Department of Biological Sciences and Tropical Marine Science Institute, National University of Singapore, Singapore1192601
Key Laboratory of Marine Biotechnology, Third Institute of Oceanography, State Oceanic Administration, Xiamen 361005, Peoples Republic of China2
Author for correspondence: Choy Hew. Fax +65 7792486. e-mail dbshewcl{at}nus.edu.sg
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Abstract |
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Introduction |
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Methods |
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Clones containing 8002000 bp inserts were selected from the cDNA library of WSSV by digestion with EcoRI+HindIII. The corresponding plasmids were prepared and purified with resin (Sambrook et al., 1989 ). Subsequently, sequencing was performed using pBluescript ks and sk primers in an automatic DNA sequencer. The DNA sequences generated and the deduced amino acid sequences were respectively analysed using DNASIS and PROSIS software (Hitachi Software Engineering, version 4). The deduced amino acid sequences were subjected to BLAST analysis in the GenBank and SWISS-PROT databases.
Expression of the vp28 gene in E. coli.
The vp28 gene was cloned and expressed in pBV220DH5 (Zhang et al., 1990
). PCR was performed using primers synthesized (Shanghai Beckman Life Science and Technology Laboratory) for amplification of the vp28 gene. The forward primer used was 5' AGAGAATTCATGGATCTTTCTTTCAC 3' (EcoRI site in italics) and the reverse primer was 5' CACGTCGACTTACTCGGTCTCAGTGC 3' (SalI site in italics). The amplified DNA and plasmid vector pBV220 were digested with EcoRI+SalI. After purification and ligation of DNA fragments, the vp28 gene was inserted into pBV220 vector downstream of its promoter. The resulting plasmid was named pBV28. Competent cells of E. coli DH5
were transformed with pBV28 and colonies containing transformants were screened by PCR. The presence of pBV28 was verified by digestion with EcoRI+SalI and DNA sequencing. The following treatments were conducted for the expression of vp28:
A, pBV28DH5 (containing the vp28 gene) induced;
B, pBV28DH5 non-induced;
C, pBV220DH5 (the vector only as a control) induced;
D, pBV220DH5 non-induced.
After incubation at 30 °C overnight, pBV28DH5 and pBV220DH5
were inoculated into new medium at a ratio of 1:100. The cultures were grown to an OD600 of 0·5 at 30 °C and then transferred immediately for induction for an additional 6 h at 42 °C. The induced and non-induced bacteria were analysed by SDSPAGE.
Purification of expressed protein.
The recombinant pBV28DH5 was inoculated and incubated in LB medium at 30 °C overnight. Cells were transferred into 1000 ml fresh LB medium and incubated at 30 °C. When the OD600 was 0·5, the culture was moved immediately to 42 °C for 6 h. The induced bacteria were spun down (5000 r.p.m.) at 4 °C, followed by suspension in lysis buffer (50 mM TrisHCl, 1 mM EDTA, 100 mM NaCl, 2% Triton X-100, 2 M urea, 1 mM PMSF, pH 8·0) and sonication for 30 s on ice. The sonicated sample was treated with DNase I at room temperature for 1 h and then spun down at 30000 g for 30 min. The pellet was resuspended in Milli-Q water. After addition of an equal volume of TE buffer (10 mM TrisHCl, 1 mM EDTA, 1 M NaCl, 8 M urea, 1% SDS, pH 8·0), the mixture was placed at room temperature for 1 h and then centrifuged at 100000 g for 10 min. The supernatant was dialysed against 0·05 M TrisHCl (pH 8·0) and subjected to SDSPAGE.
After running the extract on SDSPAGE, a section of the gel was stained in 0·3 M CuSO4 to determine the position of the expressed VP28 protein. The remaining, non-stained portion of the gel at the position corresponding to VP28 was then excised and transferred into dialysis tubing. After electrophoresis in SDSPAGE electrode buffer for 1 h, the eluate in the tubing was dialysed against 0·05 M TrisHCl, pH 8·0.
Preparation of antibody.
Mice were immunized by intradermal injection of the purified VP28 protein fortnightly over an 8-week period. Five µg antigen (VP28) was mixed with an equal volume of Freunds complete adjuvant (Sigma) for the first injection. Subsequent injections were conducted using 5 µg antigen mixed with an equal volume of Freunds incomplete adjuvant (Sigma). Four days after the last injection, mice were exsanguinated and antisera were collected. The titres of anti-VP28 sera were 1:20000, as determined by ELISA. The IgG fraction was purified with protein ASepharose (Bio-Rad) (Sambrook et al., 1989 ) and stored at -70 °C. The optimal dilution of purified IgG, after serial dilutions, was 1:1000 as determined by ELISA. Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG was obtained from Sigma. Antigen was replaced by PBS in control assays.
Transcriptional analysis of vp28 gene
Shrimp infection with WSSV.
Infected tissue from a Penaeus monodon shrimp with a pathologically confirmed infection was homogenized in TN buffer (20 mM TrisHCl, 400 mM NaCl, pH 7·4) at 0·1 g/ml. After centrifugation at 2000 g for 10 min, the supernatant was diluted to 1:100 with 0·9% NaCl and filtered (0·22 µm filter). Aliquots (0·2 ml) of the filtrate were injected intramuscularly into healthy shrimps (as determined by PCR) in the lateral area of the fourth abdominal segment. At various times post-infection (p.i.), four specimens were selected at random and their haemolymph was collected. The collected haemolymph samples were frozen immediately and stored at -70 °C.
RTPCR.
