Construction, expression, and purification of rE74-118 protein
Truncated rE74-118 gene was successfully cloned into pET-32b plasmid between the BamHI and XhoI restriction sites located before histidine tag sequence at the C terminal as shown in Figure 1A. The correction of inserted gene was confirmed by sequencing and double digestion (data not shown). To maximize the soluble protein yield of this construct , we optimize expression (37 °C for 2-4 hours, 30 °C for 4-6 hours, 22-25 °C for 6-20 hours and 12-15 °C overnight using 0.1 mM IPTG) and found that the best condition to express rE74-118 in E. coli (SHuffle) system is the induction with 0.1 mM IPTG, at 25 °C, for 20 hours (data not shown). The expected size of expressed protein is approximately 17 kDa with small amount of insoluble part and high amount of soluble part as shown in figure 1B. After that, we purified this protein by using His-Tag protein purification column and demonstrated each protein fraction with anti-E huMAbs staining as shown in figure 1C. The concentration of purified protein in elute 1 and 2 fractions were calculated by Bradford assay and the total concentration is 5.8 mg/ml obtained from 200 ml culture.
Figure 1A: Map of rE74-118 in pET-32b plasmid
Figure 1B: SDS-PAGE of rE74-118 protein
Figure 1C: Western analysis of purified rE74-118 protein
In vitro binding activity of rE74-118 protein
Expressed rE74-118 protein was tested for specific binding activity with anti-dengue E huMAbs, anti-dengue prM, anti-dengue NS1, and dengue patient sera in acute phase of DENV-2 infection as shown in figure 2. D23-1B3B9, D23-1G7C2, D23-1C2D2 anti-E huMAbs and dengue patient sera (D22, D25, D26, D27, D28, D29, D30, D32 and D33) showed strong reactivity with rE74-118. D22-2G4E3, D30-3B10E7 anti-E huMAbs and D23 patient serum showed weak binding activity. However, this rE74-118 did not bind with anti-dengue E huMAbs (D30-1E7B8, D32-2D1G5, D33-3E4H10, D23-1A10H7) and anti-dengue prM (D25-4D4F10) and anti-dengue NS1 huMAb (D26-5A2B12).
Figure 2: Binding activity of rE74-118 protein with dengue patient sera, anti-dengue E, anti-dengue prM, anti-NS1 huMAbs.
Immunogenicity of rE74-118 protein in BALB/c mice
Purified rE74-118 protein was tested in vivo activity by injecting 100 µg of rE74-118 combined with Gerbu adjuvant (ratio = 1:1) into the BALB/c mice for 3 doses (2 weeks apart) and it can induce dengue IgG antibodies after second boosting with the titer of 1:204,800 similar to whole DENV-2 immunization, and PBS with Gerbu regimen could not induce any dengue IgG antibody (data not shown). Neutralizing activity of sera immunized with rE74-118 was detected in Vero cell and calculated as FRNT50 around 1:16 (data not shown). By ADE assay using K562 cell, mouse sera immunized with DENV-2 showed enhancing activities against all 4 serotypes, but no enhancing activity of mouse sera immunized with rE74-118 with any serotype as shown in figure 3.
Figure 3: ADE activity of mouse sera against 4 serotypes of DENV
DENV E protein is one of several antigens could be designed for vaccine, inhibitor, or diagnostic agent. It can induce both of protective and pathological effects against DENV infection8. The expression and well-characterization of truncated E protein is still limited. This study aimed to express and characterize a short E protein, rE74-118, and its binding, neutralizing, and enhancing activities.
We successfully expressed rE74-118 in soluble form by using E. coli (SHuffle) with the high amount purified protein of 5.8 mg/ml from 200 ml bacterial culture and showed basic functions (e.g. binding activities, some neutralizing activities, and no ADE) similar to other expression system9. For binding activity, the short rE74-118 protein showed strong binding to dengue patient sera and most of anti-dengue E huMAbs, but no reactivity to anti-dengue prM huMAbs, and anti-dengue NS1 huMAbs, which is good for diagnostic application. Several anti-dengue E huMAbs (D30-1E7B8, D32-2D1G5, D33-3E4H10, D23-1A10H7) could not bind to rE74-118 because these antibodies might bind to the conformational epitopes4, 10, 11. Purified rE74-118 protein can stimulate humoral immune response in BALB/c mice due to the evidence that we can detect high amount of dengue IgG in mouse sera. The protective activity of rE74-118 was also investigated in our present study. We found that sera from mice immunized with rE74-118 show the low neutralizing activity against DENV-2, but no ADE activity with any serotype, which is better than the whole DENV-2 immunogen.
In conclusion, our rE74-118 protein showed both antigenic and immunogenic properties, that might be a promising target for various applications such as subunit vaccine, biological inhibitor, or diagnostic applications4, 6, 8.
This research was supported by Thailand Research Fund Grant for New Researcher (TRG, Grant Number TRG5980015 for Chonlatip Pipattanaboon).
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