Enzootic bovine leukosis (EBL) is an infectious lymphoproliferative
disease of cattle, caused by the retrovirus Bovine leukemia virus
(BLV) (Bicka et al., 2001).
Like other complex reteroviruses the BLV genome contains the gag,
pol and env structural genes and regulatory genes (Sagata et
al., 1985; Bicka et al., 2001; De Giusppe et al., 2004;
Van den Heuvel et al., 2003, 2005). Most of the structural protein
of BLV are immunogenic but the naturally infected animals develop antibodies
to env encoded glycoproteins gp51 and gp30 as well as to gag
encoded proteins p 24 and p15 (Bicka et al., 2001; Deshayes et
Since the presence of antibodies to BLV is a constant and early feature
of BLV infection, serological examination of cattle sera is the best method
for detection of infected animals. Most commonly used serological tests
are the Agar Gel Immuno Diffusion (AGID) and the Enzyme Linked Immuno
Sorbent Assay (ELISA). However, variable results have been obtained by
the two methods using the p24 and gp51 antigen. The disparities were often
the result of differences in specificity and sensitivity of the tests
used. AGID is less sensitive and is not useful for detection of antibodies
to p24 antigen. ELISA has been shown to detect both anti-gp51 and anti-p24
antibody with equal sensitivity but this method is prone to generate nonspecific
reactions. While in the ELISA the non specific reactions are difficult
to distinguish from specific ones, western blot analysis allows precise
resolution of the two reactions. So far limited studies have been performed
to confirm the usefulness of the immunoblotting assay in the routine serological
detection of BLV antibodies (Bicka et al., 2001; Kittelberger et
al., 1999; Simard et al., 2000). Recently, the recombinant
viral proteins have been found to be widely applicable in immunoassays
for detection of specific antibodies. In particular, the use of the recombinant
proteins synthesized in E. coli has been well documented in retroviral
serology (Bicka et al., 2001; De Giusppe et al., 2004; Van
den Heuvel et al., 2003, 2005).
In this study, the gag gene of BLV which encodes protein p24 from
Iranian isolated virus was cloned and expressed as 6xHis-p24 fusion protein
in E. coli. The preparing recombinant protein will be applied in
near future for designing Dot-ELISA kit for detection of antibodies against
p24 antigen of Bovine leukemia virus in infected and vaccinated
MATERIALS AND METHODS
This study was conducted from April 2007 to February 2008 in Faculty
of Veterinary Medicine, Islamic Azad University of Shahrekord.
Sample, plasmids and bacterial strains: The extracted DNA from
buffy coat of one of the BLV infected cows which had previously shown
positive molecular and serological results based on ELISA and PCR was
selected to be cloned (Momtaz and Hemmatzadeh, 2003). Plasmid pTZ57R/T
(Ins T/A clone PCR Cloning kit, Fermentas) and E. coli strain JM107
(Fermentas) were used for initial cloning, sequencing and maintenance
of DNA fragment. For recombinant protein production, a prokaryotic expression
vector pET-28(a) (Novagen) was used. This vector can express a fusion
protein with a six histidin tag (6xHis), a thrombin recognition site and
a T7 tag at the N-terminus. The recombinant pET-28(a) (pET-28-gag) is
transformed into E. coli BL21 (DE3) (Fermentas) as host strain.
The required antibiotics were added to LB media according to the reference
recommendation (Sambrook et al.,2001).
Primers design: Perimers were designed according to the published
sequence for gag gene of BLV (accession No.: M10987) (Rice et
al., 1985). The forward primer, gag F: 5-GGC AGA TCT
TGG GAA ATT CCC CCT CCT ATA-3 contain BglÃ? site. Reverse primer,
gag R:5-CCG CTG GAG TAG TTT TTT GAT TTG AGG GTT GG-3 contain
recognition site for XhoI . The restriction enzyme sites (underlined)
were added to the primers for subsequent cloning procedure.
Gene amplification of gag ( encoding the p24 protein):
PCR was performed in a 50 Î¼L total volume containing 1 Î¼g of
template DNA, 1.5 Î¼M of each primer, 1.5 mM MgCl2 , 150
Î¼M dNTP, 1x PCR buffer and 1.5 unit of Taq DNA polymerase (Sigma).
The following conditions were used for amplification: initial denaturation
at 95Â°C for 4 min, followed by 30 cycles of denaturation at 94Â°C
for 1 min, annealing at 56Â°C for 1 min and extension at 72Â°C for
50 sec. The program followed by a final extension at 72Â°C for 6 min.
