Open Journal of Veterinary Medicine, 2013, 3, 309-313
Published Online November 2013 (
Open Access OJVM
Expression of Nucleoprotein Gene of CTN Strain Rabies
Virus from China in E. coli with Antigenicity
Wangbin Cao, Ying He*, Qiumei Shi, Cairan Yang, Yanying Zhang
Department of Animal Science, Hebei Key Laboratory of Preventive Veterinary, Hebei Normal
University of Science & Technology, Qinhuangdao, China
Email: *,,,,
Received August 28, 2013; revised September 28, 2013; accepted October 4, 2013
Copyright © 2013 Wangbin Cao et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
The nucleoprotein (NP) gene of rabies CTN strain isolated from China was recombined into pMal-c2x. The antigenicity
of the recombined MBP-NP fusion proteins was examined by western blotting and by enzyme linked immunosorbent
assay (ELISA). The results demonstrated that the recombined protein possesses predominant antigenicity.
Keywords: Rabies Virus; Nucleoprotein; CTN Strain; Antigencity; Expression
1. Introduction
Rabies in humans remains a serious public health prob-
lem in many countries, where rabies virus transmission
among wildlife or domestic animal populations continues
to threaten humans exposed to rabic animals [1]. Dogs
play an important role in rabies occurrence; human rabies
transmitted by rabid dogs accounted for 83% of regis-
tered cases in China from 1980 to 1994 [2].
Rabies is a fatal viral zoonosis caused by viruses of the
Lyssavirus genus of the Rhabdoviridae family. The ra-
bies virus is a nonsegmented negative-strand RNA virus
encoding five monocistronic mRNAs encoding the nu-
cleoprotein (N), phosphoprotein (P), matrix protein (M),
the transmembrane glycoprotein (G), and the viral RNA-
dependent RNA polymerase (L) [3,4]. Nucleoprotein
forms the ribonucleoprotein which completely and
tightly encapsulates the genomic and antigenomic RNA
of the rabies virus. The N gene, which consists of 1353
nucleotides and encodes 450 amino acids, is well con-
served among lyssaviruses [5]. Moreover, nucleoprotein
is an important antigen protein with conserved epitopes,
which can activate cell immunity in the body [6-8]. So
gene products can be a feasible alternative to develop
genetically engineering vaccines and diagnostic re-
In this study, the nucleoprotein gene of a CTN strain
rabies virus was expressed. This work not only provides
a useful understanding of the nucleoprotein of the rabies
virus, but also opens up new possibilities for the devel-
opment of novel research and clinical applications. The
recombinant fusion protein with antigenicity can be used
for a safe, stable and easy method for detecting an anti-
rabies antibody in dogs and it can be used to understand
the rabies epizootic in wild and domestic animals.
2. Materials and Methods
2.1. Plasmids, Cell and Serum Samples
The pGEM-T/NP from CTN strain Rabies virus was
stored in our lab. Bacterial cell JM109 was cultured in
LB medium, and plasmids pGEM-T and pMal-c2x were
purchased from Promega and NEB. Polyclonal an-
ti-serum was obtained from the biology lab of the Insti-
tute of Lanzhou in China. The serum samples were taken
from 68 domestic dogs that had been vaccinated, and 3
dogs that had no history of vaccination, based on records
from the Veterinary Medicine Hospital of China Agri-
cultural University. Standard Positive and negative serum
were kindly given by the Veterinary Institute of the Mil-
itary Medical Academy of Sciences. Anti-rabies serum
was obtained from the Lanzhou Veterinary Research
Institute Chinese Academy of Agricultural Sciences.
*Corresponding author.
2.2. Expression of Rabies Virus N Gene in E. coli
and Purification the N Protein
The N sequences were amplified from pGEM-T/NP with
GTCACTCGAATATGT3’), digested with Ecol I and Pst
I, and recloned into pMal-c2x initially. The recombinant
plasmids were named pMal-NP and transformed into
JM109 cells, and the bacteria were grown in LB contain-
ing 0.2% glucose grown to 2 × 108 cells/ml (OD 0.5/A
600). IPTG was added to a final concentration of 0.3 mM
and the cells were incubated at 37˚C for 2 h. Boiled bac-
terial lysates were analyzed by 12% sodium dodecyl sul-
fate-polyacrylamide gel electrophoresis (SDS-PAGE).
