Cloning, Expression and Purification of Recombinant Envelope Protein VP36A of White Spot Syndrome Virus
White spot syndrome is a viral infection of penaeid shrimps which is highly
infectious and lethal, terminating shrimps rapidly. Outbreaks of this disease
can wipe out entire shrimp populations within a few days. To reduce the mortality
of shrimps and increase the production of marine industry, the research focused
on the changes on the viruss proteins. The research is aimed at the molecular
reconstruction of one of the important proteins from the White Spot Syndrome
Virus (WSSV). By exploring the unique DNA sequences as well as the corresponding
protein, the initial target shall be achieved soon. According to the published
gene sequence of White Spot Syndrome Virus (WSSV), a pair of specific primers
were designed. Using an isolate of WSSV collected in Ganyu, Jiangsu Province,
China, as template, a gene fragment of VP36A was amplified by Polymerase Chain
Reaction (PCR). The PCR product was firstly TA-cloned into a pMD-18T vector
and then inserted into a pET-28a plasmid to create a recombinant plasmid and
finally transformed into the host strain BL21. The recombinant protein was expressed
in the form of inclusion bodies with 1.0 mmol L-1 Isopropyl-β-D-thiogalactopyranoside(IPTG)
at 37°C. According to the results of an Sodium Dodecyl Sulfate Polyacrylamide
Gel Electrophoresis (SDS-PAGE), the recombinant protein had the expected size
of 36 kDa. The purified recombinant protein was tested in a Western blot to
confirm that the target protein had been successfully expressed. This showed
that the special DNA in WSSV can be replicated and the protein can be expressed
by a very cheap and sufficient way in a very limiting time, which will be utilized
in the further experiments related to the functional identification of protein
Received: August 13, 2012;
Accepted: November 03, 2012;
Published: November 19, 2012
In 1992, reports from Taiwan showed the first epidemic outbreak due to White
Spot Syndrome Virus (WSSV) (Chen, 1995), followed by
losses reported from China in 1993 (Huang et al.,
1995), where it led to significant losses of the shrimp farming industry.
From then on, outbreaks expanded to territorial waters in Japan and Korea also
in 1993 and then shrimp farming in East and South Asia were severely affected.
In late 1995, the disease was reported in the U.S. and 1998 in Central and South
America, followed by European countries. So far, the disease has been present
in shrimp-growing regions all over the world in addition to Australia. White
spot syndrome virus can severely infect a wide range of hosts, such as farmed
and wild shrimp and wild crabs and also lead to a high level of mortality up
to 100% (Lo et al., 1996). Thus, the disease
has caused a virtual collapse in global shrimp fishery as well as of shrimp
farms and hatcheries (Flegel, 1997; Tsai
and Huang, 1995), which can also threaten the balance of entire marine ecosystems
(Jory, 1999). Transmission of the virus occurs mainly
through oral ingestion and water-borne routes in farms (horizontal transmission)
(Kanchanaphum et al., 1998) or from infected
mother prawns in the case of shrimp hatcheries (vertical transmission) (Huang
and Song, 1999).
