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Characterization of glutathione S-transferase from dust mite, Der p 8 and its immunoglobulin E cross-reactivity with cockroach glutathione S-transferase

Huang CH, Liew LM, Mah KW, Kuo IC, Lee BW, Chua KY

Clin Exp Allergy, 2006 Mar;36(3):369-76.
PMID: 16499649

Sensitization to mite and cockroach allergens is common, and diagnosis and therapy of allergy can be further complicated by the presence of allergen isoforms and panallergens. Purified recombinant and native allergens are useful for studies to resolve such problems.

Matched MeSH terms: Recombinant Proteins/immunology
Molecular cloning and immunoglobulin E reactivity of a natural rubber latex lecithinase homologue, the major allergenic component of Hev b 4

Sunderasan E, Bahari A, Arif SA, Zainal Z, Hamilton RG, Yeang HY

Clin Exp Allergy, 2005 Nov;35(11):1490-5.
PMID: 16297147 DOI: 10.1111/j.1365-2222.2005.02371.x

BACKGROUND:
Hev b 4 is an allergenic natural rubber latex (NRL) protein complex that is reactive in skin prick tests and in vitro immunoassays. On SDS-polyacrylamide gel electrophoresis (SDS-PAGE), Hev b 4 is discerned predominantly at 53-55 kDa together with a 57 kDa minor component previously identified as a cyanogenic glucosidase. Of the 13 NRL allergens recognized by the International Union of Immunological Societies, the 53-55 kDa Hev b 4 major protein is the only candidate that lacks complete cDNA and protein sequence information.

OBJECTIVE:
We sought to clone the transcript encoding the Hev b 4 major protein, and characterize the native protein and its recombinant form in relation to IgE binding.

METHODS:
The 5'/3' rapid amplification of cDNA ends method was employed to obtain the complete cDNA of the Hev b 4 major protein. A recombinant form of the protein was over-expressed in Escherichia coli. The native Hev b 4 major protein was deglycosylated by trifluoromethane sulphonic acid. Western immunoblots of the native, deglycosylated and recombinant proteins were performed using both polyclonal antibodies and sera from latex-allergic patients.

RESULTS:
The cDNA encoding the Hev b 4 major protein was cloned. Its open reading frame matched lecithinases in the conserved domain database and contained 10 predicted glycosylation sites. Detection of glycans on the Hev b 4 lecithinase homologue confirmed it to be a glycoprotein. The deglycosylated lecithinase homologue was discerned at 40 kDa on SDS-PAGE, this being comparable to the 38.53 kDa mass predicted by its cDNA. Deglycosylation of the lecithinase homologue resulted in the loss of IgE recognition, although reactivity to polyclonal rabbit anti-Hev b 4 was retained. IgE from latex-allergic patients also failed to recognize the non-glycosylated E. coli recombinant lecithinase homologue.

CONCLUSION:
The IgE epitopes of the Hev b 4 lecithinase homologue reside mainly in its carbohydrate moiety, which also account for the discrepancy between the observed molecular weight of the protein and the value calculated from its cDNA.

Matched MeSH terms: Recombinant Proteins/immunology
Detection of immunoglobulin antibodies in the sera of patients using purified latex allergens

Kurup VP, Yeang HY, Sussman GL, Bansal NK, Beezhold DH, Kelly KJ, et al.