The PCR product was analyzed by electrophoresis in 1% agarose gel in 1X
TBE buffer and visualized by ethidium bromide staining on UV transilluminator.
The PCR product was purified by High pure PCR product purification kit
(Roche applied science) according to the manufacturer recommendation.
Cloning of gag gene: The PCR product was digested with
BglII and XhoI and ligated to pTZ57R/T and pET-28(a), which
were digested by the same restriction enzymes, using T4 DNA ligase (Invitrogen)
at 14Â°C over night.
E. coli JM107 and E. coli BL21(DE3) competent cells were
prepared by calcium chloride method and were used for transformation of
pTZ57R/T-p24 and pET-28(a)-p24 vectors, respectively. The transformed
bacteria were selected by screening the colonies on LB media containing
antibiotic. The suspected colony was further analyzed by restriction enzyme
digestion and PCR (Sambrook et al., 2001).
Expression and purification of recombinant p24 protein: E.
coli BL21 (DE3) was transformed with pET-28 (a)-p24 and grown in
LB broth supplemented with kanamycin (50 mg mL-1) at 37Â°C
with agitation in order to optimize the expression condition, different
concentrations of IPTG (0.5, 0.8, 1 and 1.5 mM) at different bacterial
growth rates (OD600 = 0.5, 0.7 and 1) were tested for 3 h and
analyzed on 17% SDS-PAGE (Laemmili, 1970). The expressed protein was purified
using Ni-NTA column (Qiagene) according to manufacture instructions. Quantity
of the purified recombinant p24 protein was analyzed by Bradford methods
and subsequently it,s quality was assayed by SDS-PAGE 17% (2.5
Î¼g well-1). In order to analyze the cross-reaction between
fused segment of p24 protein with infected sera, an E. coli BL21(DE3)
containing pET-28(a) a vector was induced by IPTG
Immunoblot analysis: For western blot analysis, 0.5 Î¼g of
purified recombinant p24 protein was used per well. As a negative control,
the bacterial lysate from induced E. coli BL21 (DE3) contain pET-28
(a) vector was analyzed by western blot. The gel was blotted on to Polyvinylidine
difluoride (PVDF Membrane, Roche Diagnostics GmbH) membrane using transfer
buffer containing 25 mM Tris (pH = 8.3), 192 mM glycine and 20% methanol
at 55v for 1 h at 4Â°C. The blotted membrane was blocked with 3% (w/v)
BSA in TBST buffer (0.5 M NaCl, 0.02 M Tris pH = 8.5, 0.05% Tween 20)
for 1 h at room temperature (RT). Membrane was incubated for 2 h at 37Â°C
with BLV-infected cow serum, diluted 1:25, respectively. Negative serum
from disinfected cow was used as control. After reaction the primary antibody,
the blotted membranes were washed three times with TBST and incubated
with peroxidase conjugated anti-bovine IgG (Sigma) at a 1:2500 dilution
in TBST. The blots were then washed three times with TBST and reaction
were developed by diamino benzidine (DAB) solution (Sigma).
RESULTS AND DISCUSSION
The proviral DNA of BLV virus from buffy coat of one of the BLV infected
cows in Iran was prepared and used as template for amplification and cloning
of the gag gene encoding the protein p24. The amplified fragment
had the expected size of 1180 bp comparing to 1kb DNA ladder (Fermentas)
||p24 gene amplification by PCR. Lane 1: Molecular weigh
marker 1 kb DNA ladder. ; Lane 2: Negative control; Lane 3: Amplified
||Restriction enzyme analysis of recombinant pTZ57R/T
and pET-28(a) plasmids Lane 1: Molecular weight marker 1 kb DNA ladder;
Lane 2: Digestion of recombinant pTZ57-R/T plasmid; Lane 3: Digestion
of recombinant pET-28(a) plasmid
The purified PCR product was cloned in pTZ57R/T vector and digestion
with BglII and XhoI enzymes. Figure 2 shows recombinant
plasmids after digestion.
The recombinant plasmid (pTZ57R/T-p24) was sequenced by specific primers
and Sanger sequencing method (Macrogen, Korea). The sequencing result
was confirmed by comparing with databases and using basic local alignment
search tool (BLAST) software (data not shown).
Expression of pET-28(a)-p24 in E. coli BL21(DE3) induced and the
expressed protein was purified by Ni-NTA column (Fig. 3).
