Expression of Biologically Active Measles Virus Hemagglutinin Glycoprotein by a Recombinant Baculovirus
In this study, one of the measles virus membrane
proteins, named hemagglutinin (H) which has a key role in tropism, receptor
binding, hemagglutinating activity and also induction of protective immunity
against viral infection, was expressed by the baculovirus expression system
using specific plasmid (pDONR221) to produce entry clone. Measles Virus
(AIK-C strain) genome was extracted from infected Vero cells. H gene was
amplified by specific primers during RT-PCR reaction and inserted into
the specific plasmid (pDONR221) using BP recombination reaction. Recombinant
baculovirus harboring H gene was consequently constructed by LR reaction.
Insect cells (Sf9) were infected with recombinant baculovirus.
In order to increase viral titer, recombinant baculoviruses were passaged
four times in Sf9 cells. Synthesis of H protein was verified by
SDS-PAGE, western-blot and indirect immunoflourescene using goat polyclonal
antibody against Measles Virus. The results showed that H protein was
partially glycosylated, but it appeared to be active in hemagglutination
Measles Virus (MV) a member of Morbilivirus genus in the Paramyxoviridae
family is an enveloped virus with a non-segmented negative strand RNA
genome (Griffin, 2007). It has two envelope glycoproteins, the Hemagglutinin
(H) and the Fusion (F) protein. H is responsible for receptor binding
and hemagglutinating activity. In this regard it could be used for recombinant
protein preparation in order to induce the immune system (Huang et
al., 2001; VanBinendijk et al., 1997). MV causes a common childhood
disease with high fever and a typical skin rash. Patients with measles
develop profound immunosuppression, often leading to secondary infections.
MV also causes postinfectious encephalitis, measles inclusion encephalitis
and subacute sclerosing panencephalitis (Yanagi et al., 2006).
Despite the availability of effective live vaccines against Measles, it
still claims lives of over 700000 infants and children around the world
annually (Premenko-Lanier et al., 2006). Furthermore, they are
ineffective in infants under 9 to 15 months of age. This ineffectiveness
of the vaccines is due to maternally acquired measles virus specific antibodies
which readily neutralize this parentally administered attenuated vaccine
virus (Skiadopoulos et al., 2001; Moss et al., 1999). As
a result, the need for a measles virus vaccine to protect children less
than 15 months of age is the highest in the regions of the world where
there is a high prevalence of infection with human immunodeficiency virus
(Premenko-Lanier et al., 2006).
The baculovirus expression system employing Autographa californica
Nuclear Polyhedrosis Virus (AcNPV) and Spodoptera frugiperda (Sf9)
insect cells in culture is very popular for high level expression of heterologous
genes (Hu, 2005). In such a system, the strong, efficient promoter of
polyhedrin gene directs transcription of the foreign gene. Polyhedrin
is not essential for the infection process, so it is possible to replace
the polyhedrin gene with a heterologous gene while retaining the polyhedrin
regulatory signals (O`Reilly et al., 1992).
We constructed the recombinant baculovirus containing His-tagged Measles
Virus hemagglutinin gene by Gateway baculodirect expression system and
expressed the protein in insect cells (Sf9). The hemagglutinin
was partially glycosylated and biologically active in hemagglutination
MATERIALS AND METHODS
Cells and viruses: Measles Virus AIK-C vaccine strain kindly
provided by Dr. Abbas Shafyi (Razi Vaccine and Serum Research Institute).
Vero cells were cultivated in Dulbecco`s Modified Eagle Medium supplemented
with 5% Fetal Calf Serum, L-glutamine and antibiotics, used to support
virus growth. Cells were incubated until cytopathic effect was observed,
then infected cells were harvested and stored at -70°C. Spodoptera
frugiperda insect cells and linear DNA of Autographa californica
Nuclear Polyhedrosis Virus were obtained from Invitrogen Company, USA.