Clin Exp Allergy, 2000 Mar;30(3):359-69.
PMID: 10691894

BACKGROUND: Latex allergy is largely an occupational allergy due to sensitization to natural rubber latex allergens present in a number of health care and household products. Although several purified allergens are currently available for study, information on the usefulness of these purified, native or recombinant allergens in the demonstration of specific immunoglobulin (Ig) E in the sera of patients is lacking.
OBJECTIVE: To evaluate the purified latex allergens and to demonstrate specific IgE antibody in the sera of health care workers and spina bifida patients with clinical latex allergy.
METHODS: Two radioallergosorbent and an enzyme-linked immunosorbent assay (ELISA) using latex proteins Hev b 1, 2, 3, 4, 6 and 7 along with two glove extracts and Malaysian nonammoniated latex (MNA) were evaluated to demonstrate IgE in the sera of health care workers and spina bifida with latex allergy and controls with no history of latex allergy.
RESULTS: ELISA using the purified latex allergens demonstrated specific IgE in 32-65% health care workers and 54-100% of spina bifida patients with latex allergy. The corresponding figures for RAST were 13-48 and 23-85 for RAST-1 and 19-61 and 36-57 for RAST-2. These results were comparable with the results obtained with glove extracts and crude rubber latex proteins.
CONCLUSIONS: When used simultaneously, latex proteins Hev b 2 and Hev b 7 reacted significantly with specific serum IgE in 80% of health care workers and 92% of spina bifida patients with latex allergy by ELISA technique, while this combination gave lower positivity when the RASTs were used. By the addition of Hev b 3, specific IgE was detected in all spina bifida patients with latex allergy. Both RASTs failed to show specific IgE in the control subjects, while the ELISA showed significant latex-specific IgE in 22% of controls.

Matched MeSH terms: Recombinant Proteins/immunology
Fulltext Sustained Virologic Response to a Dual Peginterferon alfa-2a and Ribavirin in Treating Chronic hepatitis C Infection: A Retrospective Cohort Study

Naing C, Sitt T, Aung AT, Aung K

Medicine (Baltimore), 2015 Jul;94(30):e1234.
PMID: 26222859 DOI: 10.1097/MD.0000000000001234

In Myanmar, hepatitis C virus (HCV) infection prevalence is 2%. A combination therapy of pegylated interferon alfa-2a and ribavirin (PEG-IFNa/RBV) is a standard treatment, but the effect of this antiviral therapy needs evaluation as to determine the efficacy and safety of dual PEG-IFNa/RBV therapy in treating patients infected with HCV in Myanmar.This was a retrospective analysis of data from a single clinic exclusively for gastrointestinal diseases in Yangon, Myanmar. We assessed treatment responses at the defined time points and stratified by genotypes of HCV. We also determined incidences of adverse events (AEs). We investigated independent predictors of sustained virologic response (SVR) in the participants.A total of 362 HCV-infected cases were included in this study. The majority were females (51.7%) with mean age of 47.12 years (±11.6) and noncirrhosis patients (82%). Rapid virologic response (RVR), early virologic response (EVR), end of treatment response (ETR), and SVR 24 weeks after completion of the dual treatment were 50.3% (178/362), 88% (314/357), 80.1% (286/357), and 85.6% (167/195), respectively. The most frequently reported AEs were nausea/anorexia (72.8%) and flu-like symptoms (62.4%). In multivariate analysis, 4 factors were independently associated with SVR; SVR to genotype 3 (odds ratio [OR] 2.4, 95% CI: 1.24-4.62), EVR (OR 0.54, 95% CI: 0.3-0.95), and duration of treatment (OR 1.52, 95% CI: 1.18-1.98). Study limitations were acknowledged.The efficacy and safety of the dual therapy in treating HCV-infected patient in Myanmar was acceptable. We recommend a prospective randomized control trial looking at duration of therapy and rates of achieving SVR, which could significantly impact the care of HCV-infected patients in Myanmar and perhaps other countries as well.

Matched MeSH terms: Recombinant Proteins/administration & dosage
New advances and potentials of fungal immunomodulatory proteins for therapeutic purposes

Ejike UC, Chan CJ, Okechukwu PN, Lim RLH

Crit Rev Biotechnol, 2020 Dec;40(8):1172-1190.
PMID: 32854547 DOI: 10.1080/07388551.2020.1808581

