Displaying publications 221 - 240 of 288 in total

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  1. Chem YK, Chua KB, Malik Y, Voon K
    Trop Biomed, 2015 Jun;32(2):344-51.
    PMID: 26691263 MyJurnal
    Monoclonal antibody-escape variant of dengue virus type 1 (MabEV DEN-1) was discovered and isolated in an outbreak of dengue in Klang Valley, Malaysia from December 2004 to March 2005. This study was done to investigate whether DEN152 (an isolate of MabEV DEN-1) is a product of recombination event or not. In addition, the non-synonymous mutations that correlate with the monoclonal antibody-escape variant were determined in this study. The genomes of DEN152 and two new DEN-1 isolates, DENB04 and DENK154 were completely sequenced, aligned, and compared. Phylogenetic tree was plotted and the recombination event on DEN152 was investigated. DEN152 is sub-grouped under genotype I and is closely related genetically to a DEN-1 isolated in Japan in 2004. DEN152 is not a recombinant product of any parental strains. Four amino acid substitutions were unique only to DEN 152. These amino acid substitutions were (Ser)[326](Leu), (Ser)[340](Leu) at the deduced E protein, (Ile)[250](Thr) at NS1 protein, and (Thr)[41](Ser) at NS5 protein. Thus, DEN152 is an isolate of the emerging monoclonal antibody-escape variant DEN-1 that escaped diagnostic laboratory detection.
    Matched MeSH terms: Viral Envelope Proteins/immunology*
  2. Gebriel AM, Subramaniam G, Sekaran SD
    Trop Biomed, 2006 Dec;23(2):194-207.
    PMID: 17322822 MyJurnal
    The detection of leptospires in patient blood in the first week of the disease using PCR provides an early diagnostic tool. PCR using two sets of primers (G1/G2 and B64-I/B64-II) tested with samples seeded with 23 leptospiral strains from pathogenic and non-pathogenic strains was able to amplify leptospiral DNA from pathogenic strains only. Of the 39 antibody negative samples collected from patients suspected for leptospirosis, only 1 sample (2.6%) was PCR positive. Using LSSP-PCR, the G2 primers allowed the characterization of Leptopira species to 10 different genetic signatures which may have epidemiological value in determining species involved in outbreaks. Leptospiral outer membrane proteins from three strains were purified and reacted against patients sera and gave rise to different profiles for pathogenic and non-pathogenic strains. Lymphocytes of mice injected with OMPs proliferated and released IFN(-3) when stimulated in vitro using Leptospira OMP as antigens. This suggests that an immune response could be established using leptospiral OMPs as a putative vaccine. OMPs were also used in a Dot-ELISA to detect antibodies against Leptospira pathogens in humans.
    Matched MeSH terms: Bacterial Outer Membrane Proteins/immunology*
  3. Pickering BS, Hardham JM, Smith G, Weingartl ET, Dominowski PJ, Foss DL, et al.
    Vaccine, 2016 09 14;34(40):4777-86.
    PMID: 27544586 DOI: 10.1016/j.vaccine.2016.08.028
    Hendra virus (HeV) and Nipah virus (NiV) are members of the genus Henipavirus, within the family Paramyxoviridae. Nipah virus has caused outbreaks of human disease in Bangladesh, Malaysia, Singapore, India and Philippines, in addition to a large outbreak in swine in Malaysia in 1998/1999. Recently, NiV was suspected to be a causative agent of an outbreak in horses in 2014 in the Philippines, while HeV has caused multiple human and equine outbreaks in Australia since 1994. A swine vaccine able to prevent shedding of infectious virus is of veterinary and human health importance, and correlates of protection against henipavirus infection in swine need to be better understood. In the present study, three groups of animals were employed. Pigs vaccinated with adjuvanted recombinant soluble HeV G protein (sGHEV) and challenged with HeV, developed antibody levels considered to be protective prior to the challenge (titers of 320). However, activation of the cell-mediated immune response was not detected, and the animals were only partially protected against challenge with 5×10(5) PFU of HeV per animal. In the second group, cross-neutralizing antibody levels against NiV in the sGHEV vaccinated animals did not reach protective levels, and with no activation of cellular immune memory, these animals were not protected against NiV. Only pigs orally infected with 5×10(4) PFU of NiV per animal were protected against nasal challenge with 5×10(5) PFU of NiV per animal. This group of pigs developed protective antibody levels, as well as cell-mediated immune memory. Peripheral blood mononuclear cells restimulated with UV-inactivated NiV upregulated IFN-gamma, IL-10 and the CD25 activation marker on CD4(+)CD8(+) T memory helper cells and to lesser extent on CD4(-)CD8(+) T cells. In conclusion, both humoral and cellular immune responses were required for protection of swine against henipaviruses.
    Matched MeSH terms: Recombinant Proteins/immunology
  4. Idris ZM, Chan CW, Mohammed M, Kalkoa M, Taleo G, Junker K, et al.
    Parasit Vectors, 2017 Apr 26;10(1):204.
    PMID: 28441959 DOI: 10.1186/s13071-017-2139-z
    BACKGROUND: Seroepidemiology can provide evidence for temporal changes in malaria transmission and is an important tool to evaluate the effectiveness of control interventions. During the early 2000s, Vanuatu experienced an acute increase in malaria incidence due to a lapse in funding for vector control. After the distribution of subsidised insecticide-treated nets (ITNs) resumed in 2003, malaria incidence decreased in the subsequent years. This study was conducted to find the serological evidence supporting the impact of ITN on exposure to Anopheles vector bites and parasite prevalence.