The cell extract was isolated from a 100 ml bacterial
culture. Then the culture was quicked-chilled in ice water
and the cells were harvested by centrifugation and frozen
in column buffer (20 mM Tris-HCI, 200 m MNaCI, 1
mM EDTA, 1 mM sodium azide and 10 mM ß-mercap-
tothanole) overnight at 20˚C. The MBP fusion protein
in the soluble fraction was purified using an amylose
resin column in affinity chromatography and cleavaged
with factor Xa following the manufacturer’s instructions.
The fusion protein was eluted with elution buffer (col-
umn buffer with 10 mM maltose).
2.3. Western Blotting
The protein purified by affinity chromatography was
separated in 12% SDS-PAGE and transferred to a PVDF
membrane (Bio-rad). Blots were blocked for 1 h in 4%
FBS in PBS (pH 7.4), then washed three times using
western blot washing solution and incubated with a poly-
clonal house anti-serum (1:800) for 1 h at room tem-
perature. Blots were then washed three times with wash-
ing buffer, and secondary rabbit anti-horse IgG (1:10000)
conjugated to horseradish peroxidase was added. Blots
were then incubated for 1 h at room temperature, and then
washed for 15 minutes three times with washing buffer.
After this, DAB was added to produce some strips and
the reaction was stopped.
2.4. ELISA Method
Ninety-six-well plates (Clustar) were coated with the
antigen in each well and incubated overnight at 4˚C.
Plates were blocked by incubating for 30 min at 37˚C
using 5% glutin in PBS followed by three washings in
0.1% PBS-Tween80. Serum dilutions (1:50) were pre-
pared and added to the wells and incubated for 1 h at
room temperature followed by three more washings in
0.1% PBS-Tween80. Secondary antibody, goat anti-dog
IgG horseradish peroxidase (1:5000) was added and the
samples were incubated for 1 h at room temperature fol-
lowed by three more washings in 0.1% PBS-Tween.
O-Phenylendiamine dihydrochloride was used to develop
horseradish peroxidase-conjugated antibodies for 15 min
at 37˚C and stopped by adding 1 M H3PO4. Plates were
read for optical density at 490 nm using a kinetic micro-
plate reader.
3. Results
3.1. Expression of Rabies N in E. coli and
Purification of the MBP-NP Fused Protein
The recombinant plasmid pMal-NP was checked by re-
striction enzyme analysis and sequencing (Figures 1 and
2), and then transformed into E. coli JM109. The JM109
cells were induced with IPTG and subjected to
SDS-PAGE. The results indicated that the rabies nucleo-
protein gene could be highly expressed in the competent
cells of E. coli JM109 (about 25% in total cell proteins)
(Figure 3).
By using an amylose resin column in affinity chroma-
tography, NP-MBP fusion protein was purified; the nu-
cleoprotein was obtained by cleaving NP-MBP fusion
protein with factor Xa and western blotting using poly-
clonal anti-serum of rabies identified both MBP-NP fu-
sion protein and NP (Figures 4 and 5)
3.2. ELISA to Determine Anti-Rabies Antibody
Level in Dog Using NP-MBP Fusion Protein
A series of indirect ELISAs were conducted to establish
an ELISA method for detecting the antirabies antibody
level in dogs using NP-MBP fusion protein as antigen.
The recombinant protein possesses good antigenticity.
Figure 1. Identification of plasmid pMal-NP by PCR. Line
1-3: PCR product of positive; Line M: λDNA/EcoRI + Hind
III marker.
Figure 2. Identification of plasmid pMal-NP by digested by
EcoRI and PstI. Line 1-3 pMal-NP digested by EcoRI and
PstI; Line M: λDNA/EcoRI + Hind III marker.
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Figure 3. SDS-PAGE analysis of MBP-NP fusion protein expressed in E .coil; 2, 3 pMal-c2x E. coil lysate before and after
IPTG induction; 1, 9, 10 JM109 lysate before and after PTG induction; 4, 5, 6, 7 pMal-NP E. coil lysate at 1 h, 2 h, 4 h after
or before IPTG induction 8MW marker.
Figure 4. SDS-PAGE analysis of purified MBP_NP and MBP-NP cleavage; 1 MW marker 2 purified MBP-NP; 3, 4, 5,6, 7, 0
h, 2 h, 4 h, 8 h and 24 h after Factor Xa cleavage.
Figure 5. Western blotting analysis of purified MBP-NP fusion protein and product after factor Xa cleavage.
4. Discussion
The best quantity of antigen coated in each well was 6ug
MBP-NP fusion protein. Positive and negative serum
testing showed that the method had high specificity.