Fungal immunomodulatory proteins (FIPs) are fascinating small and heat-stable bioactive proteins in a distinct protein family due to similarities in their structures and sequences. They are found in fungi, including the fruiting bodies producing fungi comprised of culinary and medicinal mushrooms. Structurally, most FIPs exist as homodimers; each subunit consisting of an N-terminal α-helix dimerization and a C-terminal fibronectin III domain. Increasing numbers of identified FIPs from either different or same fungal species clearly indicates the growing research interests into its medicinal properties which include immunomodulatory, anti-inflammation, anti-allergy, and anticancer. Most FIPs increased IFN-γ production in peripheral blood mononuclear cells, potentially exerting immunomodulatory and anti-inflammatory effects by inhibiting overproduction of T helper-2 (Th2) cytokines common in an allergy reaction. Recently, FIP from Ganoderma microsporum (FIP-gmi) was shown to promote neurite outgrowth for potential therapeutic applications in neuro-disorders. This review discussed FIPs' structural and protein characteristics, their recombinant protein production for functional studies, and the recent advances in their development and applications as pharmaceutics and functional foods.

Matched MeSH terms: Recombinant Proteins/metabolism
Cloning and characterization of cDNAs encoding three isoforms of phospholipase A2 in Malayan spitting cobra (Naja naja sputatrix) venom

Armugam A, Earnest L, Chung MC, Gopalakrishnakone P, Tan CH, Tan NH, et al.

Toxicon, 1997 Jan;35(1):27-37.
PMID: 9028006

cDNAs encoding three phospholipase A2 (PLA2) isoforms in Naja naja sputatrix were cloned and characterized. One of them encoded an acidic PLA2 (APLA) while the others encoded neutral PLA2 (NPLA-1 and NPLA-2). The specific characteristics of APLA and NPLA were attributed to mutations at nt139 and nt328 from G to C and G to A, respectively, resulting in amino acid substitutions from Asp20 and 83 in APLA to His20 and Asn83 in NPLA. Amino acid sequencing of purified protein also showed the presence of this Asp20 and His20 in APLA and NPLA, respectively. The cDNA encoding one of the PLA2 (NAJPLA-2A), when expressed in Escherichia coli, yielded a protein that exhibited PLA2 activity.

Matched MeSH terms: Recombinant Proteins/metabolism
Fulltext Protection against henipaviruses in swine requires both, cell-mediated and humoral immune response

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
Synthesis, SAR elucidations and molecular docking study of newly designed isatin based oxadiazole analogs as potent inhibitors of thymidine phosphorylase

Javid MT, Rahim F, Taha M, Nawaz M, Wadood A, Ali M, et al.

Bioorg Chem, 2018 09;79:323-333.
PMID: 29803079 DOI: 10.1016/j.bioorg.2018.05.011

Thymidine phosphorylase is an enzyme involved in pyrimidine salvage pathway that is identical to platelet-derived endothelial cell growth factor (PD-ECGF) and gliostatin. It is enormously up regulated in a variety of solid tumors. Furthermore, surpassing of TP level protects tumor cells from apoptosis and helps cell survival. Thus TP is identified as a prime target for developing novel anticancer therapies. A new class of exceptionally potent isatin based oxadiazole (1-30) has been synthesized and evaluated for thymidine phosphorylase inhibitory potential. All analogs showed potent thymidine phosphorylase inhibition when compared with standard 7-Deazaxanthine, 7DX (IC50 = 38.68 ± 1.12 µM). Molecular docking study was performed in order to determine the binding interaction of these newly synthesized compounds, which revealed that these synthesized compounds established stronger hydrogen bonding network with active site of residues as compare to the standard compound 7DX.

Matched MeSH terms: Recombinant Proteins/metabolism
Fulltext Identification and validation of a novel panel of Plasmodium knowlesi biomarkers of serological exposure

Herman LS, Fornace K, Phelan J, Grigg MJ, Anstey NM, William T, et al.