WSSV is the only member of the family Nimaviridae that discovered so far. It
is symbolized as a large, rod-shaped, double-stranded DNA virus (Nadala
Jr. et al., 1998). It has an outer lipid bilayer membrane envelope
and cannot form an inclusion body during its infectious period (Mayo,
2002). WSSV genomic DNA was purified from infected prawns in 1997 and was
fully sequenced in 2001. Nearly 180 Open Reading Frames (ORFs) of 50 or more
amino acids have been found within the complete virus genome (Yang
et al., 1997; Van Hulten et al., 2001;
Yang et al., 2001). Recently, studies of the
pathogenicity of WSSV are mostly focusing on viral genes such as the function
of viral proteins. The transcription and translation of WSSV-VP36A are conducted
by genes in the ORF WSSV134, encoding a total of 297 amino acids. The theoretical
molecular weight of VP36A is 33.1 kDa, while a dimensional electrophoresis profile
revealed a weight of 36 kDa. VP36A is one of the membrane proteins that functions
in the early phase of infection and the usage of its antibody can significantly
delay the initial infection in Procambarus shrimps (Li
et al., 2006a). Envelope proteins play an important role during the
processes of adsorption, invasion, packaging and release of enveloped viruses
like WSSV (Chiu and Chang, 2002; Hsiao
et al., 1999; Lin et al., 2000; Chazal
and Gerlier, 2003). VP36A also contains a Arginine-Glycine-Aspartic acid
(RGD) locus (Tsai et al., 2004), was justified
by analyzing the function of VP36A-related factors during the viral infectious
progress. In addition, the recombined envelope protein will provide advantageous
experimental material with high purity and quantity, which would be needed in
further research about the role of VP36A in initial infection (Li
et al., 2006b).
In the present study, the T7-RNA polymerase based pET system was employed to
express the viral envelope protein in Escherichia coli. The ORF-WSSV134
fragment was cloned into a pET-28a (+) vector, in which the genes of interest
were cloned downstream to the E. coli thioredoxin (TRX) chimera in order
to increase the solubility of the target protein and tagged with an N-terminal
His tag to allow easy purification (Rosenberg et al.,
1996). The recombinant protein was then purified with a purification column.
In addition, SDS-PAGE and Western blot were applied in order to verify the obtained
protein. Therefore, a foundation is established to discern the functional mechanisms
between WSSV envelope proteins, such as VP36A and shrimp cells in future studies.
MATERIALS AND METHODS
This research was initiated in 2011. The WSSV strain used in the experiments
was obtained from Ganyu, Jiangsu Province in 2011 and kept in the Microbiology
and Immunology Laboratory, College of Veterinary Medicine, Nanjing Agricultural
University, China. E. coli strain DH5-a competent cells and BL21 (DE3)
competent cells were purchased from Tiangen. Restriction endonucleases EcoRI
and XholI, pMD18-T Simple Vector and T4 DNA ligase were obtained from Takara.
T7 polymerase expression vector pET-28a (+) was stored in the laboratory. A
PCR-product purification kit, DNA purification kit and agarose gel DNA purification
kit were purchased from Geneaid, while a 2xTaqMaster Mix was purchased
from Dongsheng Biotech. A protein purification kit (HisTrapTM HP)
was purchased from GE Healthcare, HRP secondary antibodies were from Boster
Biotech. 3,3,5,5-Tetramethylbenzidine (TMB) was from Tiangen. Other
reagents were imported or domestic analytical reagents.
DNA purification: Gills of contagious shrimps stored at 40°C
were ground in Phosphate Buffer Sline (PBS). The liquid was frozen at 20°C
for 2 h and thoroughly thawed at room temperature. This freeze-thaw move was
repeated twice. Afterwards, the grinding liquid was centrifuged in a 2 mL EP
tube for 10 min at 13,000 rpm (Sambrook and Russell, 2001).
Following the instructions of the Viral Nucleic Acid Extraction Kit II manual
(Geneaid), total DNA was extracted from the supernatant of the centrifuged liquid.
Cloning of the VP36A gene: Primers were designed by software Primer
5.0 according to the cDNA sequences of WSSV ORF 134 within white spot syndrome
virus Thailand strain (GenBank accession No.: AF369029) and were synthesized
by Invitrogen. VP36A was amplified by using the following primers:
Forward: 5'-GGAATTCGCATTACAGGAAAAGGATAT- 3'
Containing an EcoRI (forward, underlined) and an XholI site (reverse, underlined),
respectively. The following PCR protocol was used: pre-denaturation at 94°C
for 5 min; denaturation at 94°C for 30 sec annealing at 51°C for 60
sec, extension at 72°C for 30 sec, 30 cycles, followed by an extension at
72°C for 10 min. The PCR product was verified by 1% Agarose Gel Electrophoresis
(AGE) and then extracted by a Gel/PCR DNA Fragments Extraction kit (Geneaid)
following the manuals instructions.