Compared to the purchased ELISA kit with rabies virion
antigen, specificity of the fusion nucleoprotein was better
and the sensitivity was higher (Figure 6). Testing 68 sera
of vaccinated dogs and 3 sera of nonvaccinated dogs , the
specificity was 100%.
Nucleoprotein is a nucleocapsid protein in the rabies vi-
rus and the major internal component of the rabies virion
[3]. The N gene is the most conservative of the five
structure genes [9,10]. The results of the present study
confirmed this. The average conservation of the nucleo-
tide sequence in the N gene and amino acid sequence
among the ten fixed rabies srains is 91.2%, and 96.4%. t
Figure 6. Comparison between the Established ELISA method with MBP-NP as coated antigen and purchased ELISA kit
The distance of different strains was exhibited in the time
in which the virus was isolated. The CTN rabies strain
has more similarity with the India strain. Moreover, both
the 3 aG and 5 aG vaccine strains from China have less
similarity with the CTN strain.
Flamand has shown that the N protein has group-spe-
cific antigenic determinants that are shared by all rabies
viruses [6].The reports have shown that antigenic sites I
and IV, and antigenic sites II and III on the NP are com-
posed of linear- and conformation-dependent epitopes,
respectively [11-13]. The amino acid sequence of anti-
genic site I of NP in all rabies strains is the same; but
there is more variation in antigenic site IV. Residue 379
in antigenic site IV of CTN was leucine, which was dif-
ferenct from the other strains, in which the residue is
To futher understand the molecular properties of the N
protein of the CTN rabies strain, a great quantity of re-
combinant fusion MBP-NP was produced in E. coli. As it
is difficult to directly purify the N protein from rabies
virion, recombinant N protein subunit was the best option.
The two linear epitopes and non-glycosylation in the NP
makes it possible to produce nucleoprotein with a bio-
logical function in E. coli. The expression quantity of N
protein in proeukaryote cells is high, purification is easy,
and MBP-NP fusion protein still exhibited good antigen-
ticity and antigenic specificity. Therefore, the NP subunit
protein expressed from the CTN rabies strain in the pre-
sent study can be used as a diagnostic antigen and im-
munogen for the prevention and control of rabies. Also,
the NP subunit protein can be an alternative to manipu-
lating live viruses in diagnostic tests and vaccine produc-
An enzyme linked immunosorbent assay (ELISA) has
been used successfully for the qualitative assessment of
rabies virus-specific antibodies in serum samples from a
cohort of vaccinated dogs and cats [14]. Cliquet et al.
showed that data generated using this prototype ELISA
indicate a strong correlation for specificity when com-
pared to the gold standard fluorescent antibody virus
neutralisation (FAVN) test. Although the ELISA has a
lower sensitivity than the FAVN test, it is a useful tool
for rapidly screening serum samples from vaccinated
companion animals [14]. In the present study, an ELISA
test using MBP-NP fusion protein as coated antigen was
constructed to determine the properties of the NP in the
CTN rabies strain.
His-tagged recombinant N-protein was used as a safe
and stable antigen for FELISA test, Inouse et al. [15]
showed that the virus neutralization titers by rapid fluo-
rescence focus inhibition test correlated well with the
FELISA results (r = 0.616) and the sensitivity and speci-
ficity of the FELISA were 91.7 and 100%. A 100% spe-
cificity was obtained by using sera of vaccinated and
nonvaccinated dogs in our study. The safe and conven-
ient test using MBP-NP would contribute to our under-
standing of the status of herd immunity among not only
domestic dogs but also wild animals.
Antigenic proteins of whole virion have been used as
diagnostic antigen and immunogens for the control of
viral diseases. However, the whole virus proteins some-
times exhibit low specificity in diagnostic tests and im-
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W. B. CAO ET AL. 313
mune responses. Therefore, futher efforts should be
geared toward genetically engineered subunit proteins to
overcome these problems.
5. Conclusion
The nucleoprotein (NP) gene of a CTN strain rabies virus
was expressed. The antigenicity of the recombined
MBP-NP fusion proteins was examined by western blott-
ing and by enzyme linked immunosorbent assay (ELISA).
An enzyme linked immunosorbent assay (ELISA) has
been used successfully for the qualitative assessment of
rabies virus-specific antibodies in serum samples.
6. Acknowledgements
The authors thank supported by Grant of Hebei Educa-
tion Department (No Z2007212).
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