    METHODS: On Ambae Island, blood samples were collected from 231 and 282 individuals in 2003 and 2007, respectively. Parasite prevalence was determined by microscopy. Antibodies to three Plasmodium falciparum (PfSE, PfMSP-119, and PfAMA-1) and three Plasmodium vivax (PvSE, PvMSP-119, and PvAMA-1) antigens, as well as the Anopheles-specific salivary antigen gSG6, were detected by ELISA. Age-specific seroprevalence was analysed using a reverse catalytic modelling approach to estimate seroconversion rates (SCRs).

    RESULTS: Parasite rate decreased significantly (P 

    Matched MeSH terms: Insect Proteins/immunology
  5. Jesse FF, Ibrahim HH, Abba Y, Chung EL, Marza AD, Mazlan M, et al.
    BMC Vet Res, 2017 Apr 05;13(1):88.
    PMID: 28381248 DOI: 10.1186/s12917-017-1010-y
    BACKGROUND: Hemorrhagic septicemia is a fatal disease of cattle and buffaloes caused by P. multocida. Although the pathogenesis of the bacteria has been well established in literature, there is a paucity of information on the possible role of the bacteria and its immunogens; lipopolysaccharide (LPS) and outer membrane proteins (OMPs) on the reproductive capacity of buffalo heifers.

    METHODS: In this study, twenty one healthy prepubertal female buffaloes aged 8 months were divided into seven groups of 3 buffaloes each (G1-G7). Group 1 (G1) served as the negative control group and were inoculated orally with 10 mL sterile Phosphate Buffer Saline (PBS), groups 2 (G2) and 3 (G3) were inoculated orally and subcutaneously with 10 mL of 10(12) colony forming unit (cfu) of P.multocida type B: 2, while groups 4 (G4) and 5 (G5) received 10 mL of bacterial LPS orally and intravenously, respectively. Lastly, groups 6 (G6) and 7 (G7) were orally and subcutaneously inoculated with 10 mL of bacterial OMPs. Whole blood was collected in EDTA vials at stipulated time points (0, 2, 4, 6, 8, 10, 12, 24, 36, 48, 72, 120, 168, 216, 264, 312, 360, 408, 456 and 504 h), while tissue sections of the pituitary glands were collected and transported to the histopathology laboratory in 10% buffered formalin for processing and Hematoxylin and eosin staining. Plasma levels of luteinizing hormone (LH), follicle stimulating hormone (FSH), progesterone (PG), estradiol (EST) and gonadotrophin releasing hormone (GnRH) were determined.

