HYBRIDOMA Volume 30, Number 5, 2011 ª Mary Ann Liebert, Inc. DOI: 10.1089/hyb.2011.0027
A Monoclonal Antibody Against PrM/M Protein of Japanese Encephalitis Virus Rong-Hong Hua and Zhi-Gao Bu
Japanese encephalitis virus ( JEV) is a major public health threat in the Asia-Pacific region. The pre-membrane (PrM) protein of Japanese encephalitis virus is cleaved during maturation by the cellular protease into the structural protein M and a pr-segment. Here, we describe a procedure to generate monoclonal antibody (MAb) against JEV PrM/M protein and investigate its characteristics. Western blot analysis showed that the MAbs produced in this study were against JEV PrM/M specifically. Indirect immunofluorescence assay demonstrated that they could recognize native PrM/M protein in JEV-infected BHK-21 cells. Preliminary studies identified the epitope of the MAb with a set of synthesized overlapping peptides covering the whole length of PrM protein of JEV. The MAbs reported here may provide valuable tools for the further exploration of biological properties and functions of PrM/M protein and may also be developed for potential clinical applications.
Introduction
J
apanese encephalitis ( JE), the most common mosquitoborne encephalitis, is widely prevalent in Asia and in parts of Oceania.(1,2) Globally, up to 50,000 human cases are reported every year, of which as many as 10,000 result in fatality, and around half of JE survivors have neurological sequelae.(3,4) In addition to humans, many animal species can be infected by JEV, such as swine, equine, and birds.(5,6) JEV belongs to the family Flaviviridae, genus Flavivirus. It possesses a single-strand, positive sense, approximate 11 Kb RNA genome that contains a long, open reading frame. A polyprotein, encoded by the ORF, is cleaved co- and posttranslationally into three structural proteins (C, prM/M, and E) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5).(7) The PrM protein has a length of 167 amino acids, with a molecular weight of about 20 to 26 kDa.(8) PrM is the precursor of the membrane-anchored and virion-associated M protein (about 8 kDa). After synthesis of the nascent polyprotein of PrM and E, the signal cleavages generate the N and C termini of PrM. The C terminus containing the adjacent transmembrane domains allows PrM to anchor itself to the membrane and serves as the signal sequence for the translocation of E.(7,9) PrM is also associated with the E protein folding into the native conformation.(10) Additionally, PrM interacts with E to form PrM-E heterodimers and formed immature virions.(11) When the virus is released from the cell, the PrM protein is cleaved by furin or related protease in the
trans-Golgi apparatus and the proteolytic cleavage of PrM to M protein generates mature virions.(12) However, the functional role of PrM in replication, virus infectivity, and induction of neutralizing activity is not fully understood. In order to develop a useful tool for studying PrM/M protein of flavivirus, in the present study we have generated a hybridoma cell line secreting monoclonal antibodies (MAb) against JEV PrM/M protein and have investigated some of its characteristics. Materials and Methods Cell lines, virus, and other reagents Baby hamster kidney (BHK-21) cells and SP2/0 myeloma cells were cultured in RPMI-1640 medium (Hyclone, Beijing, China) supplemented with 10% fetal calf serum (PAA, Somerset, United Kingdom) and antibiotics (100 IU/mL penicillin and 0.1 mg/mL streptomycin). All cells were maintained in humidified 5% CO2 atmosphere at 37C. The JEV strain SA1414-2 was propagated in BHK-21 cells to prepare the antigen for WB and immunofluorescence assays. The JEV-positive sera were obtained from pigs reared in pig farms. The sera were first tested by indirect ELISA and latex agglutination test (LAT) for detection of antibodies to JEV. Animals Eight-week-old female BALB/c mice were purchased from the Experimental Animal Center of Harbin Veterinary
State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, P.R. China.
451
452
HUA AND BU
Total RNA was extracted from JEV-infected BHK-21 cells via TRIzol (Invitrogen, Carlsbad, CA), according to standard protocol, and the cDNA was produced with SuperscriptII RNAse H(-) reverse transcriptase (Invitrogen). In order to amplify the full-length gene of PrM from the cDNA template, a pair of primers, containing BamH I and Xho I sites, were designed according to the sequence of JEV SA14-14-2 strain (GenBank accession no. AF315119). The upstream primer was 5¢-GCAGGATCCATGAAGTTGTCGAATTTCC-3¢ and the downstream primer was 5¢-AATCTCGAGTTAACTGTAAG CCGGAGCGACCAACAGCAG-3¢ (restriction enzyme sites are underscored). The PCR products were purified with the TIANgel Midi Purification kit (Tiangen, Beijing, China), digested with BamH I and Xho I, and subsequently cloned into pET-30a (Novagen, Darmstadt, Germany). The resulting recombinant plasmid was confirmed by enzyme digestion and nucleotide sequencing.