Total RNA was isolated from WSSV-infected shrimp haemolymph according to the manufacturers instruction (Macherey-Nagel). RTPCR was performed with primers 5' AGAGAATTCATGGATCTTTCTTTCAC 3' and 5' CACGTCGACTTACTCGGTCTCAGTGC 3' by using the TITANIUM One-step RTPCR kit (Clontech). The RTPCR program was as follows: 50 °C for 1 h followed by 94 °C for 5 min; 30 cycles of 94 °C for 30 s, 65 °C for 30 s and 68 °C for 1 min and, finally, 68 °C for 2 min.
Western blot.
Infected shrimp haemolymph samples from various time-points were analysed in 12% SDSPAGE gel. Proteins were visualized by Coomassie brilliant blue staining. The proteins were transferred onto nitrocellulose membrane (Bio-Rad) in electroblotting buffer (25 mM TrisHCl, 190 mM glycine, 20% methanol) for 3 h. The membrane was immersed in blocking buffer (3% BSA, 20 mM TrisHCl, 0·9% NaCl, 0·1% Tween 20, pH 7·2) at 4 °C overnight followed by incubation with polyclonal mouse anti-VP28 IgG for 3 h. Subsequently, the membrane was incubated in HRP-conjugated goat anti-mouse IgG (Sigma) for 1 h and detected with substrate solution (4-chloro-1-naphthol, Sigma).
WSSV and immuno-electron microscopy (IEM).
Infected tissue from P. monodon shrimp was homogenized and centrifuged as above and the supernatant was injected (1:100 dilution in 0·9% NaCl) intramuscularly into healthy Cambarus clarkii crayfish from Singapore in the lateral area of the fourth abdominal segment. Four days later, haemolymph, freshly extracted from infected crayfish, was layered on the top of a 1040% (w/v) continuous sodium bromide gradient and centrifuged at 110000 g using an RP40-T rotor in a Prespin ultracentrifuge (Shimadzu Model MSE-75) for 2 h at 4 °C. Virus bands were collected by side puncture, diluted 1:10 in TNE buffer (50 mM TrisHCl, 100 mM NaCl and 1 mM EDTA, pH 7·4) and pelleted at 119000 g for 1 h at 4 °C. The resulting pellets were resuspended in TNE as intact WSSV virions (Huang et al., 2001 ). Some intact WSSV particles were treated with 0·51·0% Triton X-100 for 30 min at room temperature and then centrifuged at 119000 g using an SW41-Ti rotor (Beckman). The pellet was resuspended in 0·1xTNE buffer and centrifuged at 119000 g. After several repeats to remove the Triton X-100 completely, the resulting WSSV nucleocapsids were resuspended in TNE. Virus samples were examined under transmission electron microscope (JEOL 100 cxII) for purity.
The purified WSSV virion suspension and nucleocapsids were mounted on carbon-coated nickel grids (200 mesh) and incubated for 1 h at room temperature. After washing with PBS, the grids were blocked in 3% BSA for 1 h. The grids were rinsed with PBS and incubated in anti-VP28 IgG or anti-grouper iridovirus IgG (kindly provided by Qiwei Qin, Tropical Marine Science Institute, National University of Singapore) as a negative control for 1 h at room temperature, followed by washing with PBS. Next, 15-nm-gold-labelled anti-mouse goat IgG (Sigma) was added to the grids and incubated for 1 h at room temperature. After negative staining with 2% phosphotungstic acid, the specimens were examined with a transmission electron microscope.
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Results |
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Expression and purification of the vp28 gene product
The vp28 gene was cloned into the pBV220 vector containing the promoter PRPL. The recombinant plasmid was named pBV28. pBV28 and pBV220 were transformed into DH5. After induction at 42 °C, induced and non-induced pBV28DH5
and pBV220DH5
were analysed by SDSPAGE (Fig. 2
). A band corresponding to the VP28 protein was observed in the induced pBV28DH5
(Fig. 2
, lane 4). No protein was found at the same positions in the non-induced pBV28DH5
(Fig. 2
, lane 2) or in induced and non-induced pBV220 cells (Fig. 2
, lanes 3 and 5). This showed that the vp28 gene was expressed. The VP28 protein was extracted and purified from the inclusion bodies by SDSPAGE (Fig. 2
, lane 6).
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Transmission electron microscopy studies
Crayfish (Cambarus clarkii) were infected with WSSV by intramuscular injection of a virus preparation from P. monodon shrimp. Four days after infection, the virus was isolated and purified from the haemolymph. As a negative control, haemolymph was also taken from healthy crayfish. These preparations were observed under transmission electron microscopy for the presence and purity of WSSV virions. No virus particles were found in the healthy crayfish samples but, in the infected samples, numerous enveloped and rod-shaped virions were found (Fig. 4a).
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Discussion |
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Temporal analysis revealed that vp28 might be a late gene of WSSV, but the conserved motif (ATAAG) present in late genes of insect baculoviruses could not be found in the DNA sequence of the vp28 gene. This suggests a difference between WSSV and baculoviruses. Analysis showed that the 5' non-coding region of the gene was longer than the corresponding region of genes from baculoviruses and WSSV. A minicistron was found in the leading sequence of vp28 (Fig. 1), which might affect translation of the vp28 gene. The significance of this observation is presently unknown.
Previously, anti-VP28 IgG served successfully as a specific diagnostic reagent in the detection of WSSV in shrimp by ELISA (Zhang et al., 2001b ). The IEM result confirmed the immunoassay detection. Moreover, because the VP28 protein is one of the major envelope proteins of WSSV, it could be used for further analysis, in particular, to study its relatedness to structural proteins of other viruses, including baculoviruses. It could also be used to examine the infection process of WSSV in shrimp by hybridization in situ and to study whether the VP28 protein has an effect on WSSV infection. Such studies would be helpful to reveal the function of the gene.
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Acknowledgments |
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Footnotes |
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References |
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Received 22 October 2001;
accepted 14 January 2002.