    RESULTS: The histopathological lesions observed in the pituitary gland included hemorrhage, congestion, inflammatory cell infiltration, hydropic degeneration, necrosis and edema. These changes were higher (p 

    Matched MeSH terms: Bacterial Outer Membrane Proteins/immunology*
  6. Haque E, Banik U, Monwar T, Anthony L, Adhikary AK
    PLoS One, 2018;13(3):e0194516.
    PMID: 29590206 DOI: 10.1371/journal.pone.0194516
    Human adenovirus type 3 (HAdV-3) respiratory infections occurs worldwide in both children and adults, leading to severe morbidity and mortality, particularly in the paediatric age group and especially in neonates. During HAdV infection, neutralizing antibodies are formed against the epitopes located in the hyper variable regions (HVRs) of the hexon protein. These neutralizing antibodies provide protection against reinfection by viruses of the same type. Therefore it is reasonable to speculate that variations of HAdV-3 in the HVRs could impair the immunity acquired by previous infection with a different strain with variation in its HVRs. HAdV-3 has recently become the major agent of acute respiratory infection worldwide, being responsible for 15% to 87% of all adenoviral respiratory infections. However, despite the increased prevalence of HAdV-3 as respiratory pathogen, the diversity of hexon proteins in circulating strains remains unexplored. This study was designed to explore the variation in HVRs of hexon among globally distributed strains of HAdV-3 as well as to discover possible relationship among them, thus possibly shedding light on the cause for the increased prevalence of HAdV-3. In this study, for the first time we analysed the hexon proteins of all 248 available strains of HAdV-3 from the NCBI database and compared them with those of the HAdV-3 prototype (GB stain). We found that the HVRs of HAdV-3 strains circulating worldwide were highly heterogeneous and have been mutating continuously since -their original isolation. Based on their immense heterogeneity, the strains can be categorized into 25 hexon variants (3Hv-1 to 3Hv-25), 4 of which (3Hv-1 to 3Hv-4) comprises 80% of the strains. This heterogeneity may explain why HAdV-3 has become the most prevalent HAdVs type worldwide. The heterogeneity of hexon proteins also shows that the development of a vaccine against HAdV-3 might be challenging. The data on hexon variants provided here may be useful for the future epidemiological study of HAdV-3 infection.
    Matched MeSH terms: Capsid Proteins/immunology
  7. Kang AY, Park AY, Shin HJ, Khan NA, Maciver SK, Jung SY
    Exp Parasitol, 2018 Sep;192:19-24.
    PMID: 30031120 DOI: 10.1016/j.exppara.2018.07.009
    Amoebae from the genus Acanthamoeba are facultative pathogens of humans and other animals. In humans they most frequently infect the eye causing a sight threatening infection known as Acanthamoeba keratitis (AK), and also cause an often fatal encephalitis (GAE). A mannose-binding protein (MBP) has been identified as being important for Acanthamoeba infection especially in AK. This lectin has previously been characterized from Acanthamoeba castellanii as consisting of multiple 130 kDa subunits. MBP expression correlates with pathogenic potential and is expressed in a number of Acanthamoeba species. Here we report the purification of a similar lectin from Acanthamoeba culbertsoni and the production of a monoclonal antibody to it. The A. culbertsoni MBP was isolated by affinity chromatography using α-D-mannose agarose and has an apparent molecular weight of 83 kDa. The monoclonal antibody is an IgM that is useful in both western blots and immunofluorescence. We expect that this antibody will be useful in the study of the pathology of A. culbertsoni and in its identification in clinical samples.
    Matched MeSH terms: Protozoan Proteins/immunology*
  8. Habib MA, Yuen GC, Othman F, Zainudin NN, Latiff AA, Ismail MN
    Biochem. Cell Biol., 2017 04;95(2):232-242.
    PMID: 28177774 DOI: 10.1139/bcb-2016-0144
    The natural rubber latex extracted from the bark of Hevea brasiliensis plays various important roles in today's modern society. Following ultracentrifugation, the latex can be separated into 3 layers: C-serum, lutoids, and rubber particles. Previous studies have shown that a large number of proteins are present in these 3 layers. However, a complete proteome for this important plant is still unavailable. Protein sequences have been recently translated from the completed draft genome database of H. brasiliensis, leading to the creation of annotated protein databases of the following H. brasiliensis biosynthetic pathways: photosynthesis, latex allergens, rubberwood formation, latex biosynthesis, and disease resistance. This research was conducted to identify the proteins contained within the latex by way of de novo sequencing from mass spectral data obtained from the 3 layers of the latex. Peptides from these proteins were fragmented using collision-induced dissociation, higher-energy collisional dissociation, and electron-transfer dissociation activation methods. A large percentage of proteins from the biosynthetic pathways (63% to 100%) were successfully identified. In addition, a total of 1839 unique proteins were identified from the whole translated draft genome database (AnnHBM).
    Matched MeSH terms: Plant Proteins/immunology
  9. Pushparajah V, Fatima A, Chong CH, Gambule TZ, Chan CJ, Ng ST, et al.
    Sci Rep, 2016 07 27;6:30010.
    PMID: 27460640 DOI: 10.1038/srep30010
    Lignosus rhinocerotis (Tiger milk mushroom) is an important folk medicine for indigenous peoples in Southeast Asia. We previously reported its de novo assembled 34.3 Mb genome encoding a repertoire of proteins including a putative bioactive fungal immunomodulatory protein. Here we report the cDNA of this new member (FIP-Lrh) with a homology range of 54-64% to FIPs from other mushroom species, the closest is with FIP-glu (LZ-8) (64%) from Ganoderma lucidum. The FIP-Lrh of 112 amino acids (12.59 kDa) has a relatively hydrophobic N-terminal. Its predicted 3-dimensional model has identical folding patterns to FIP-fve and contains a partially conserved and more positively charged carbohydrates binding pocket. Docking predictions of FIP-Lrh on 14 glycans commonly found on cellular surfaces showed the best binding energy of -3.98 kcal/mol to N-acetylgalactosamine and N-acetylglucosamine. Overexpression of a 14.9 kDa soluble 6xHisFIP-Lrh was achieved in pET-28a(+)/BL21 and the purified recombinant protein was sequence verified by LC-MS/MS (QTOF) analysis. The ability to haemagglutinate both mouse and human blood at concentration ≥0.34 μM, further demonstrated its lectin nature. In addition, the cytotoxic effect of 6xHisFIP-Lrh on MCF-7, HeLa and A549 cancer cell lines was detected at IC50 of 0.34 μM, 0.58 μM and 0.60 μM, respectively.
    Matched MeSH terms: Fungal Proteins/immunology*
  10. Chua CL, Sam IC, Merits A, Chan YF
    PLoS Negl Trop Dis, 2016 08;10(8):e0004960.
    PMID: 27571254 DOI: 10.1371/journal.pntd.0004960
    BACKGROUND: Chikungunya virus (CHIKV) is a re-emerging mosquito-borne virus which causes epidemics of fever, severe joint pain and rash. Between 2005 and 2010, the East/Central/South African (ECSA) genotype was responsible for global explosive outbreaks across India, the Indian Ocean and Southeast Asia. From late 2013, Asian genotype CHIKV has caused outbreaks in the Americas. The characteristics of cross-antibody efficacy and epitopes are poorly understood.