Freund’s complete adjuvant (Sigma). The mice were then given two booster injections of purified protein emulsified with Freund’s incomplete adjuvant. A final intraperitoneal booster immunization of soluble PrM protein alone was given 3 days prior to the harvest of spleen cells for hybridoma fusion. General MAb preparation procedures were performed as described previously with minor modifications. Splenocytes from immunized animals were harvested aseptically and fused to the myeloma cell line SP2/0 with polyethylene glycol 50% (w/v) PEG 4000 (Sigma) at a splenocyte-myeloma cell ratio of 5:1. The fused cells were cultured and selected in HAT medium (RPMI medium with 10% fetal bovine serum, 100 mg/mL streptomycin, 100 IU/mL penicillin, 100 mM hypoxanthine, 16 mM thymidine, and 400 mM aminopterin); aminopterin was omitted from the medium on the twelfth day. Cell culture supernatants from the surviving clones were screened by indirect enzyme-linked immunosorbent assay (ELISA), and positive cell lines were subcloned thrice by limiting dilution method. To produce large quantities of the MAbs, selected hybridoma cells were injected intraperitoneally into pristine-primed mice, and ascitic fluids were collected 10 days after injections. Isotype of the MAbs was determined with a mouse MAb isotyping kit according to the manufacturer’s instructions (Hycult Biotechnology, Uden, The Netherlands).
Expression and purification of PrM/M protein
Western blot analysis
The confirmed recombinant plasmids were transformed into Escherichia coli BL21(DE3) (Novagen) cells for expression. Overnight cultures of the transformed cells were diluted 1:100 in 50 mL Luria-Bertani (LB) broth containing 100 mg/mL kanamycin at 37C. When OD600 reached 0.6, 1 mM isopropyl-D-thiogalactopyranoside (IPTG; Sigma, St. Louis, MO) was added to the broth to induce expression and the cells were incubated for a further 3 h with agitation. Thereafter, bacterial cells were removed from the growth medium by centrifugation at 5000 g for 15 min and lysed by sonication in cold phosphate-buffered saline (PBS; pH 7.4). To investigate whether the recombinant protein was expressed as soluble or inclusion body, sodium dodecyl sulfate-polyarcylamide gel electrophoresis (SDS-PAGE, 12%) was performed. Results showed that the His-tagged protein expressed predominantly as an inclusion body. To prepare antigen for immunization, we purified fusion protein by SDS-PAGE. Briefly, the inclusion bodies were resuspended in 2 mL PBS; the samples were then mixed with an equal volume of sample loading buffer (50 mM Tris-HCl [pH 6.8], 100 mM DTT, 2% SDS, 0.1% bromophenol blue, and 10% glycerol) and subjected to SDS-PAGE. By soaking in 0.25 M potassium chloride for a few minutes, a clear, white band representing NS1 protein can be seen on the gel. The band was then excised, triturated, and added to a proper volume of sterilized PBS. After freeze-thawing three times, supernatant was then centrifuged at 10000 r/min for 15 min collected and used for immunization and ELISA coating antigen.
MAbs were analyzed by Western blot analysis with infected cells and recombinant proteins expressed in E. coli to determine their specificity and reactivity. Samples were mixed with an equal volume of 2 · SDS sample loading buffer, boiled for 5 min, separated on 5% stacking/12% separating SDS polyacrylamide gels in a Tris-glycine buffer (0.025 M Tris base, 0.25 M glycine, 0.1% SDS), and transferred onto a nitrocellulose membrane with a Transblot apparatus (Bio-Rad, Hercules, CA). The membrane was washed three times in PBST with shaking, blocked with 5% skim milk at 4C overnight, and probed with MAbs at 37C for 1 h. Membranes were washed three times with PBST (0.1% Tween-20 in PBS) and incubated with anti-mouse Alexa Fluor 680-conjugated secondary antibodies (Invitrogen, Carlsbad, CA) for 1 h. Membranes were washed three times with PBST, then protein bands were detected with the Li-Cor Odyssey system (Li-Cor Biosciences, Lincoln, NE) and quantified using the Odyssey infrared imaging software (Li-Cor Biosciences).