PLoS Negl Trop Dis, 2018 Jun;12(6):e0006457.
PMID: 29902183 DOI: 10.1371/journal.pntd.0006457

BACKGROUND: Plasmodium knowlesi is the most common cause of malaria in Malaysian Borneo, with reporting limited to clinical cases presenting to health facilities and scarce data on the true extent of transmission. Serological estimations of transmission have been used with other malaria species to garner information about epidemiological patterns. However, there are a distinct lack of suitable serosurveillance tools for this neglected disease.
METHODOLOGY/PRINCIPAL FINDINGS: Using in silico tools, we designed and expressed four novel P. knowlesi protein products to address the distinct lack of suitable serosurveillance tools: PkSERA3 antigens 1 and 2, PkSSP2/TRAP and PkTSERA2 antigen 1. Antibody prevalence to these antigens was determined by ELISA for three time-points post-treatment from a hospital-based clinical treatment trial in Sabah, East Malaysia (n = 97 individuals; 241 total samples for all time points). Higher responses were observed for the PkSERA3 antigen 2 (67%, 65/97) across all time-points (day 0: 36.9% 34/92; day 7: 63.8% 46/72; day 28: 58.4% 45/77) with significant differences between the clinical cases and controls (n = 55, mean plus 3 SD) (day 0 p<0.0001; day 7 p<0.0001; day 28 p<0.0001). Using boosted regression trees, we developed models to classify P. knowlesi exposure (cross-validated AUC 88.9%; IQR 86.1-91.3%) and identified the most predictive antibody responses.
CONCLUSIONS/SIGNIFICANCE: The PkSERA3 antigen 2 had the highest relative variable importance in all models. Further validation of these antigens is underway to determine the specificity of these tools in the context of multi-species infections at the population level.

Matched MeSH terms: Recombinant Proteins/immunology
Recombinant VP9-based enzyme-linked immunosorbent assay for detection of immunoglobulin G antibodies to Banna virus (genus Seadornavirus)

Mohd Jaafar F, Attoui H, Gallian P, Isahak I, Wong KT, Cheong SK, et al.

J Virol Methods, 2004 Mar 01;116(1):55-61.
PMID: 14715307

Banna virus (BAV, genus Seadornavirus, family Reoviridae) is an arbovirus suspected to be responsible for encephalitis in humans. Two genotypes of this virus are distinguishable: A (Chinese isolate, BAV-Ch) and B (Indonesian isolate, BAV-In6969) which exhibit only 41% amino-acid identity in the sequence of their VP9. The VP7 to VP12 of BAV-Ch and VP9 of BAV-In6969 were expressed in bacteria using pGEX-4T-2 vector. VP9 was chosen to establish an ELISA for BAV, based mainly on two observations: (i). VP9 is a major protein in virus-infected cells and is a capsid protein (ii). among all the proteins expressed, VP9 was obtained in high amount and showed the highest immuno-reactivity to anti-BAV ascitic fluid. The VP9s ELISA was evaluated in three populations: French blood donors and two populations (blood donors and patients with a neurological syndrome) from Malaysia, representing the region where the virus was isolated in the past. The specificity of this ELISA was >98%. In mice injected with live BAV, the assay detected IgG-antibody to BAV infection 21 days post-injection, which was confirmed by Western blot using BAV-infected cells. The VP9 ELISA permits to determine the sero-status of a population without special safety precautions and without any requirements to propagate the BAV. This test should be a useful tool for epidemiological survey of BAV.

Matched MeSH terms: Recombinant Proteins/immunology
Display of the VP1 epitope of foot-and-mouth disease virus on bacteriophage T7 and its application in diagnosis

Wong CL, Sieo CC, Tan WS

J Virol Methods, 2013 Nov;193(2):611-9.
PMID: 23933075 DOI: 10.1016/j.jviromet.2013.07.053

Foot-and-mouth disease (FMD) is a highly contagious epidemic disease threatening the cattle industry since the sixteenth century. In recent years, the development of diagnostic assays for FMD has benefited considerably from the advances of recombinant DNA technology. In this study, the immunodominant region of the capsid protein VP1 of the foot-and-mouth disease virus (FMDV) was fused to the T7 bacteriophage and expressed on the surface of the bacteriophage capsid protein. The recombinant protein of about 42 kDa was detected by the anti-T7 tag monoclonal antibody in Western blot analysis. Phage ELISA showed that both the vaccinated and positive infected bovine sera reacted significantly with the recombinant T7 particle. This study demonstrated the potential of the T7 phage displaying the VP1 epitope as a diagnostic reagent.

