to the final concentration of 1 mM and the medium was further incubated at 16 °C shaken at 100 rpm overnight to produce soluble chimeric protein. The induced culture was then centrifuged at 4700 g for 10 min at 4 °C and the cell pellet formed was collected. The harvested pellet was resuspended in 20 mL of IMAC5 buffer (20 mM Na2HPO4, 1 M NaCl, 10% [v/v] glycerol, and 5 mM imidazole), including 0.1 mM of phenylmethyl sufonyl fluoride (PMSF) (Bio Basic Inc.) and lysed by sonication in an ice bath. The cell pellet was disrupted 10 times with pulse on at 40% amplitude for 30 s and then pulse off for 30 s. The soluble fraction was separated by centrifugation at 7000 g for 45 min at 4 °C and 2 mL of equilibrated TALON™ resins (Clontech Laboratories Inc., Mountain View, CA, USA) and IMAC5 was added to this fraction to purify the fusion protein by immobilized metal affinity chromatography (IMAC). After incubation for 16–18 h at 4 °C under orbital agitation, the column was set up and continuously washed with 40 mL of IMAC5, 20 mL of IMAC10, 20 mL of IMAC15, and, finally, with 20 mL of IMAC20. All ingredients of IMAC5, IMAC10, IMAC15, and IMAC20 were the same, except for imidazole at 5 mM, 10 mM, 15 mM, and 20 mM, respectively. The bound chimeric proteins were eluted with 10 mL of IMAC400 and concentrated using the Amicon™ Ultra Centrifugal Filter (Millipore Corporation, Burlington, Massachusetts, USA). The eluted protein concentration was determined using the Bradford protein assay by measuring the absorbance at 595 nm (Bio-Rad Laboratories Inc., Hercules, California, USA) and stored at − 80 °C until further use.
To verify the chimeric protein, SDS-PAGE and western blotting were performed. The purified proteins were: (1) separated on 12% polyacrylamide gels by electrophoresis and stained with Coomassie brilliant blue R-250 for SDS-PAGE analysis, and (2ii) transferred onto a polyvinylidene difluoride (PVDF) membrane (Pall Corporation, Port Washington, New York, USA) for western blot hybridization. The blotted membrane was washed twice with TBS buffer (10 mM Tris–HCl; pH 7.5, 150 mM NaCl) and soaked with a blocking buffer (20 mM Tris–HCl; pH 7.5, 500 mM NaCl 0.05% [v/v] Tween 20, 0.2% [v/v] Triton X-100 [TBST] and 5% non-fat dry milk) for 1 h at room temperature. After twice washing with TBST buffer and once with TBS, 1:1000 diluted anti-His horseradish peroxidase [HRP] conjugate (Qiagen, Hilden, Germany), the blocking buffer was added and the mixture was incubated for 1 h. The membrane was washed as in the previous step and visualized for immunoreactivity with 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (KPL, Gaithersburg, MD, USA).
In addition, the chimeric proteins were investigated by western blotting hybridization. The blotted membranes were separately probed with human antibodies from 1:200 dilution of pooled positive sera from the melioidosis patients (n = 5) and pooled normal sera from healthy donors (n = 5). The secondary antibodies from 1:2000 diluted goat anti-human IgG/IgM/IgA HRP conjugate (KPL) were added to react with the immune complex and detected by adding the TMB substrate.
Determination of the specific binding of chimeric protein with melioidosis antibodies by indirect ELISA
Each serum sample from the three groups (i.e., PS, NS, and DS) was tested in duplicate using the 96-well Microlon™ plate (Greiner bio-one, Kremsmünster, Austria). 100 μL of 10 μg/mL purified protein in the coating buffer (0.05 M carbonate buffer, pH 9.6) was added to the microtiter plate and incubated at 4 °C for 16–18 h. The plate was rinsed with PBST buffer (0.15 M PBS, 0.1% Tween 20, pH 7.4) and incubated with 200 μL of the blocking buffer (2% BSA in 0.15 M PBS, pH 7.4) for 1 h at room temperature to prevent nonspecific binding, followed by washing again with PBST. The immunoreactivity was tested according to the following protocol: 100 μL of 1:3200 diluted sample in the serum diluent (1% BSA in PBST) was added and incubated at 37 °C for 1 h and then rinsed with PBST. 100 μL of 1:10,000 diluted goat anti-human IgG HRP conjugate (KPL) was added to the mixture and then incubated at 37 °C for 1 h. Detecting IgG is better than IgM to diagnose melioidosis by indirect ELISA. The reaction time of 10 min was maintained after adding 100 μL of the TMB substrate and the reaction was later stopped with 50 μL of 2 M H2SO4. The resultant color product was determined under OD450nm reading with a microplate reader (Bio-Rad model 550, Bio-Rad Laboratories Inc.). For each experimental plate, positive (pooled melioidosis patient sera), negative (pooled normal sera), and direct conjugate controls (diluent without serum) were used.
Statistical analysis
Data analysis and graphing were performed using the statistical software package SPSS 16.0 for Windows (SPSS) and Stata version 13.0 (StataCorp LP, College Station, Tx). Mean and standard deviation (SD) optical densities (OD) of normal serum samples were calculated. The upper limit of normal was determined as mean plus 2SD and serum samples exceeding this value were considered positive. Using the bacterial culture results as the reference method (“gold standard”) and the NS and DS groups as true negatives, the diagnostic indices (sensitivity, specificity, accuracy) of this developed assay were determined. The independent t-test was used to analyze the difference in the mean OD of each group. A p value < 0.05 was considered to be statistically significant.