Sf9 cells were cultured in Grace medium (GIBCO, Grand Island, N.Y.)
supplemented with 10% fetal calf serum, Yeastolate (GIBCO, Grand Island,
N.Y.), lactalbumin hydrolysate, 50 `μg of gentamycin sulfate per
mL at 27°C by the procedures of Summers and Smith (1987).
RNA extraction: Infected cell cultures were centrifuged at low
speed for 20 min. RNA was extracted from clarified supernatant using RNA
extraction kit (RNAfast manufactured in National Institute of Genetic
Engineering and Biotechnology). The extracted RNA was stored at -70°C.
Primers: Primers used for RT-PCR and PCR were designed based on
published MV sequences (GenBank Accession No.AF266286) and contained recombinant
sites (attB1 and attB2 for Forward and Reverse primers respectively shown
with Bolded nucleotides) for cloning into pDONR221 vector. The stop codon
in the reverse primer was omitted in order to be in frame with C-terminal
6xHis in the BaculoDirectTM Linear DNA for tagging and purifying
our recombinant protein. The sequence of the primers were as follow:
H-Forward: 5` GGGGACAAGTTTGTACAAAAAAGCACT
Amplification of H gene: Extracted RNA was used as template for
reverse transcription reaction. The RT-PCR reaction was performed in total
of 20 `μL final volume using 2.5 mM of each dNTPs, 100 `ρmol
of each primer, reaction buffer (containing 250 mM Tris HCl, 250 mM KCl,
20 mM MgCl2, 50 mM DTT), 40 U RNase Inhibitor, 200 U M-MuLV
Reverse Trancriptase (Fermentas) and nuclease free water. Thermocycler
was programmed for one cycle at 70°C for 5 min, followed by one cycle
at 37°C for 5 min and one cycle at 42°C for 60 min. The reaction
was stopped at 70°C for 10 min and then the microtube was chilled
on ice (Verma, 1981). PCR test was performed in total of 50 `μL final
volume, using 1 ng cDNA, 10 `ρmol of each primer, 2.5 mM of each
dNTPs, 2.5 mM MgSO4 and 2.5 units of pfu DNA polymerase (Lundberg,
1991). Thermocycler was programmed for one cycle at 94°C for 5 min
followed by thirty cycles at 95°C for 1 min, 50°C for 1 min, 72°C
for 70 sec and one cycle of final extension at 72°C for 10 min. The
PCR product were electrophoresed in 1% (w/v) agarose gel and stained by
ethidium bromide and visualized by UV transilluminator.
Construction of H entry vector: The pDONR221 plasmid and Omnimax
chemically competent E. coli (Invitrogen) were used for cloning.
The full coding region sequence of H protein (AIK-C Stain) with attB sites
was inserted into pDONR221 containing attP sites (Fig. 1),
using the BP recombination reaction by BP clonase (Invitrogen). Bacterial
cells were transformed according to the published method (Sambrook and
Russel, 2001). Transformation mix was spread on a prewarmed selective
plate (LB+50 `μg mL-1 Kanamycin) and incubated overnight
at 37°C. The presence of the ccdB gene as controller of cell death
gene in pDONR221 allows negative selection of the donor vectors in E.
coli following recombination and transformation. The ccdB protein
interferes with E. coli DNA gyrase, thereby inhibiting growth of
most E. coli strains (Invitrogen).
Colonies carrying entry clone were picked up and suspended in 5 mL of
LB broth with appropriate kanamycin concentration. Plasmids were purified
from the bacteria using plasmid purification kit (Roche). Plasmid constructs
were confirmed by restriction endonucleases digestion and sequenced with
M13 primers in both direction by a dye terminator method, using the ABI
3130XL sequencer (Griffin and Griffin, 1993). The sequencing results were
compared with other sequences deposited in the Genbank by the BLAST software
(Atschul et al., 1997).
||Schematic presentation of entry clone construct (pDONR221
+ H gene of Measles Virus) with some restriction sites
Construction of recombinant baculovirus: Recombinant baculovirus
harboring H gene was constructed by LR reaction between entry clone and
BaculoDirect Linear DNA containing thymidine kinase locus of Herpes Virus
flank to the polyhedrin promoter which substitute by foreign gene and
could help to selection after recombination, in the presence of ganciclovir.