Construction of recombined plasmid pET- VP36A: After purification from
the agarose gel, the amplified fragment was ligated into a pMD18-T vector to
produce pMD-VP36A. E. coli DH5-a was transformed with the ligative mixture
and grown at 37°C after plating on an LB agar medium containing 100 μg
mL-1 ampicillin. Positive clones were selected and identified with
PCR and restriction enzyme digestion. DNA sequences were verified by Invitrogen
Biotechnology. The TA-cloned pMD-VP36A was digested with EcoRI/XholI and followed
by ligation with EcoRI/Xhol-digested pET-28a (+) to produce pET-VP36A. The expression
vector was transformed into E. coli BL21 (DE3). The positive clones were
identified by PCR and restriction enzyme digestion and their DNA sequences were
verified by Invitrogen Biotech.
Expression of VP36A: The E. coli BL21(DE3) harboring pET-P36A
were inoculated at a proportion of 1:100 into LB containing 100 μg mL-1
kanamycin (1%), then incubated at 37°C with shaking overnight. The overnight
culture was inoculated into LB with 100 μg mL-1 kanamycin (1%)
for continuous amplification until the OD600 value reached about
0.6 (which took less than 3 h). After instantly adding IPTG at a final concentration
of 1 mmol L-1, the culture was incubated for another 4 h to induce
the expression of the target protein. Before and during the period of inducement,
1 mL of the bacterium culture was collected every hour. After high speed centrifugation
of the collected cultured liquid, an SDS-PAGE was performed to identify the
optimum time point for the expression of the target protein. The empty vector
pET-28a (+) cultured at the same conditions was treated as control.
Examination of the expression site in bacteria: To obtain sufficient
quantity of bacteria for further analysis, more than 400 mL of LB with 1% 100
μg mL-1 kanamycin was used in culturing E. coli BL21
(DE3) that carried pET-P36A at the optimum induced expression. The cultured
liquid was collected for high-speed centrifugation at 10,000 g for 10 min at
4°C, with the supernatant being discarded while the sediment was washed
three times with sterile PBS. The bacteria were resuspended in the same PBS
and then sonicated (at 200 W for 4 sec work time and 8 sec interval) until the
resuspension became more transparent. The supernatant and sediment were collected
after a centrifugation of sonicated resuspension at 4°C for 20 min at 10,000
g. After an SDS-PAGE analysis of both supernatant and sediment, the VP36A protein
was shown to be expressed either in the form of solution or inclusion bodies.
Purification inclusion bodies and renaturation of recombined protein:
After verifying the expression of VP36A in the inclusion bodies, the sediment
was washed by inclusion body purgation buffer and centrifuged at 10,000 g at
4°C for 10 min. The sediment was resuspended with binding buffer in a 30°C
water bath for 1 h and the resuspension was centrifuged at 10,000 g at 4°C
for 10 min. The supernatant was filtered by a 0.45 μm membrane and then
purified by a His trap affinity column (GE Healthcare). The purified protein
was dialyzed by PBST containing urea of a concentration subsequently decreasing
from 6 to 4 to 2 to 0 M. After a thorough dialysis the protein solution was
collected and stored at 4°C.
Verification by Western blot analysis: The renatured protein was analyzed
by SDS-PAGE to isolate the fused protein. The gel was then subjected to Western
blotting; proteins were transferred onto nitrocellulose membranes using a semi-dry
electroblot apparatus (Bio-Rad) in electroblotting buffer (25 mM Tris, 190 mM
glycine, 20% methanol) at a constant voltage of 100 V for 2 h. The membrane
was immersed in blocking buffer (PBST containing 5% (w/v) skimmed milk) at 4°C
overnight, followed by incubation with polyclonal mouse anti (His)-HRP
(1:3000) (Invitrogen) for 2.5 h in a shaker (50 rpm, 37°C). The membrane
was washed three times with PBST for 5 min each at 37°C with gentle rotation.