Matched MeSH terms: Recombinant Proteins/genetics
Quantitative autoradiographic studies of relaxin binding in rat atria, uterus and cerebral cortex: characterization and effects of oestrogen treatment

Tan YY, Wade JD, Tregear GW, Summers RJ

Br J Pharmacol, 1999 May;127(1):91-8.
PMID: 10369460

The binding characteristics of the relaxin receptor in rat atria, uterus and cortex were studied using a [33P]-labelled human gene 2 relaxin (B33) and quantitative receptor autoradiography. The binding kinetics of [33P]-human gene 2 relaxin (B33) were investigated in slide-mounted rat atrial sections. The binding achieved equilibrium after 60 min incubation at room temperature (23+/-1 degrees C) and dissociated slowly. The association and dissociation rate constants were 4.31+/-0.34x10(8) M(-1) x min(-1) and 1.55+/-0.38x10(-3) min(-1) respectively. Thus, the kinetic dissociation constant was 3.46+/-0.59 pM. Binding was saturable to a single population of non-interacting sites throughout atria, in uterine myometrium and the 5th layer of cerebral cortex. The binding affinities (pK(D)) of [33P]-human gene 2 relaxin (B33) were 8.92+/-0.09 in atrial myocardium and 8.79+/-0.04 in cerebral cortex of male rats, and 8.79+/-0.10 in uterine myometrium. Receptor densities in the cerebral cortex and atria were higher than in uterine myometrium, indicating that relaxin also has important roles in non-reproductive tissues. In male rats, treatment with 17beta-oestradiol (20 microg in 0.1 ml sesame oil s.c., 18-24 h) significantly decreased the density of relaxin receptors in atria and cerebral cortex. Identical treatment in female rats had no significant effect in atria and cerebral cortex, but it significantly increased the density of relaxin receptors in uterine myometrium. Relaxin binding was competitively displaced by porcine and rat native relaxins. Porcine native relaxin binds to the relaxin receptor in male rat atria (8.90+/-0.02), and cerebral cortex (8.90+/-0.03) and uterine myometrium (8.89+/-0.03) with affinities not significantly different from human gene 2 (B33) relaxin. Nevertheless, rat relaxin binds to the receptors with affinities (8.35+/-0.09 in atria, 8.22+/-0.07 in cerebral cortex and 8.48+/-0.06 in uterine myometrium) significantly less than human gene 2 (B33) and porcine relaxins. Quantitative receptor autoradiography is the method of choice for measurement of affinities and densities of relaxin receptor in atria, uterine myometrium and cerebral cortex. High densities were found in all these tissues. 17beta-oestradiol treatment produced complex effects where it increased the densities of relaxin receptors in uterus but decreased those in atria and cerebral cortex of the male rats, and had no effect on the atria and cerebral cortex of the female rats.

Matched MeSH terms: Recombinant Proteins/metabolism
Fulltext Cloning and expression of highly pathogenic avian influenza virus full-length nonstructural gene in Pichia pastoris