Research Institute (Chinese Academy of Agricultural Sciences, Harbin, China). The experiments using animals were licensed by the Animal Ethics Committee of Harbin Veterinary Research Institute. Construction of recombinant plasmid bearing PrM/M gene of JEV
Generation, selection, and purification of PrM/M specific MAb Eight-week-old, female BALB/c mice were immunized subcutaneously with recombinant protein emulsified with
Indirect immunofluorescence assays BHK-21 cells were cultured as monolayers and infected with JEV. When 60 - 70% CPE was observed, cells were harvested and washed in PBS. Smears were coated with infect and mock-infect cells, air dried, and fixed with acetone. The MAbs were added to glass slides as primary antibody. The slides were incubated for 1 h at 37C in a moist chamber. After washing three times with gentle shaking in PBS, 100 mL FITC-labeled goat anti-mouse IgG (Sigma), diluted 1:200 in PBS, was added and incubated at 37C for 30 min. The smears were washed three times as described above, air dried, and mounted in glycerol. Finally, the cells were observed under the fluorescence microscopy (IMT2 Olympus, Tokyo, Japan).
MAb AGAINST JEV PrM/M PROTEIN
453
Epitope mapping of MAb To map the epitope of MAb against PrM/M protein of JEV, a set of 20 partially overlapping in length of 16 amino acids (with the exception of the last one, which was 15 in amino acid length) short peptides covering the whole PrM/M protein were synthesized (Table 1). The epitope peptide was screened by indirect ELISA using synthesized peptides as the coating antigen. Results Expression of PrM/M protein To detect the expression of JEV PrM in E. coli, the bacteria lysate were analyzed by SDS-PAGE. Results indicated that the recombinant protein was successfully expressed after IPTG induction, which mainly expressed in the form of an inclusion body. Western blot analysis results showed that the recombinant PrM protein could be recognized by sera against JEV (Fig. 1). Preparation of anti-PrM/M protein MAb Five female BALB/c mice were immunized with the fusion protein. After thrice immunization, the mouse that presented the highest antibody titer to the immunogen was selected and given a booster injection so that its spleen cells could be used in cell fusion. For screening with ELISA, three positive clones were obtained. In order to avoid crossreaction with His tag, His-E, a recombinant protein of His tag and JEV E protein prepared by our laboratory (data not published), was used as control. After three rounds of subcloning, one strain showed strong and specific reactivity to PrM protein and was designated as 3C8. After isotyping, it was found that 3C8 was of IgG1 type, and the light chains were k type (Fig. 2).
FIG. 1. Expression and Western blot analysis of JEV PrM protein. The bacteria harboring the recombinant plasmid were induced with IPTG and the bacterial protein was analyzed by SDS-PAGE. After transferring the protein to the NC membrane, it was probed with JEV-positive swine sera. M, protein marker; lane 1, bacterial protein harboring empty plasmid; lane 2, bacterial protein harboring PrM protein expressing plasmid; lane 3, bacterial protein harboring empty plasmid probed with JEV-positive swine sera; lane 4, bacterial protein harboring PrM protein expressing plasmid probed with JEV-positive swine sera.
Table 1. Epitope Screening of MAb 3C8 Against PrM/M Protein of JEV by ELISA Peptide sequence MKLSNFQGKLLMTINN KLLMTINNTDIADVIV TDIADVIVIPTSKGEN IPTSKGENRCWVRAID RCWVRAIDVGYMCEDT VGYMCEDTITYECPKL ITYECPKLTMGNDPED TMGNDPEDVDCWCDNQ VDCWCDNQEVYVQYGR EVYVQYGRCTRTRHSK CTRTRHSKRSRRSVSV RSRRSVSVQTHGESSL QTHGESSLVNKKEAWL VNKKEAWLDSTKATRY DSTKATRYLMKTENWI LMKTENWIIRNPGYAF IRNPGYAFLAAVLGWM LAAVLGWMLGSNNGQR LGSNNGQRVVFTILLL VVFTILLLLVAPAYS Negative control (no peptide coating)
Value of OD450 0.056 0.049 0.054 0.055 0.056 0.061 0.063 0.058 0.054 0.060 0.059 0.054 0.061 1.073 0.057 0.055 0.056 0.062 0.059 0.055 0.054
FIG. 2. Isotyping of monoclonal antibody. The isotype of the MAb 3C8 was identified with mouse MAb isotyping kit. The isotype is shown on the left. The developed dark point was the positive signal.