The reaction was performed in a total volume of 10 `μL using 2 `μL
entry clone (50-150 ng/reaction), 5 `μL BaculoDirect Linear DNA (150
ng/reaction), 1 `μL TE buffer, PH 8.0 and 2 `μL LR Clonase II
mix. The tubes containing reaction mix were incubated at 25°C for
18 h, then 1 `μL of the 2 `μg `μL-1 proteinase
K was added to each reaction and the tubes incubated for additional 10
min at 37°C.
Transfection of the Sf9 cells: The BaculoDirectTM
Linear DNA Expression System uses Gateway®7 Technology to facilitate
direct transfer of the foreign gene into the baculovirus genome without
the need for additional cloning or recombination in bacterial or insect
cells. Transfection of recombinant baculovirus into cultured Sf9
cells was achieved using cellfectin. LR recombination reaction were composed
of two mixture: mixture A containing 5 `μL LR recombination reaction
and 100 `μL unsupplemented Grace`s Insect Medium and mixture B containing
6 `μL cellfectin reagent and 100 `μL unsupplemented Grace`s
Insect Medium. The two mixtures were combined and incubated for 45 min
at RT. Then 800 `μL unsupplemented Grace`s Insect Medium was added
to transfection mix and mixed gently. The Sf9 cells were washed
with fresh unsupplemented Grace`s Insect Medium and the entire transfection
mix were added onto the cells and incubated for 5 h at 27°C. Then
transfection mixture were removed and 2 mL of complete Grace`s insect
medium with antibiotics (100 U mL-1 penicillin and 100 `μg
mL-1 streptomycin) and 100 `μM ganciclovir were added
to each well (Invitrogen). The cells incubated at 27°C until CPE appeared
and then were harvested and pelleted by centrifuge at 3000 rpm for 5 min.
The supernatant which was the P1 viral stock was stored at 4°C and
protected from light. The pellet was washed 2 times with PBS and kept
for detection of recombinant protein. In order to increase viral titer,
recombinant baculoviruses were passaged 4 times in Sf9 cells.
Immunoflourescence assay: Recombinant baculovirus protein expression
was assayed in acetone-fixed infected Sf9 cells with a 1:5 dilution
goat antibody specific for measles virus (prepared from Razi Vaccine and
Serum Institute) and rabbit anti goat flourescein isothiocyanate-conjugated
antibody (Koma Biotech Inc.).
Polyacrylamide gel electrophoresis and immunoblot: Cellular proteins
were analysed using electrophoresis lysis mix containing 0.5M Tris-hydrochloride
pH: 6.8, 2% SDS, 10% Glycerol, 2% `β-mercaptomethanol, 1% bromophenolblue
and boiled for 5 min. Samples were applied to 10% acrylamide gels (acrylamide/bisacrylamide
weight ratio, 50:1.6) and subjected to electrophoresis at 140 V, 4 h by
the methods of Laemmli. Protein bands were detected by coomassie blue
Hemgglutination assay: Cells expressing H protein were serially
twofold diluted in 96-well round-bottom microdilution plates in 25 `μL
volumes. A 1% suspension of monkey erythrocytes was prepared in PBS and
added to each well in 50 `μL volumes. The plates were kept at 4°C
overnight and the results were recorded after 16 h.
RESULTS AND DISCUSSION
Amplification of H gene: In order to obtain Measles Virus
H gene with attachment sites for recombination reaction, PCR was performed
using specific primers containing attachment sites. As it shown in Fig.
2 DNA band of approximately 1950 base pair was achieved (Lane 2).
In control reaction no DNA band was detected (Lane 1).