    METHODOLOGY/PRINCIPAL FINDINGS: We characterized human immune sera collected during two independent outbreaks in Malaysia of the Asian genotype in 2006 and the ECSA genotype in 2008-2010. Neutralizing capacity was analyzed against representative clinical isolates as well as viruses rescued from infectious clones of ECSA and Asian CHIKV. Using whole virus antigen and recombinant E1 and E2 envelope glycoproteins, we further investigated antibody binding sites, epitopes, and antibody titers. Both ECSA and Asian sera demonstrated stronger neutralizing capacity against the ECSA genotype, which corresponded to strong epitope-antibody interaction. ECSA serum targeted conformational epitope sites in the E1-E2 glycoprotein, and E1-E211K, E2-I2T, E2-H5N, E2-G118S and E2-S194G are key amino acids that enhance cross-neutralizing efficacy. As for Asian serum, the antibodies targeting E2 glycoprotein correlated with neutralizing efficacy, and I2T, H5N, G118S and S194G altered and improved the neutralization profile. Rabbit polyclonal antibody against the N-terminal linear neutralizing epitope from the ECSA sequence has reduced binding capacity and neutralization efficacy against Asian CHIKV. These findings imply that the choice of vaccine strain may impact cross-protection against different genotypes.

    CONCLUSION/SIGNIFICANCE: Immune serum from humans infected with CHIKV of either ECSA or Asian genotypes showed differences in binding and neutralization characteristics. These findings have implications for the continued outbreaks of co-circulating CHIKV genotypes and effective design of vaccines and diagnostic serological assays.