The HRP secondary antibody was then added and incubated at 37°C in a shaker
for 1 h. The membrane was again washed as described above. Subsequently, detection
was performed with chromogenic substrate TMB (4,4'-bi-2,6-xylidine, Tiangen)
Cloning of the WSSV-VP36A gene: Using the DNA of WSSV as template, the
products of PCR were separated by 1.5% agarose gel electrophoresis. PCR amplification
of the WSSV-VP36A gene yielded an 891 bp DNA fragment with the expected sequence
Construction and identification of recombinant vectors: The fragments
of WSSV-VP36A and pMD18-T vector were ligated to a plasmid pMD-VP36A, which
was identified by PCR (the same primers used in amplifying the template). The
results showed a band between 750 and 1000 bp as expected (Fig.
2). The plasmids pET-28a (+) and pMD-VP36A were digested with EcoRI and
XholI, ligated by T4 DNA ligase and then transformed by E. coli DH5-a.
The recombinants were selected and identified by PCR and double digestion.
||PCR products of VP36A gene. M: DNA marker, Lane 1: PCR products
of gene VP36A (891 bp)
||Identification of recombinant plasmid pMD-VP36A by PCR, M:
DNA marker, Lane 1: pMD-VP36A plasmid used as template (891 bp), Lane 2:
Blank control sample
The expected bands were verified through AGE (Fig. 3, 4).
The sequencing results revealed that neither pre-termination nor frame shift
had occurred and that the sequence matched the complete genome in GenBank, which
indicates the successful recombined plasmid construction.
||Identification of recombinant plasmid pET-VP36A by PCR, M:
DNA marker, Lane 1: pET-VP36A plasmid used as template (891 bp), Lane 2:
Blank control sample
||Identification of recombinant plasmid pET-VP36A by restriction
enzyme, M: DNA marker, Lane 1: pET-VP36A digested with EcoRI and XholI (4.9+0.89
Expression of recombined plasmid and renaturation of inclusion bodies:
The expression of recombined plasmid was detected after inducing the pET-VP36A
that transformed into competent cell E. coli BL21 (DE3) by IPTG (final
concentration 1 mM L-1) for 6 h.
||Analysis of recombinant protein pET-VP36A by SDS-PAGE, M:
Molecular weight protein marker, Lane 1: pET-VP36A induced by IPTG for 6
h, Lane 2: pET-28a (+) induced by IPTG for 6 h, Lane 3: pET-28a (+) before
||Analysis of recombinant protein pET-VP36A induced at different
times by SDS-PAGE, M: Molecular weight protein marker, Lane 1: pET-VP36A
before induction, Lanes 2-7: pET-VP36A induced by IPTG for 1, 2, 3, 4, 5,
6 h, respectively
The weight of the expressed protein was between 35.0 and 45.2 kDa, thus, matching
the expected weight of around 36.0 kDa (Fig. 5, 6).
Both the supernatant and sediment of centrifuged sonicated bacteria were analyzed
in an SDS-PAGE assay. The results showed that the fused protein was virtually
expressed in the inclusion body and was almost absent in the supernatant (Fig.
7). After being purified by urea-containing PBST, the WSSV envelope protein
VP36A was analyzed by SDS-PAGE. The resulting band of around 36 kDa size indicated
that the target protein was obtained (Fig. 8).
||Analysis bacteria lysates of recombinant protein by SDS-PAGE,
M: Molecular weight protein marker, Lane 1: Precipitation of bacteria with
recombinant pET-VP36A after ultrasonic treatment, Lane 2: Supernatant of
bacteria with recombinant pET-VP36A following ultrasonic treatment
||Analysis of purified recombinant protein pET-VP36A by SDS-PAGE,
M: Molecular weight protein marker, Lane 1: Purified recombinant protein
Western blot of fused protein: After renaturation, the recombined protein
was analyzed by Western blot to identify the expression level of the fused protein.
