Abstract: Rift Valley Fever Virus (RVFV), a phlebovirus of the family Bunyaviridae is a major public health threat in Egypt and sub-Saharan Africa. RVFV possesses a single stranded segmented RNA genome composed of a Large (L), a Medium (M) and a Small (S) segment. The M segment codes for a polyprotein which is the precursor to the glycoproteins G1 and G2 and two nonstructural proteins. The present study aimed to study the possibility of production a subunit recombinant viral G1 protein of RVFV, to use it as alternative immunogen. To produce subunit protein of RVFV, a seed stock of RVFV pantropic Menya strain (M/S/258) strain was obtained then titrated. A virus seed stock was prepared using Chicken Embryo Related Cells (CER) cell line. Specific primers for G1 gene containing BamHI and KpnI restriction sites were designed and used to excise the gene using RT-PCR technique. The PCR products were purified and ligated into plasmid (pCR®II-TOPO®) cloning vector. Transformed colonies were selected and tested for the presence of rG1 gene using miniprep, followed by restriction endonuclease digestion. Positive plasmids containing insert were subjected to DNA sequence analysis to confirm that the insert DNA is G1 gene. Insert 2 was prepared by digesting the expression vector pQE-30 with BamHI and KnpI restriction enzymes. Rapid screening of small expression cultures showed the ability of selected colonies to express rG1 protein. Time course and Isopropylthio-β-D-galactoside (IPTG) concentrations were tested to determine optimum time and IPTG concentration to be used in large-scale expression culture. Bacterial cultures revealed the presence of specific band at approximately 52 Kda in induced culture. Western blot analysis verified the presence and antigenicity of rG1. Large-scale production and purification of rG1 resulted in 6 mg protein. The produced recombinant viral antigenic subunit protein is a step to develop new, safe and effective vaccine.
INTRODUCTION
Rift Valley Fever Virus (RVFV), a Phlebovirus, belongs to the family Bunyaviridae. Bunyaviruses are RNA spherical, enveloped particles 90 to 100 nm in diameter and display surface glycoprotein projections of 5 to 10 nm which are embedded in lipid bilayered envelope approximately 5 to 7 nm thick (Schmaljohn and Hooper, 2001). The spikes are generally thought to consist of heterodimers of the viral G1 and G2 glycoproteins (Freiberg et al., 2008).
RVFV contains three single-stranded RNA genome segments designated as large (L), medium (M) and small (S) in which their 3' and 5' termini have the same complementary nucleotides. Non covalently closed circular RNAs and panhandle structures may be formed due to base pairing of the termini (Ikegami et al., 2009). The M segment gene of RVFV has 5'-NSm-G2-Gl-3' cRNA gene order that encodes the two envelop glycoproteins G1 and G2, in a single Open Reading Frame (ORF) of cRNA (Collett et al., 1985).
The first epidemic and epizootic occurrence of RVF outside sub-Saharan Africa was in Egypt in 1977 (Zeller et al., 1997; Ding et al., 2005) which infected 200,000 people of which 600 died (McElroy et al., 2009; Pepin et al., 2010). In regions where cattle, sheep, goats and mosquitoes are abundant; the RVF invasion is of a high probability as indicated from the outbreaks of this disease in Egypt, Saudi Arabia and Yemen. Furthermore, RVFV can be aerosolized which has raised concerns of it being used as a bioterrorist agent (Fisher et al., 2003; McElroy et al., 2009).
The major way to control RVF is by vaccine (Sall et al., 1998). Live attenuated viruses based on the mouse neuroadapted Smithburn RVF strain are used in Kenya and South Africa. In Egypt and South Africa, formalin-inactivated wild-type viruses have been used (Nichol, 2001). Although the Smithburn strain is the only veterinary vaccine widely available, it has serious drawbacks as it has been proven to be teratogenic, cause abortions and encephalitis in young lambs (Sall et al., 1998). Efficient vaccines and antiviral drugs are not available yet which are essential for the suppression of outbreaks and treatment of RVF patients, respectively (Tetsuro and Shinji, 2011).
In the present study, this work aimed in the isolation of G1 gene from RVFV M segment, the expression and cloning of G1 gene in bacterial vector to produce a recombinant glycoprotein. The antigenicity of the recombinant RVFV glycoprotein was evaluated and compared with the viral authentic protein.