The result showed that the best conditions for recombinant p24 protein
expression can be achieved when 1 mM of IPTG and OD600 = 0.7
for 3 h was used.
To determine the reactivity of recombinant protein p24, the purified
recombinant protein was assayed by western blotting method. The five infected
cattle serum (which had previously shown positive serological result based
on ELISA and AGID) were used. A negative serum from disinfected cattle
used as a control. Figure 4 shows the specific interaction between positive
sera and purified recombinant p24 protein. There was no reaction between
the expressed pET-28(a) in E. coli BL21(DE3) and BLV infected sera
(Lane 10 in Fig. 4).
Recognition and study of retrovirus infections are critical in different
aspects. The occurrence of mutations consequently genotypic and phenotypic
diversity is frequent in retroviruses because of their natural characteristics
which is resulted from reverse transcription from their genomes. This
feature is that makes the diagnostic value of most of the experimental
tests uncertain. There are lots of studies that notice to the special
figures and numbers as sensitivity and special quality in diagnostic tests.
The repeat of these tests by other researchers usually had different results
and it is reported that the reason can be found in genetic diversity of
these viruses (Bunqer et al., 1994; Grover and Guillemain, 1992;
Bicka et al., 2001).
Anyway, one of the main goals of this examination which was tracing of
the coding gene of p24 protein of BLV in the infected samples to this
virus, achieved for the first time in Iran and the presence of the corresponded
gene was confirmed with the help of sequencing of the fragment.
With respect to this point that primers applied for identification of
the gag gene in this study involve the main part of encoding frame
of the gene thus, from the beginning the primers were designed for cloning
and gene expression of gag in the way that the amplified fragment
could be able to be cloned in different vectors such as cloning and expressing
The second goal of this study was cloning of the mentioned gene in each
of the cloning vector (pTZ57R/T vector) and expressing vector (pET-28(a)).
The cloning of this gene in the cloning vector after sequencing and comparing
resulted sequences to other known sequences of the gag gene available
in Genebank indicates the success in cloning the gene into the related
vector. Such vector have the capacity to be proliferated in the competent
bacterial cells, to be digested because of several sites for restriction
enzymes, to be extracted and to be inserted in the expressing vectors.
The last finding was derived by cloning the coding gene of p24 protein
of BLV in the expressing vector of pET-28(a) for the first time in Iran
and the presence of expressing protein was confirmed through SDS-PAGE
and immunoblotting system.
||Expression of recombinant p24 protein and its purification
Lane 1: Protein marker; Lane 2: pET-28(a)-p24 before induction; Lane
3: pET-28(a)-p24 after induction; Lane 4: Purified p24 recombinant
||Western blot analysis against recombinant p24 protein
by BLV-infected sera Line 1: Protein marker; Lane 2: Western blotting
pET-28(a)-p24 before induction; Lane 3: Western blotting pET-28(a)-p24
after induction; Lane 4: Western blotting by negative control serum;
Lane 5-9: Western blotting by infected sera; Lane 10: Western blotting
reaction between the expressed pET-28(a) and positive serum
Many researchers show that p24 and gp51 proteins which are the products
of the gag and env genes of BLV, are strong antigens and
usually the first serological responses will be against these antigens.
As the gp51 antigen is a very suitable candidate for manufacturing the
recombinant vaccines because of its glycoprotein structure and positioning
in the membrane of virus. So, Altaner et al. (1991) and Kono et
al. (1986 ) have started to design diagnostic methods based on tracing
of antibody for p24 antigen and currently several institutions have designed
and supplied ELISA and immunoblot methods specific to p24.
With respect to the remarkable frequency of infection to BLV in Iran
and the necessity of controlling it through vaccination with recombinant
vaccines of gp 51, manufacturing and applying the recombinant p24 protein
are vital goals in recognition and distinction between infection and responses
caused by vaccine. As the amplified fragment by PCR involves all the domains
of p24 and be placed in the expressing frame based on first designs of
primers and has successfully been cloned in the expressing vector of pET-28(a)
so, the expression of this gene and the preparing recombinant protein
will be applied in near future for designing Dot-ELISA kit for detection
of antibodies against p24 antigen of bovine leukemia virus in infected
and vaccinated cows.
We thank Dr. A. Sharifzadeh, Dr. S. Nekoie, Dr. M. Rohani and Dr. S.
Nejat for their cooperation. This study was supported by Grant No. 19594
from the Islamic Azad University of Shahr-e-kord Branch in Iran.