Abubakar MB, Aini I, Omar AR, Hair-Bejo M

J Biomed Biotechnol, 2011;2011:414198.
PMID: 21541235 DOI: 10.1155/2011/414198

Avian influenza (AI) is a highly contagious and rapidly evolving pathogen of major concern to the poultry industry and human health. Rapid and accurate detection of avian influenza virus is a necessary tool for control of outbreaks and surveillance. The AI virus A/Chicken/Malaysia/5858/2004 (H5N1) was used as a template to produce DNA clones of the full-length NS1 genes via reverse transcriptase synthesis of cDNA by PCR amplification of the NS1 region. Products were cloned into pCR2.0 TOPO TA plasmid and subsequently subcloned into pPICZαA vector to construct a recombinant plasmid. Recombinant plasmid designated as pPICZαA-NS1 gene was confirmed by PCR colony screening, restriction enzyme digestion, and nucleotide sequence analysis. The recombinant plasmid was transformed into Pichia pastoris GS115 strain by electroporation, and expressed protein was identified by SDS-PAGE and western blotting. A recombinant protein of approximately ~28 kDa was produced. The expressed protein was able to bind a rabbit polyclonal antibody of nonstructural protein (NS1) avian influenza virus H5N1. The result of the western blotting and solid-phase ELISA assay using H5N1 antibody indicated that the recombinant protein produced retained its antigenicity. This further indicates that Pichia pastoris could be an efficient expression system for a avian influenza virus nonstructural (NS1).

Matched MeSH terms: Recombinant Proteins/metabolism
The capacity for long-chain polyunsaturated fatty acid synthesis in a carnivorous vertebrate: Functional characterisation and nutritional regulation of a Fads2 fatty acyl desaturase with Δ4 activity and an Elovl5 elongase in striped snakehead (Channa striata)

Kuah MK, Jaya-Ram A, Shu-Chien AC

Biochim. Biophys. Acta, 2015 Mar;1851(3):248-60.
PMID: 25542509 DOI: 10.1016/j.bbalip.2014.12.012

The endogenous production of long-chain polyunsaturated fatty acids (LC-PUFA) in carnivorous teleost species inhabiting freshwater environments is poorly understood. Although a predatory lifestyle could potentially supply sufficient LC-PUFA to satisfy the requirements of these species, the nutrient-poor characteristics of the freshwater food web could impede this advantage. In this study, we report the cloning and functional characterisation of an elongase enzyme in the LC-PUFA biosynthesis pathway from striped snakehead (Channa striata), which is a strict freshwater piscivore that shows high deposition of LC-PUFA in its flesh. We also functionally characterised a previously isolated fatty acyl desaturase cDNA from this species. Results showed that the striped snakehead desaturase is capable of Δ4 and Δ5 desaturation activities, while the elongase showed the characteristics of Elovl5 elongases. Collectively, these findings reveal that striped snakehead exhibits the genetic resources to synthesise docosahexaenoic acid (DHA; 22:6n-3) from eicosapentaenoic acid (EPA; 20:5n-3). Both genes are expressed at considerable levels in the brain and the liver. In liver, both genes were up-regulated by dietary C18 PUFA, although this increase did not correspond to a significant rise in the deposition of muscle LC-PUFA. Brain tissue of fish fed with plant oil diets showed higher expression of fads2 gene compared to fish fed with fish oil-based diet, which could ensure DHA levels remain constant under limited dietary DHA intake. This suggests the importance of DHA production from EPA via the ∆4 desaturation step in order to maintain an optimal reserve of DHA in the neuronal tissues of carnivores.

Matched MeSH terms: Recombinant Proteins/genetics; Recombinant Proteins/metabolism
Fulltext Helicobacter pylori recombinant UreG protein: cloning, expression, and assessment of its seroreactivity

Khalilpour A, Osman S, Yunus MH, Santhanam A, Vellasamy N, Noordin R

BMC Res Notes, 2014;7:809.
PMID: 25406411 DOI: 10.1186/1756-0500-7-809

Helicobacter pylori is a human pathogen and during the process of infection, antigens from the bacterium elicit strong host humoral immune responses. In our previous report, native H. pylori UreG protein showed good reactivity with sera from H. pylori patients. This study was aimed at producing the recombinant form of the protein (rUreG) and determining its seroreactivities.