MATERIALS AND METHODS
Rift valley fever virus strain: A seed stock of RVFV pantropic Menya (M/S/258) strain was kindly provided by Dr. El Karamany, Ex-General manger of research and development sector VACSERA, Egypt. The strain had a titre of 109.7 mice Intracerebral Lethal Dose50/mL (MICLD50/mL) of suckling mice and 107.5/mL mice Intraperitoneal Lethal Dose50/mL (MIPLD50/mL). RVF virus seed was diluted to 10-2 using sterile Phosphate-buffered Saline solution (PBS), pH 7.2 (0.5 M NaH2PO4, 0.5 M Na2HPO4, 1 M NaCl). Ten suckling (six to eight weeks old out bred female Swiss Webster) mice groups, 7 each, were inoculated intracerebrally (i.c.) at a dose of 0.01-0.03 mL of RVFV. Brains of infected mice were aseptically aspirated, homogenized and then clarified by high speed cold centrifugation at 10000 xg for 30 min. MICLD50 was calculated according to Reed and Muench (1938).
Preparation of master virus seed stock: Chicken Embryo Related Cells (CER) were maintained in MEM medium (GIBCO), supplemented with 200 mM L-glutamine, 10% (v/v) foetal calf serum (Sigma Chemical Co., St Louis, MO, USA) and 100 IU mL-1 and 100 mg mL-1 penicillin/streptomycin solution (Invitrogen) in 25 cm2 tissue culture flasks then incubated at 37°C in a moist atmosphere containing 5% (v/v) CO2 incubator until monolayer was formed in the flask. Flasks that developed CPE were freezed and thawed three times to release the virus from cells according to Bussereau et al. (1982).
Preparation of insert 1: RNA sequence of RVFV virus glycoprotein 1 (G1) was obtained from GenBank Accession No: NC_002044. The primers were designed to flank sequence encoding part of G1 gene which contains the antigenic determination and glycolisation sites (Keegan and Collett, 1986). The sequence of sense primer was 5’-TGTTCAGAACTGATTCAGG-3’ while the reverse primer was 5’-TTTACACGTACTGATCTATGAG-3’. Restriction site that does not cut inside target sequence was chosen to be added to the reverse primers. kpnI (complementary strand 5’-GGTACC-3’) site was added to the 5’ end of the reverse primer to be 5’ GGTACC-TTTACACGTACTGATCTATGAG-3’.
Cloning of insert 1 into pCR® II-TOPO vector: RVFV-RNA extraction and polymerase chain reaction were carried out according to Sambrook et al. (1989). The generated DNA fragment was ligated into pCR® II-TOPO cloning vector (Pharmacia) and transformed into DH5α ultracompetent® cells according to pCR®II-TOPO cloning kit instruction manual. Produced clones harboring the plasmid of interest were validated by color screening of LB amp agar plates containing X-gal. White colonies were chosen to be tested for the presence of plasmid and insert. Plasmid miniprep was done using QIAprep® Spin Miniprep Kit (Qiagen). Aliquots of the eluted plasmids were checked for the presence of insert using restriction digestion (BamH I and Kpn I enzymes) and DNA sequence analysis according to Sambrook et al. (1989).
Cloning of insert 2 into pQE 30 vector: The QIAexpressionest kit was prepared (according to the instruction manual of pQE-30® vector) by first dissolving in 10 μL TE buffer to yield 0.5 μg μL-1 of plasmid. A 2 μL aliquot was linearized using BamHI and KpnI restriction enzymes. The reaction mixture was prepared by mixing 2 μL pQE-30® vector, 1 μL 10X buffer REact 4® (Invitrogen), 1 μL BamH I enzyme (10 U μL-1, Invitrogen), 1 μL Kpn I enzyme (10 U μL-1, Invitrogen) and 2 μL H2O in 0.5 mL tube. The linearized pQE-30® was electrophoresed on preparative agarose gel and purified. The ligation of insert 2 with digested pQE-30® vector was carried out using Ready-To-Go™ T4 DNA ligase kit (Pharmacia) according to instruction manual.
The produced clones were followed by the expression of 6xHis-tagged proteins and purification on Nickel-Nitrilotriacetic Acid (Ni-NTA) matrices. Isopropylthio-β-D-galactoside (IPTG) was used for protein induction to a final concentration of 1 mM and incubated for 4 h before the cells were harvested by centrifugation (Sambrook et al., 1989). Rapid screening of small expression cultures and SDS PAGE took place using the methods described by Sambrook et al. (1989).