454
HUA AND BU
FIG. 3. Titration of MAb 3C8 by ELISA. The ascites of MAb 3C8 was titered with indirect ELISA. PBS was used as the negative control. The dilution titer of MAb 3C8 ascites was over 204,800.
Biological activity of MAb 3C8 Indirect ELISA was performed to measure the MAb titers. Results showed that the antibody titers of ascites were over 204,800 (Fig. 3). The specificity and reactivity were further determined by Western blot analysis using JEV-infected BHK21 cells. The results of Western blotting suggested that MAb 3C8 reacted with virus-infected cells (Fig. 4). These data revealed that the MAbs have good specificity and reactivity. To identify the ability of specific MAbs to recognize the native NS1 protein in JEV-infected cells, indirect immunofluorescence assay was carried out. A bright green signal could be
detected, showing that the MAb recognized the native PrM/ M protein (Fig. 5). Epitope mapping of MAb 3C8 With a set of overlapping synthesized peptides as coating antigen, the linear epitope of MAb 3C8 was mapped. Among these 20 peptides, MAb 3C8 was only strongly reactive with VNKKEAWLDSTKATRY. Other peptides, especially those containing eight amino acids overlapping with the epitope in the N and C termini, were not at all reactive with MAb 3C8 (Table 1). Discussion
FIG. 4. Western blot analysis shows the reaction to MAb 3C8 with JEV-infected BHK-21 cell. BHK-21 cells infected with JEV or mock infected were analyzed by SDS-PAGE and transferred to NC membrane, probed with the MAb 3C8, and developed with goat anti-mouse IgG. M, protein marker; lane 1, mock-infected cell lysate; lane 2, BHK-21-infected cel lysate.
Although JEV is mainly prevalent in tropical and subtropical regions of Asia, the epidemiological trend of the JEV outbreak has changed since the virus was isolated in Australia.(1,2) This suggests that JEV has spread to regions beyond its ecological boundaries and there is concern that JEV might become a global threat. In fact, it is not unusual to find two or more flaviviruses co-circulating in one area. In Southeast Asia, the most important flaviviruses are JEV and dengue virus (DENV).(13) In northern Australia, Kunjin virus is found to co-circulate with JEV.(14) In Vladivostok, Russia, studies have reported the detection of WNV in birds.(15) In addition, there is evidence of WNV infection in India from Japanese encephalitis endemic and nonendemic areas.(16) Flaviviruses, such as WNV, DENV, and JEV, share some common features, such as transmission via mosquitoes, and cross-react with each other in serological tests. These crossreactive responses could confound the interpretation during serological testing, including neutralization tests and ELISA.(17) Previous reports,(18,19) however, show that in Western blot (WB) prM protein may be used to serologically differentiate individuals infected with JEV from those infected with DENV, WNV and SLEV. Hence, prM and antibodies against prM would be useful for conducting seroepidemiological studies
MAb AGAINST JEV PrM/M PROTEIN
455
FIG. 5. Indirect immunofluorescence assay analysis shows the reaction of MAb 3C8 with native PrM/M on JEV-infected cells. (A) JEV-infected BHK-21 cells incubated with MAb 3C8. (B) Uninfected BHK-21 cells incubated with MAb 3C8.