Analyzing the entry vector: After BP reaction and purification
of H containing plasmid (Entry Clone) from bacteria, they were digested
by Bgl II restriction endonuclease since H gene has two restriction sites
for Bgl II, but pDONR221 has no restriction site for this enzyme. Therefore
only two fragments with approximate molecular weight of 1100 and 3500
base pair appeared on agarose gel (Fig. 3 Lane 2, Lane
Immunoflourescence test: Immunofluorescence analysis of infected
Sf9 cells with recombinant baculovirus containing H gene using
measles virus antiserum and flourescein isothiocyanate conjugated rabbit
anti goat antibody showed that the entire infected cells were stained
indicating the presence of viral expressed proteins (Fig.
4B). Control uninfected cells processed similarly did not show any
fluorescence (Fig. 4A).
Expression of recombinant H proteins in Sf9 cells: Sf9
cells cultivated in 6-well microdilution plates (Fig. 5A)
were infected with recombinant (Fig. 5B) or non-recombinant
virus at a multiplicity of infection 5 PFU per cell. At specific times
(4, 5, 6 and 7 days) following infection, cells showing CPE were washed
twice with PBS, lysed with SDS-electrophoresis sample buffer applied to
polyacrylamide gels (10%) and subjected to
||Amplification of H gene (Measles virus AIK-C strain) on 1% agarose
gel. Lane M: DNA Molecular weight Marker (0.07-12.2 Kbp). Lane 1:
Negative control Lane 2: Amplification of H gene with specific primers
||Digestion of entry clones by BglII restriction endonuclease on 0.7%
agarose gel. Lane 1: Undigested entry clone 1, Lane 2: Digested entry
clone Lane, Lane 3: Undigested entry clone 2, Lane 4: Digested entry
clone 2. Lane M: DNA Molecular weight Marker (0.07-12.2Kbp)
electrophoresis. As it is shown in Fig. 6B many protein
bands observed in the gel which some of them were H specific proteins
as appeared by immunoblot (Fig. 6A).
Two species of H protein appeared to be synthesized: 80 and 60 kD which
probably were glycosylated and non-glycosylated forms of H protein respectively.
Lower molecular weight bands were most probably degradation products of
the H protein.
||Immunoflourescence of uninfected (A) and Infected Sf9 Cells
with recombinant baculovirus harboring H gene of Measles Virus (B)
||Uninfected (A) and infected Sf9 cells with recombinant
baculovirus harboring H gene of Measles Virus (B). The infected cells
in B showed CPE as rounding and detachment from the surface
At least 9% of the total stained proteins were H proteins as was shown
by densitometric scanning of the electrophoretic gel (data not shown).
Production of H gene products in insect cells remained stable even after
four passages of the purified recombinant virus in culture.
||Immunoblot (A) and Coomassie blue-stained gel (B) of
total proteins produced in Sf9 cells infected with either wild-type
or recombinant H virus. Sf9 cells were infected with non recombinant
or recombinant virus containing the H gene of measles virus. Total
proteins were solubilized by lysing the cells in sample buffer at
120 h postinfection after 2, 3 and 4 passage (Lane 1, 2, 3 respectively).
Lane 4: Uninfected Sf9 cell proteins. Lane 5: Low molecular
weight protein marker. Lane 6: Non-recombinant baculovirus proteins.