Preparation of anti-RVFV antibodies: Antibodies against RVFV were obtained from mouse serum. Mice were given 0.5 mL of RVFV inactivated vaccine at 0, 7 and 14 days. Mice were then challenged at 28 days post vaccination by 100 Cell Culture Infective Dose 50 (CCID50). Sera were separated, pooled, aliquot and stored according to the method described by Mohamed et al. (1996). The produced sera was used to evaluate the anti-RVFV antibody level and authenticated by Western blot analysis using the protocol described by Sambrook et al. (1989).
Large-scale production of rG1 protein: Culture growth for preparative purification (1 L) was done according to the protocol described in QIAexpressionest® kit. Purification of 6xHis-tagged proteins was done using Ni-NTA affinity chromatography as described in QIAexpressionest kit protocol. Protein content of dialyzed samples was determined according to Bradford (1976) method using BioRad® kit.
Detection of endotoxins contamination in rG1 protein: The standard endotoxin of the kit was prepared by dissolving in 1.6 mL water for bacterial endotoxins test (water for BET; LAL water) to get a stock standard solution of 10,000 Endotoxins Unit (EU) mL-1. After vigorously mixing the standard endotoxin stock solution, appropriate serial dilutions (8, 4, 2, l, 0.5 and 0.25) were prepared using LAL water. Test solutions (rG1 protein) were prepared by making 1/10th dilution using LAL water. A volume of the dissolved LAL solution (0.1 mL aliquot) was mixed with an equal volume of one of the standard solutions, LAL water (negative control) or test solutions in separate tubes. Reaction mixtures were incubated for a constant period with avoiding vibration according to the recommendations of the lysate manufacturer (37±1°C for 60±2 min). The tubes were taken in turn directly from the incubator and inverted through approximately 180° in one smooth motion. If a firm gel is formed that remains in place upon inversion, the result will be recorded as positive. A result is negative if an intact gel was not formed.
RESULTS
Virus titration: RVF virus has known to have a good reproducibility in Chicken Embryo Related (CER) cell line than other cell lines. Cytological changes were observed 24 h post infection of the cells with RVF virus. The infectivity titre recorded in infected cells was 7.6 Log (10) TCID50/0.1 mL.
Ligation of the insert 1 into pCR®II-TOPO® cloning vector and transformation of competent bacteria: Forward and reverse primers were designed to flanked part of the G1 glycoprotein gene. The forward primer was designed at the bases No. 2091-2109. Insert 1 was prepared using PCR. Taq DNA polymerase was used in the amplification resulting in sticky ends that end with A nucleotide. The expected size of the PCR products (insert), including the restriction sites added to the primers is 1545 bp Electrophoresis of PCR products revealed that a DNA band at the expected size was obtained (Fig. 1).
Cloning of insert 1 was done using pCR®II-TOPO® vector. Transformation of DH5α® gold bacteria using pUC19® resulted in a huge number of blue colonies indicating high transformation rate.
| Fig. 1: | Agarose gel electrophoresis pattern of amplified products after PCR, Lane 1: PCR marker, Lanes 2, 3 and 4: Aliquots of loaded DNA |
| Fig. 2: | Agarose gel electrophoresis of BamH I and Kpn I restriction enzyme digested 9 miniprep products of white colonies and one blue colony of pUC19 transformed bacteria, Colonies number 2, 3, 5 and 8 showed the presence of insert while the rest of colonies contain no insert, Lane M: PCR marker, Lanes 1-9: Undigested (U) and digested (D) miniprep products of colonies number 1-9, Lane 10: Undigested and digested pUC19 |
The presence of insert 1 was tested in positive colonies using both miniprep and restriction endonuclease digestion. Nine positive colonies were tested for the presence of insert 1 in the extracted recombinant plasmids. Both miniprep and enzymatic digestion were efficient as shown after electrophoresis of extracted undigested and digested pCR®II-TOPO®. Three of nine white colonies (colonies 2, 5 and 7) showed the presence of insert 1 (Fig. 2). These positive clones were subjected to DNA sequence analysis. Results obtained from DNA sequence analysis were further analyzed through sequence similarity search using blast search option of internet. The search results showed that both clones are similar to specified part of G1 gene.
Recombinant vector 2 preparation and transformation of M15 bacteria: Digestion of the recombinant vector 1 with both BamH I and Kpn I enzymes resulted in the release of insert 2. The electrophoresis products of the digestion showed a band of digested vector and released insert at approximately 1500 bp (Fig. 3). Insert 2 was ligated into pQE-30® vector. The vector was efficiently digested with the same enzymes. Both insert 2 and vector 2 were successfully ligated together and transformed into M15 bacteria. Twenty five colonies resulted after transformation. Four colonies were checked for the presence of insert 2. After miniprep and enzymatic digestion with BamH I and Kpn I, 4 colonies showed the presence of insert after electrophoresis (Fig. 4).