    Matched MeSH terms: Viral Envelope Proteins/immunology*
  11. Hempolchom C, Sookrung N, Srisuka W, Reamtong O, Sakolvaree Y, Chaicumpa W, et al.
    Parasitol Res, 2019 Aug;118(8):2353-2359.
    PMID: 31263951 DOI: 10.1007/s00436-019-06383-x
    Simulium dermatitis is an IgE-mediated skin reaction in animals and humans caused by the bites of black flies. Although Simulium nigrogilvum has been incriminated as the main human-biting black fly species in Thailand, information on its salivary allergens is lacking. Salivary gland extract of S. nigrogilvum females was subjected to sodium dodecylsulfate-polyacrylamide gel electrophoresis, and the separated components were applied onto nitrocellulose membranes for immunoblotting, which was performed by probing the protein blots with sera from 17 individuals who were allergic to the bites of S. nigrogilvum. IgE-reactive protein bands were characterized further by liquid chromatography-mass spectrometry (LC-MS/MS) analysis. Nine protein bands (79, 42, 32, 25, 24, 22, 15, 13, and 11 kDa) were recognized in the serum of the subjects. Four of the nine protein bands (32, 24, 15, and 11 kDa) showed IgE reactivity in all (100%) of the tested sera, and they were identified as salivary secreted antigen 5-related protein, salivary serine protease, erythema protein, and hypothetical secreted protein, respectively. Three other proteins, salivary serine protease (25 kDa), salivary D7 secreted protein (22 kDa), and hypothetical protein (13 kDa), reacted with > 50% of the sera. The relevance of the identified protein bands as allergens needs to be confirmed by using pure recombinant proteins, either in the in vivo skin prick test or in vitro detection of the specific IgE in the serum samples of allergic subjects. This will be useful for the rational design of component-resolved diagnosis and allergen immunotherapy for the allergy mediated by the bites of black flies.
    Matched MeSH terms: Insect Proteins/immunology
  12. Oyong DA, Wilson DW, Barber BE, William T, Jiang J, Galinski MR, et al.
    J Infect Dis, 2019 11 06;220(12):1950-1961.
    PMID: 31419296 DOI: 10.1093/infdis/jiz407
    BACKGROUND: Complement-fixing antibodies are important mediators of protection against Plasmodium falciparum malaria. However, complement-fixing antibodies remain uncharacterized for Plasmodium vivax malaria. P. vivax merozoite surface protein 3α (PvMSP3α) is a target of acquired immunity and a potential vaccine candidate.

    METHODS: Plasma from children and adults with P. vivax malaria in Sabah, Malaysia, were collected during acute infection, 7 and 28 days after drug treatment. Complement-fixing antibodies and immunoglobulin M and G (IgM and IgG), targeting 3 distinctive regions of PvMSP3α, were measured by means of enzyme-linked immunosorbent assay.

    RESULTS: The seroprevalence of complement-fixing antibodies was highest against the PvMSP3α central region (77.6%). IgG1, IgG3, and IgM were significantly correlated with C1q fixation, and both purified IgG and IgM were capable of mediating C1q fixation to PvMSP3α. Complement-fixing antibody levels were similar between age groups, but IgM was predominant in children and IgG3 more prevalent in adults. Levels of functional antibodies increased after acute infection through 7 days after treatment but rapidly waned by day 28.

    CONCLUSION: Our study demonstrates that PvMSP3α antibodies acquired during P. vivax infection can mediate complement fixation and shows the important influence of age in shaping these specific antibody responses. Further studies are warranted to understand the role of these functional antibodies in protective immunity against P. vivax malaria.

    Matched MeSH terms: Protozoan Proteins/immunology*
  13. Johnson RB, Dawkins HJ, Spencer TL, Saharee AA, Bahaman AR, Ramdani, et al.
    Res Vet Sci, 1989 Sep;47(2):277-9.
    PMID: 2508206
    ELISA and immunoblotting techniques were used to examine the humoral immune response to Pasteurella multocida, in bovine sera from Indonesia and Malaysia. Elevated levels of antibody to a crude lipopolysaccharide preparation were found in vaccinated animals. In addition to the response to lipopolysaccharide, antibodies from the vaccinated cattle strongly labelled five to six of the 40 protein bands in this organism.
    Matched MeSH terms: Bacterial Proteins/immunology
  14. Hajissa K, Zakaria R, Suppian R, Mohamed Z
    BMC Infect Dis, 2017 12 29;17(1):807.
    PMID: 29284420 DOI: 10.1186/s12879-017-2920-9
    BACKGROUND: The inefficiency of the current tachyzoite antigen-based serological assays for the serodiagnosis of Toxoplasma gondii infection mandates the need for acquirement of reliable and standard diagnostic reagents. Recently, epitope-based antigens have emerged as an alternative diagnostic marker for the achievement of highly sensitive and specific capture antigens. In this study, the diagnostic utility of a recombinant multiepitope antigen (USM.TOXO1) for the serodiagnosis of human toxoplasmosis was evaluated.