Immunoblot assay prepared from a gel which was a duplicate of the
one shown in panel B. The immunoblot was obtained by electrophoretic
transfer of proteins from gel to nitrocellulose. The H recombinant
protein products are indicated with arrows
||Hemagglutination test in different passage of recombinant
baculovirus containing H gene of Measles Virus. C+ Edmonston
Strain of Measles Virus. H2, H3, H4: HA of Sf9 cells from different
passage of recombinant baculovirus containing H gene. C-:
HA in uninfected Sf9 cells. F: HA in Sf9 cells extract
that infected by recombinant baculovirus containing F gene of Measles
Virus. NR: Sf9 cells extract that infected by non-recombinant
baculovirus. C RBC: Control of Red Blood Cells
Biological activity of recombinant H protein: Recombinant H protein
was biologically active in hemagglutination assay. Hemagglutination of
African Green Monkey erythrocytes by Sf9 cells extract which contained
recombinant H protein was evident and a reciprocal dilution of 2, 16 and
128 was obtained after two, three and four passages of recombinant baculovirus
(Fig. 7). Partial hemadsorption was also observed on
the surface of infected Sf9 cells (data not shown).
Surface antigens of measles are the main targets for recombinant expression
to develop recombinant vaccines. The simplest method is expression of
proteins in prokaryotes. However, the lack of proper conformation and
post-translational changes such as glycosylation restricts application
of these systems to research (Hu et al., 1994). An alternative
method of expression systems for measles virus proteins is mammalian system
using virus expression systems like vaccinia virus (Kidokora et al.,
2002), canarypox (Taylor et al., 1992) and Semliki forest virus
(Bouche et al., 1998a). Major drawback for virus vectors in mammalian
expression system is high cost and more importantly, safety issues regarding
unexpected consequences of these systems on human body, but can be applied
to diagnostic procedures (Bouche et al., 1998b). Insect cell expression
systems are promising alternatives in search for a suitable expression
vectors. There are only two other attempts in producing glycoprotein H
in insect cell expression system. Vialard and coworkers used `β-galactosidase
gene for screening recombinants baculovirus containing measles H protein.
In their system consequent plaque purification was required (Vialard
et al., 1990). The use of `β-galactosidase gene for screening
baculovirus recombinants has previously been developed by Kidokora
et al. (2002). This approach lacks required criteria as a candidate
for vaccine developments. The large `β-galactosidase-fused protein
will initiate production of undesired antibody development without offering
any significant advantage in downstream processing of the recombinant
protein, namely purification of the H protein. Takehara et al.
(1992) used traditional baculovirus vectors in order to construct an expression
vector for recombinant measles proteins. To examine the expression and
purification efficiency of the newly developed expression system, we sought
recombinant expression and activity of H protein of measles using BaculoDirectTM
system. In this system the resulting recombinant baculovirus DNA is transfected
directly into insect cells to generate recombinant virus. In another word,
cloning and expression of desired gene was performed without plaque purification
or selection in bacteria., thus facilitating its faster screening (Invitrogen).
In this study, the 1950 bp fragment resulted from RT-PCR was used to construct
a modified baculovirus with two defined recombination sites. Sf9
cells transfected with the newly synthesized recombinant baculovirus containing
the measles virus hemagglutinin gene, were shown to synthesize the H protein
under control of the polyhedrine promoter. Advantage of our approach compared
with previous attempts is our high yield of recombinant protein with similar
authentic viral protein as was shown in immunofluorescent and western-blot
experiments. In comparison with the prokaryotic expression system, the
post-translational modification of H protein has been reported to occur
(Hu et al., 1994). On the other hand due to presence of the His-tag
at the c-terminal of the recombinant H protein, purification of this protein
was improved compared to conventional methods (Lund and Salmi, 1981).
According to present results the antigenic sites were present in the
recombinant H protein. Levels of protein expression were higher than previous
studies (Takehara et al., 1992), since H protein could be detected
on SDS-gel stained with coomassie blue. The highest level of protein expression
was observed at day 5 post infection. Although, the H membrane protein
was shown to be only partially glycosylated, it could still agglutinate
the African Green Monkey red blood cells indicating that it was biologically
In conclusion, the above expression system developed in this study may
be helpful for the investigation of the function and structure of the
This investigation was supported by Iran University of Medical Sciences
by grant number 260 and Razi Vaccine and Serum Research Institute. The
authors thank National Institute of Genetic Engineering and Biotechnology
for their technical assistance.
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