Production, purification and characterization of polyclonal antibody against the truncated gK of the duck enteritis virus
- Shunchuan Zhang†1,
- Jun Xiang†1,
- Anchun Cheng1, 2, 3Email author,
- Mingshu Wang1, 2Email author,
- Xin Li1,
- Lijuan Li1,
- Xiwen Chen2,
- Dekang Zhu1, 2,
- Qihui Luo2 and
- Xiaoyue Chen1, 2, 3
© Zhang et al; licensee BioMed Central Ltd. 2010
Received: 20 August 2010
Accepted: 17 September 2010
Published: 17 September 2010
Duck virus enteritis (DVE) is an acute, contagious herpesvirus infection of ducks, geese, and swans, which has produced significant economic losses in domestic and wild waterfowl. With the purpose of decreasing economic losses in the commercial duck industry, studying the unknown glycoprotein K (gK) of DEV may be a new method for preferably preventing and curing this disease. So this is the first time to product and purify the rabbit anti-tgK polyclonal antibody. Through the western blot and ELISA assay, the truncated glycoprotein K (tgK) has good antigenicity, also the antibody possesses high specificity and affinity. Meanwhile the rabbit anti-tgK polyclonal antibody has the potential to produce subunit vaccines and the functions of neutralizing DEV and anti-DEV infection because of its neutralization titer. Indirect immunofluorescent microscopy using the purified rabbit anti-tgK polyclonal antibody as diagnostic antibody was susceptive to detect a small quantity of antigen in tissues or cells. This approach also provides effective experimental technology for epidemiological investigation and retrospective diagnose of the preservative paraffin blocks.
Duck virus enteritis (DVE) is an acute, contagious herpesvirus infection of ducks, geese, and swans, characterized by vascular damage, tissue hemorrhages, digestive mucosal eruptions, lesions of lymphoid organs, and degenerative changes in parenchymatous organs [1–5]. The causative agent of DVE is duck enteritis virus(DEV), composing of a linear, double-stranded DNA genome with 64.3% guanine-plus-cytosine content, which is higher than any other reported avian herpesvirus in the Alpha-herpesvirinae subfamily. In duck-producing areas of the world where the diseases has been reported, DEV has produced significant economic losses in domestic and wild waterfowl due to mortality, condemnations, and decreased egg production.
With the purpose of decreasing economic losses in the commercial duck industry, studying gK of DEV may be a new method for preferably preventing and curing this disease. Because glycoproteins are the major antigens recognized by the infected host's immune system and play an important role in mediating target cell infection, cellular entry of free viruses, and the maturation or egress of the virus [8, 9]. Glycoprotein K is one of the major glycoproteins encoded by the DEV-gK gene, which is located in the unique long region of the DEV genome. Additionally, gK is capable of inducing a protective immune response in vivo and is responsible for viral binding to the cellular receptor [10, 11].
Western blotting was used to detect the reactivity and specificity of the tgK. The purified recombinant proteins were separated on 12% SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membrane at 120 V for 1.5 h in a BioRad mini Trans-Blot electrophoretic transfer cell (BioRad, Shanghai, China) for western blot analysis. The blotted membrane was blocked at 4°C for 16 h with 10% skimmed milk in TBST (Tris-buffered saline with 0.1% Tween-20, pH 8.0). Then, the membranes were washed and incubated with rabbit anti-tgK polyclonal antibody while using the preimmune serum of normal rabbit as negative control. The membranes were then washed and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (Invitrogen) at 1:5000 of dilution in TBST buffer containing 0.5% BSA. After further washing, immunoreactive protein was visualized by using diamino benzidine (DAB). From the result, we can see the purified tgK, which was recognized by rabbit anti-tgK polyclonal antibody, was apparent on western blots (Figure 2B, lane1) as a single specific band approximately 34 kDa. Meanwhile, the rabbit preimmune serum did not show any reaction with tgK in western blots (Figure 2B, lane2). All the data indicated the tgK had good reactivity and specificity.