Rapid screening of small expression cultures: Small bacterial cultures were used to establish time course of protein expression. The presence of specific band in induced culture compared to that of the uninduced culture was checked for up to 4 h after induction. Higher expression rate was at 4 h after induction (Fig. 5). Large-scale production of rG1 protein was done using 1 mM IPTG and 4 h induction.
| Fig. 3: | Agarose preparative gel electrophoresis of recombinant TOPO vector digested with BamHI and KpnI enzymes creating insert 2, Lanes 1, 2 and 3: Purified products of BamH I and Kpn I enzymes digested insert, Lane 3: marker |
| Fig. 4: | Agarose gel electrophoresis for plasmids purified from 4 bacterial colonies resulted from transformation of M15 bacteria with recombinant vector 2 and restriction enzymes digested with BamHI and KpnI enzymes, Lanes 1, 2, 3: Purified digested vector 2, Lanes 4: Purified undigested vector 2 and Lane M: Ladder PCR marker |
| Fig. 5: | SDS-PAGE of bacterial cultures and purified bacterial products to determine the proper expression induction time of rG1 protein in M15 bacteria, Lane M: prestained molecular weight marker, Lane 1: Purified band 4 h after IPTG induction, Lane 2: Purified band 3 h after IPTG induction, Lane 3: Purified band 2 h after IPTG induction, Lane 4, 5: Purified band 1 h after IPTG induction, Lane 6: Wash, Lane 7: Flow-through, Lane 8: Induced culture |
| Table 1: | Evaluation of immunized and unimmunized antibody titre reactivity to RVFV antigen |
| OD: Optical density (it was for a pool of 6 mice sera in both immunized and unimmunized groups) | |
Evaluation of anti-RVFV antibodies: Anti-RVFV antibodies were needed to test the antigenicity of rG1 protein. Before using anti-RVFV antibodies, the antibody titer was evaluated. Both sera collected from immunized and unimmunized mice were 2-fold diluted up to 1:2048. Unimmunized sera turned negative to react with RVFV (background antibodies) at dilution of 1:32 while immunized mice sera were positive up to dilution of 1:1024 (Table 1).
Western blot analysis: Western blot analysis was done to check the presence of rG1 protein and to verify its antigenicity. The mice anti-RVFV could identify the expressed rG1 protein approximately at 52 kDa (Fig. 6). Because it was detected by anti-RVFV anti-sera, the rG1 protein was found to be antigenic after expression.
Production of recombinant rG1 protein: Large-scale production of rG1 protein was done in one to one scale culture (Fig. 7). Protein content was 410 μg mL-1 in elution 1 (10 mL total volume) and 190 μg mL-1 in elution 2 (10 mL total volume).
| Fig. 6: | Western blot analysis for detection of rG1 protein in induced and purified cultures, Lane 1: Marker, Lanes 2, 3, 4, 5, 6, 7 and 8: Purified cultures |
| Fig. 7: | SDS-PAGE of rG1 protein after purification from large-scale culture, Lane 1: Prestained marker, Lane 2: Elution 1, Lane 3: Elution 2 |
Total protein content of purified protein by this method was nearly 6 mg (4.1 mg from elution 1 and 1.9 mg from elution 2, respectively). There was no any detectable amount of endotoxin present in any of the tested protein using LAL test.
DISCUSSION
Rift Valley fever virus, a Phlebovirus, causes epizootics among domestic animals in many parts of sub-Saharan Africa and also in Egypt (Haaheim et al., 2005; Ikegami et al., 2009). Now-a-days, research is focused on the development of subunit vaccines containing the most immunogenic antigens from the particular pathogen (Rocha et al., 2004). In the present study, our work aimed to produce a recombinant viral antigenic subunit protein which is a step to develop new, safe and effective vaccine.
In the present study, PCR products which showed specific and clean amplification products were used in the subsequent cloning steps. pCR®II-TOPO cloning vector was used to clone selected sequence designed of G1 gene in the form of PCR products. The vector used has blue/white screening capabilities to facilitate selection of positive colonies that contain the insert. The inclusion of a lacZalpha Multiple Cloning Site (MCS) allows blue/white screening. Previous studies reported the cloning of RVFV genome into pET20(+)b (McElroy et al., 2009). You et al. (2010) used T-vector with further digestion using the EcoRV-linearized pCAGX with XcmI to produce T tails on both 3'-ends. This method could efficiently minimize the non-recombinant background of T-vector and eliminate the necessity of selective marker genes such as LacZ that allowed blue/white screening.