    METHODS: An indirect enzyme-linked immunosorbent assay (ELISA) was developed to evaluate the usefulness of USM.TOXO1 antigen for the detection of IgG antibodies against Toxoplasma gondii in human sera. Whereas the reactivity of the developed antigen against IgM antibody was evaluated by western blot and Dot enzyme immunoassay (dot-EIA) analysis.

    RESULTS: The diagnostic performance of the new antigens in IgG ELISA was achieved at the maximum values of 85.43% and 81.25% for diagnostic sensitivity and specificity respectively. The USM.TOXO1 was also proven to be reactive with anti- T. gondii IgM antibody.

    CONCLUSIONS: This finding makes the USM.TOXO1 antigen an attractive candidate for improving the toxoplasmosis serodiagnosis and demonstrates that multiepitope antigens could be a potential and promising diagnostic marker for the development of high sensitive and accurate assays.

    Matched MeSH terms: Recombinant Proteins/immunology
  15. Yeo CH, Hsien YC, Abdullah MS, Telesinghe PU, Ramasamy R
    Singapore Med J, 2009 Apr;50(4):371-7.
    PMID: 19421680
    Little or no information is available on the prevalence of nasopharyngeal carcinoma (NPC) among different ethnic groups in Brunei, or how useful plasma IgA antibodies are against viral capsid antigen (VCA) and early antigen (EA) in the diagnosis of NPC, even though they are routinely measured in patients suspected to have NPC.
    Matched MeSH terms: Capsid Proteins/immunology*
  16. Chen RE, Smith BK, Errico JM, Gordon DN, Winkler ES, VanBlargan LA, et al.
    Cell Host Microbe, 2021 Nov 10;29(11):1634-1648.e5.
    PMID: 34610295 DOI: 10.1016/j.chom.2021.09.006
    Although divergent dengue viruses (DENVs) have been isolated in insects, nonhuman primates, and humans, their relationships to the four canonical serotypes (DENV 1-4) are poorly understood. One virus isolated from a dengue patient, DKE-121, falls between genotype and serotype levels of sequence divergence to DENV-4. To examine its antigenic relationship to DENV-4, we assessed serum neutralizing and protective activity. Whereas DENV-4-immune mouse sera neutralize DKE-121 infection, DKE-121-immune sera inhibit DENV-4 less efficiently. Passive transfer of DENV-4 or DKE-121-immune sera protects mice against homologous, but not heterologous, DENV-4 or DKE-121 challenge. Antigenic cartography suggests that DENV-4 and DKE-121 are related but antigenically distinct. However, DENV-4 vaccination confers protection against DKE-121 in nonhuman primates, and serum from humans immunized with a tetravalent vaccine neutralize DENV-4 and DKE-121 infection equivalently. As divergent DENV strains, such as DKE-121, may meet criteria for serotype distinction, monitoring their capacity to impact dengue disease and vaccine efficacy appears warranted.
    Matched MeSH terms: Viral Envelope Proteins/immunology
  17. Fang CM, Zainuddin ZF, Musa M, Thong KL
    Protein Expr Purif, 2006 Jun;47(2):341-7.
    PMID: 16510294 DOI: 10.1016/j.pep.2005.12.007
    Tuberculosis remains a major infectious disease with over 8 million new cases and 2 million deaths annually. Therefore, a vaccine more potent than BCG is desperately needed. In this regard, an approximately 800 bp DNA encoding a mycobacterial synthetic gene designated as VacIII (containing ubiquitin gene UbGR and four immunogenic mycobacterial epitopes or genes of ESAT-6, Phos1, Hsp 16.3, and Mtb8.4) was sub-cloned into a bacterial expression vector of pRSET-B resulting in a 6 x His-VacIII fusion gene construction. This recombinant clone was over expressed in Escherichia coli BL-21 (DE-3). The expressed fusion protein was found almost entirely in the insoluble form (inclusion bodies) in cell lysate. The inclusion bodies were solubilized with 8M urea and the recombinant protein was purified by Ni-NTA column and dialyzed by urea gradient dialysis. This method produced a relatively high yield of recombinant VacIII protein and the cloned VacIII gene offers the potential development of other vaccine formats such as DNA vaccine and recombinant vaccine.
    Matched MeSH terms: Recombinant Fusion Proteins/immunology
  18. Lim CC, Woo PCY, Lim TS
    Sci Rep, 2019 Apr 15;9(1):6088.
    PMID: 30988390 DOI: 10.1038/s41598-019-42628-6