The result suggested a high level of protein expression as well as the identification
of WSSV-VP36A (Fig. 9).
|| Western blot analysis, M: Molecular weight protein marker,
Lane 1: Western blot analysis
Previous studies of WSSV have mainly focused on the tissues and range of viral
hosts as well as on DNA replication. Van Hulten et al.
(2001) and Yang et al. (1997) completed
the sequencing of the complete genome of WSSV (Van Hulten
et al., 2001; Yang et al., 2001).
In recent years, the focus of WSSV-related research has shifted to infection
mechanisms and pathogenesis, including viral proteomics (Durand
et al., 1997; Wang et al., 2000).
More than 40 kinds of proteins have been reported (Escobedo-Bonilla
et al., 2008), including structural proteins such as VP28, VP26 and
VP35 that are necessary during the formation of a morphologically mature contagious
viral particle. Other identified proteins include non-structural proteins like
thymidine kinase, thymidine kinase and ribonucleotide reductase, which play
a regulatory role during viral replication.
In this study, the non-structural protein VP36A was studied and the pET-28a
(+) plasmid of the series of pET-TRX fused expression plasmids was used in the
experiment. The Trx tag, which could enhance the solubility and stability of
exogenous proteins expressed in E. coli, has been introduced into pET-TRX
plasmids as a fusion partner. Attached to the plasmid was the protein purification
tag His tag that is conducive to the purification of recombinant proteins and
contains a T7 RNA polymerase-binding site. Recurring to the T7 RNA polymerase
on host bacteria strain E. coli BL21 and the plasmid, the target gene
can be transcribed and expressed. This method of transcription has the advantage
of being easy to control and selective (Xu, 2003). Furthermore,
fused expression could reduce the destabilization of protein products in E.
coli and improve the expression levels of target genes and the antigenicity
of expressed proteins (Novagen, 2010). As a result, the
extraneous gene was expressed efficiently and steadily during the experiment.
To design the PCR primers, both the restriction sites and protective bases
should be taken into consideration. EcoRI and XholI were chosen because they
can be easily obtained and are relatively cheap. They also both possess an optimal
digestion temperature of around 37°C, so, that synchronous digestion can
be realized and efficiency can be improved. In addition, protective bases added
to the 5-end (G added for EcoRI and GGC for XholI) can significantly influence
the binding of endonucleases to DNA duplexes and enzyme digestion of DNA. Throughout
the experiment, the designed primers acted with excellent specificity that yielded
large quantities of PCR amplification products. The SDS-PAGE also showed a protein
of expected size. The expression of the protein in the inclusion body rather
than in the supernatant of the recombinant plasmid after IPTG inducement suggested
high yields of protein products, which was also verified by Western blot.
WSSV is mainly spread between shrimps by vertical infection through the intake
of virus hosts. This characteristic of viral spread has been related to the
capsule, as the nucleocapsid of non-capsule WSSV is not infectious. This was
demonstrated by Li et al. (2006b) who performed
a neutralization test with VP36A-specific antibodies (Li
et al., 2006b).
The results showed that the antibody could delay the initial WSSV infection
of Procambarus clarkii, although the mortality of the experimental animals
was still 100% 11 days post injection. Although, WSSV-VP36A is a non-structural
protein, it still actively functions during infection, which is probably realized
by its reciprocity with other proteins. In this study, the target protein was
successfully expressed and purified, which should enable further in-depth research
on protein interactions, including structural and nonstructural proteins as
well as envelope and nucleocapsid proteins.
We are grateful to the Laboratory of Microbiology in College of Veterinary
Medicine, Nanjing Agricultural University for providing the experimental equipments
in need. This work was supported by grants from the Special Fund for Agro-scientific
Research in the Public Interest (No. 201103034) and the Priority Academic Program
Development of Jiangsu Higher Education Institutions.
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