DH5α bacteria were used as competent bacteria in cloning step. DH5α transformed with standard plasmid (pUC19) showed a huge number of blue colonies indicating high transformation rate of these bacteria. A double cassettes vector was constructed and cloned in E. coli DH5 α for the production of ghost bacteria vaccine to prevent the streptococcal disease in aquaculture fish species by Ra et al. (2009). In this study, DH5α bacteria were also transformed with ligation reaction. Large number of white colonies and little number of blue colonies indicated high transformation rate using the ligation reaction. The presence of insert 1 was checked in positive colonies using both miniprep and restriction endonuclease digestion. Restriction enzymes digestion showed the presence of insert in ligation reaction transformed bacteria. The presence of insert 1 in some of the checked colonies indicated that not all the white colonies contained the insert, some of the white colonies contained self-ligated form of the pCR®II-TOPO cloning vector. Successful transformation was supported by DNA sequence analysis and similarity search using BLAST that proved the right orientation of insert 1. Our results are in accordance with Soumare et al. (2012). pCR®II-TOPO cloning vector was intended to be used as transitional cloning step to create insert 2 cassette into pQE 30 expression vector. High level expression of 6xHis-tagged proteins in E. coli using pQE 30 vector is based on the T5 promotor transcription-translation system.
Optimal expression of recombinant proteins in various expression systems including E. coli can be easily achieved when the vectors and host cells are carefully chosen and the growth conditions are properly controlled. Fernandez-Caballero et al. (2009) showed that recombinant proteins expressed in E. coli can be produced in a soluble form, but in many cases (especially at high expression levels), they aggregate and form insoluble inclusion bodies. McElroy et al. (2009) used expression vector with the goal of achieving soluble expression of His-tagged versions of the proteins in bacteria. The N-terminus directs the expressed protein to the periplasmic space which promotes folding and disulfide bond formation and theoretically enhance solubility.
In this study, M15 [pREP4] bacteria was used for transformation. It contains pREP4 repressor plasmid that permits high level of expression of tagged recombinant proteins. The M15 bacteria lacks laclq mutation, so pREP4 plasmid is required to produce lac repressor to efficiently block transcription and ideal for storing and replicating pQE and prevent leaky expression before induction. Higher expression rate was achieved at 4 h after induction. Thus, large-scale production of rG1 protein was performed using 1 mM IPTG and 4 h induction.
Purification of rG1 protein was done under denaturing conditions using 8 M urea to make the 6His-tag exposed to facilitate the purification steps. The advantage of the 6His tag is that it allows the immobilization of the protein on the metal chelating surface (Ni-NTA). In this study the rG1 protein which is N-terminal 6His-tagged, was purified using affinity chromatography. The purified protein was reasonable in both purity and quantity. Similar results were obtained by Stuber et al. (1990).
Present results revealed the presence of specific band at approximately 52 Kda in induced culture. Large-scale production and purification of rG1 resulted in 6 mg protein. Anti-commercial RVFV vaccine antibodies were successfully prepared and used in Western blot analysis to verify the expressed rG1 protein. The sera of unimmunized animal showed background antibodies up to dilution of 1/16 serum dilution. In Western blot analysis, the anti-RVFV serum was diluted to 1:1000 to remove any probability of antibacterial toxin antibodies in the used serum.
Western blot analysis showed clear band and indicated that rG1 protein is antigenic protein that can be recognized by anti-RVFV antibodies. Several treatments during expression and purification of rG1 protein did not destroy the antigenicity of rG1 protein.
Vanlandschoot et al. (2005) showed that when proteins are purified from E. coli, the danger for contamination with endotoxins is very high and its presence should be monitored carefully. On the contrary, this study showed that recombinant protein preparations contain less than 0.05 EU mL-1 at 1/10 dilution.
In this study, the recombinant protein was produced and purified. The expressed rG1 was similar in antigenecity to the natural G1 protein. Our accomplishment in this field has brought us one step further in developing copious, safer and effective vaccine.
ACKNOWLEDGMENTS
A seed stock of RVFV pantropic Menya (M/S/258) strain was kindly provided by Dr. El Karamany, Ex-General manger of research and development sector VACSERA, Egypt.
Chicken Embryo Related Cells (CER) was kindly provided by Tissue Culture Laboratory, Virology Sector, VACSERA, Giza, Egypt.