    Antibody phage display has been pivotal in the quest to generate human monoclonal antibodies for biomedical and research applications. Target antigen preparation is a main bottleneck associated with the panning process. This includes production complexity, downstream purification, quality and yield. In many instances, purified antigens are preferred for panning but this may not be possible for certain difficult target antigens. Here, we describe an improved procedure of affinity selection against crude or non-purified antigen by saturation of non-binders with blocking agents to promote positive binder enrichment termed as Yin-Yang panning. A naïve human scFv library with kappa light chain repertoire with a library size of 109 was developed. The improved Yin-Yang biopanning process was able to enrich monoclonal antibodies specific to the MERS-CoV nucleoprotein. Three unique monoclonal antibodies were isolated in the process. The Yin-Yang biopanning method highlights the possibility of utilizing crude antigens for the isolation of monoclonal antibodies by phage display.
    Matched MeSH terms: Nucleocapsid Proteins/immunology*
  19. Chin CF, Lai JY, Choong YS, Anthony AA, Ismail A, Lim TS
    Sci Rep, 2017 05 19;7(1):2176.
    PMID: 28526816 DOI: 10.1038/s41598-017-01987-8
    Hemolysin E (HlyE) is an immunogenic novel pore-forming toxin involved in the pathogenesis of typhoid fever. Thus, mapping of B-cell epitopes of Salmonella enterica serovar Typhi (S. Typhi) is critical to identify key immunogenic regions of HlyE. A random 20-mer peptide library was used for biopanning with enriched anti-HlyE polyclonal antibodies from typhoid patient sera. Bioinformatic tools were used to refine, analyze and map the enriched peptide sequences against the protein to identify the epitopes. The analysis identified both linear and conformational epitopes on the HlyE protein. The predicted linear GAAAGIVAG and conformational epitope PYSQESVLSADSQNQK were further validated against the pooled sera. The identified epitopes were then used to isolate epitope specific monoclonal antibodies by antibody phage display. Monoclonal scFv antibodies were enriched for both linear and conformational epitopes. Molecular docking was performed to elucidate the antigen-antibody interaction of the monoclonal antibodies against the epitopes on the HlyE monomer and oligomer structure. An in-depth view of the mechanistic and positional characteristics of the antibodies and epitope for HlyE was successfully accomplished by a combination of phage display and bioinformatic analysis. The predicted function and structure of the antibodies highlights the possibility of utilizing the antibodies as neutralizing agents for typhoid fever.
    Matched MeSH terms: Hemolysin Proteins/immunology*
  20. Teoh PG, Ooi AS, AbuBakar S, Othman RY
    J Biomed Biotechnol, 2009;2009:781712.
    PMID: 19325913 DOI: 10.1155/2009/781712
    A Cucumber green mottle mosaic virus (CGMMV) was used to present a truncated dengue virus type 2 envelope (E) protein binding region from amino acids 379 to 423 (EB4). The EB4 gene was inserted at the terminal end of the CGMMV coat protein (CP) open reading frame (ORF). Read-through sequences of TMV or CGMMV, CAA-UAG-CAA-UUA, or AAA-UAG-CAA-UUA were, respectively, inserted in between the CP and the EB4 genes. The chimeric clones, pRT, pRG, and pCG+FSRTRE, were transcribed into full-length capped recombinant CGMMV transcripts. Only constructs with the wild-type CGMMV read-through sequence yielded infectious viruses following infection of host plant, muskmelon (Cucumis melo) leaves. The ratio of modified to unmodified CP for the read-through expression clone developed was also found to be approximately 1:1, higher than what has been previously reported. It was also observed that infectivity was not affected by differences in pI between the chimera and its wild counterpart. Analysis of recombinant viruses after 21-days-postinculation (dpi) revealed that deletions occurred resulting in partial reversions of the viral population to near wild type and suggesting that this would be the limiting harvest period for obtaining true to type recombinants with this construct.
    Matched MeSH terms: Viral Envelope Proteins/immunology
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