Enzyme linked immunosorbent assay (ELISA) was used to evaluate the affinity of antibody. Microplates were coated for 1 h at 37°C with 100 μl per well of truncated gK at the concentrations 5 μg/ml in 50 mM carbonate/bicarbonate buffer pH 9.6 and then coated overnight at 4°C. After this procedure, plates were washed three times in PBST (PBS buffer with 0.1% Tween-20) for 5 min each and blocked with 110 μl per well of PBST with 1% BSA for 1 h at 37°C. The sample of the rabbit anti-tgK positive serum was diluted with 11 gradients ranging from 1:800 to 1:819200 and incubated for 1 h at 37°C. After incubating antiserum, plates were washed and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (Invitrogen) at working concentration 1:5000 for 1 h at 37°C. After washing 3 times, 100 μl TMB (3,3',5,5'-tetramethyl-benzidine) was added to the plates followed by exposure for 8 minutes. The reaction was terminated with 2 M H2SO4 and the OD450 value was then read with Elx800 Universal Microplate Reader (Bio-Tek Instruments, Inc., Winooski, VT, USA). Also, other plates incubated with rabbit preimmune serum had the same procedures with those plates incubated with rabbit anti-tgK positive serum. The result of ELISA showed a minimum detection limit of the duck anti-tgK positive sera was 1:409600. The higher the titer, the stronger is the affinity. So the affinity of the antiserum collected from rabbits was so good.
The neutralization titer of the rabbit anti-tgK polyclonal antibody was evaluated by micro neutralization test. First of all, duck embryo fibroblasts (DEF) were prepared in 96-well cell culture plate and each well had 250 μl cell suspension. Then, inactivated test sera rabbit anti-tgK (56°C for 30 min) were serially diluted twofold from 1:1 to 1:64. The 200TCID50 virus, which was diluted from the virus stock suspension (TCID50 = 10-5.567), in a 25 μl volume was mixed with an equal volume of serum dilution and incubated at 35°C for 1 h. Also, each serum dilution had 6 duplications. When the cells grew as monolayer, then 50 μl of the incubated mixture was inoculated onto the cells. After a 1 h adsorption period at 37°C, the cells were overlaid with the modified eagle's medium. Meanwhile, seven contrast controls were set up for later observation:1) blank control was normal cells; 2)100TCID50, 10TCID50, 1TCID50 and 0.1TCID50 without incubating with diluted positive serum was respectively added to the cells in cell culture plate, used as controls; 3) cells incubated only with high concentration positive serum or negative serum were used as controls. Through observation, the 50% serum neutralized destination was calculated by Reed-muench method. The neutralization titer of the rabbit anti-gK polyclonal antibody was 1:5.623. The result indicated the gK may possess the functions of neutralizing DEV and anti-DEV infection, also has the potential to produce subunit vaccines.
In conclusion, this is the first time to product the rabbit anti-tgK polyclonal antibody and purify the antibody by ion exchange column chromatography. Through the western blot and ELISA assay, the tgK has good antigenicity, and the antibody possesses high specificity and affinity. Meanwhile the rabbit anti-tgK polyclonal antibody has the potential to produce subunit vaccines, and possesses the functions of neutralizing DEV and anti-DEV infection because of its neutralization titer.
Meanwhile, this study showed indirect immunofluorescent microscopy using the purified rabbit anti-tgK polyclonal antibody as diagnostic antibody could be used to detect DEV and antigen location in organs, provide a new diagnostic method to detect DEV, provide useful method and data for researching and clarifying the morbigenous mechanism of DEV.
Until now, there is no report about indirect immunofluorescent microscopy using the purified rabbit anti-tgK polyclonal antibody as diagnostic antibody to detect the antigen locations of DEV in the infected ducks. Indirect immunofluorescent microscopy combines together the special immunoreaction, the good cells morphous maintained in paraffin section with the illuminant easily descried in the black background, which was susceptive to detect a small quantity of antigen in tissues or cells. This approach also provides effective experimental technology for epidemiological investigation and retrospective diagnose of the preservative paraffin blocks.
The research was supported by grants from the Changjiang Scholars and Innovative Research Team in University (PCSIRT0848), the earmarked fund for Modern Agro-industry Technology Research System (nycytx-45-12).
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