of flavivirus infections in the regions that have prevalence of more than one flavivirus. One of the most powerful tools in the revelation of protein properties represents the use of MAbs, which have been successfully applied for antigenic analysis, clinical diagnosis, and treatments. There are few reports about the production of JEV PrM specific MAbs. In this study, using a prokaryotic expressed recombinant protein as antigen to immunize the mice, we developed an MAb against JEV PrM protein using the hybridoma technique. Our results indicated that the resulting MAb 3C8 recognized the PrM/M protein specifically. Indirect immunofluorescence assay showed that the MAb could react with native PrM/M protein in JEV-infected BHK21 cells. We believe that the MAb generated in this study would be important for delineating undiscovered characteristics and functions of PrM/M protein, and may also be useful for clinical application in the diagnosis and therapeutics for controlling JE. Acknowledgments This work was supported by a grant from the National Natural Science Foundation of China (30700027) and a special fund for agro-scientific research in the public interest of China (200803015). Author Disclosure Statement The authors have no financial interests to disclose. References 1. Hanna JN, Ritchie SA, Phillips DA, Shield J, Bailey MC, Mackenzie JS, Poidinger M, McCall BJ, and Mills PJ: An outbreak of Japanese encephalitis in the Torres Strait, Australia, 1995. Med J Aust 1996;165:256–260. 2. van den Hurk AF, Nisbet DJ, Johansen CA, Foley PN, Ritchie SA, and Mackenzie JS: Japanese encephalitis on Badu Island, Australia: the first isolation of Japanese encephalitis virus from Culex gelidus in the Australasian region and the role of mosquito host-feeding patterns in virus transmission cycles. Trans R Soc Trop Med Hyg 2001;95:595–600.
3. Solomon T, and Vaughn DW: Pathogenesis and clinical features of Japanese encephalitis and West Nile virus infections. Curr Top Microbiol Immunol 2002;267:171–94. 4. Solomon T: Control of Japanese encephalitis––within our grasp? N Engl J Med 2006;355:869–871. 5. Endy TP, and Nisalak A: Japanese encephalitis virus: ecology and epidemiology. Curr Top Microbiol Immunol 2002;267:11–48. 6. Vaughn DW, and Hoke CH Jr: The epidemiology of Japanese encephalitis: prospects for prevention. Epidemiol Rev 1992;14:197–221. 7. Chambers TJ, Hahn CS, Galler R, and Rice CM: Flavivirus genome organization, expression, and replication. Annu Rev Microbiol 1990;44:649–688. 8. Kim JM, Yun SI, Song BH, Hahn YS, Lee CH, Oh HW, and Lee YM: A single N-linked glycosylation site in the Japanese encephalitis virus prM protein is critical for cell type-specific prM protein biogenesis, virus particle release, and pathogenicity in mice. J Virol 2008;82:7846–7862. 9. Heinz FX, and Allison SL: Structures and mechanisms in flavivirus fusion. Adv Virus Res 2000;55:231–269. 10. Lorenz IC, Allison SL, Heinz FX, and Helenius A: Folding and dimerization of tick-borne encephalitis virus envelope proteins prM and E in the endoplasmic reticulum. J Virol 2002;76:5480–5491. 11. Wang S, He R, and Anderson R: PrM- and cell-binding domains of the dengue virus E protein. J Virol 1999;73:2547– 2551. 12. Stadler K, Allison SL, Schalich J, and Heinz FX: Proteolytic activation of tick-borne encephalitis virus by furin. J Virol 1997;71:8475–8481. 13. Mackenzie JS, Gubler DJ, and Petersen LR: Emerging flaviviruses: the spread and resurgence of Japanese encephalitis, West Nile and dengue viruses. Nat Med 2004;10: S98–109. 14. Hall RA, Scherret JH, and Mackenzie JS: Kunjin virus: an Australian variant of West Nile? Ann NY Acad Sci 2001;951: 153–160. 15. Kitai Y, Shoda M, Kondo T, and Konishi E: Epitope-blocking enzyme-linked immunosorbent assay to differentiate west nile virus from Japanese encephalitis virus infections in equine sera. Clin Vaccine Immunol 2007;14:1024–1031.
456 16. Thakare JP, Rao TL, and Padbidri VS: Prevalence of West Nile virus infection in India. Southeast Asian J Trop Med Publ Health 2002;33:801–805. 17. Kuno G: Serodiagnosis of flaviviral infections and vaccinations in humans. Adv Virus Res 2003;61:3–65. 18. Cardosa MJ, Wang SM, Sum MS, and Tio PH: Antibodies against prM protein distinguish between previous infection with dengue and Japanese encephalitis viruses. BMC Microbiol 2002;2:9. 19. Oceguera LF III, Patiris PJ, Chiles RE, Busch MP, Tobler LH, and Hanson CV: Flavivirus serology by Western blot analysis. Am J Trop Med Hyg 2007;77:159–163.
HUA AND BU Address correspondence to: Dr. Zhi-Gao Bu Harbin Veterinary Research Institute Chinese Academy of Agricultural Sciences Maduan Street #427 Nangang District Harbin 150001 P.R. China E-mail:
[email protected] Received: March 4, 2011 Accepted: May 9, 2011