Matched MeSH terms: Recombinant Proteins/genetics; Recombinant Proteins/immunology
Immunogenic characterization of the chimeric surface antigen 1 and 2 (SAG1/2) of Toxoplasma gondii expressed in the yeast Pichia pastoris

Lau YL, Thiruvengadam G, Lee WW, Fong MY

Parasitol Res, 2011 Sep;109(3):871-8.
PMID: 21455621 DOI: 10.1007/s00436-011-2315-6

In this study, we successfully expressed a chimerical surface antigen 1 and 2 (SAG1/2) of Toxoplasma gondii in Pichia pastoris. Eighty human serum samples, including 60 from confirmed cases of toxoplasmosis, were tested against the purified recombinant SAG1/2 in Western blots. Results of Western blots targeted at Toxoplasma IgG and IgM showed that the recombinant SAG1/2 reacted with all sera from the toxoplasmosis cases but none with the Toxoplasma-negative serum samples. These results showed that the P. pastoris-derived recombinant SAG1/2 was sensitive and specific and suitable for use as antigen for detecting anti-Toxoplasma antibodies. To further investigate the immunological characteristic of the recombinant protein, the recombinant SAG1/2 was injected subcutaneously into BALB/c mice, and their serum was tested against total protein lysate of T. gondii. Mice immunized with the recombinant SAG1/2 reacted specifically with the native SAG1 and SAG2 of T. gondii. Significant proliferation of splenocytes stimulated with tachyzoite total protein lysate was observed in vaccinated BALB/c mice but not in those from negative control mice. Specific production of IFN-γ, the Th1-type cytokines, was also found in stimulated splenocytes from vaccinated mice. These results show that the chimeric protein recombinant SAG1/2 can elicit a Th1-associated protection against T. gondii infections in mice. Finally, vaccinated mice were significantly protected against lethal challenge with live T. gondii RH strain tachyzoites (P

Matched MeSH terms: Recombinant Proteins/genetics; Recombinant Proteins/immunology
Immunogenicity and in vitro protective efficacy of recombinant Mycobacterium bovis bacille Calmette Guerin (rBCG) expressing the 19 kDa merozoite surface protein-1 (MSP-1(19)) antigen of Plasmodium falciparum

Nurul AA, Norazmi MN

Parasitol Res, 2011 Apr;108(4):887-97.
PMID: 21057812 DOI: 10.1007/s00436-010-2130-5

Vaccine development against the blood-stage malaria parasite is aimed at reducing the pathology of the disease. We constructed a recombinant Mycobacterium bovis bacille Calmette Guerin (rBCG) expressing the 19 kDa C-terminus of Plasmodium falciparum merozoite surface protein-1 (MSP-1(19)) to evaluate its protective ability against merozoite invasion of red blood cells in vitro. A mutated version of MSP-1(19), previously shown to induce the production of inhibitory but not blocking antibodies, was cloned into a suitable shuttle plasmid and transformed into BCG Japan (designated rBCG016). A native version of the molecule was also cloned into BCG (rBCG026). Recombinant BCG expressing the mutated version of MSP-1(19) (rBCG016) elicited enhanced specific immune response against the epitope in BALB/c mice as compared to rBCG expressing the native version of the epitope (rBCG026). Sera from rBCG016-immunized mice contained significant levels of specific IgG, especially of the IgG2a subclass, against MSP-1(19) as determined by enzyme-linked immunosorbent assay. The sera was reactive with fixed P. falciparum merozoites as demonstrated by indirect immunofluorescence assay (IFA) and inhibited merozoite invasion of erythrocytes in vitro. Furthermore, lymphocytes from rBCG016-immunized mice demonstrated higher proliferative response against the MSP-1(19) antigen as compared to those of rBCG026- and BCG-immunized animals. rBCG expressing the mutated version of MSP-1(19) of P. falciparum induced enhanced humoral and cellular responses against the parasites paving the way for the rational use of rBCG as a blood-stage malaria vaccine candidate.

Matched MeSH terms: Recombinant Proteins/genetics; Recombinant Proteins/immunology
Efficacy of VP2 protein expressed in E. coli for protection against highly virulent infectious bursal disease virus

Omar AR, Kim CL, Bejo MH, Ideris A

J Vet Sci, 2006 Sep;7(3):241-7.
PMID: 16871018

The ability of a heat-inactivated whole virus from a highly virulent infectious bursal disease virus (hvIBDV) and VP2 protein from hvIBDV expressed in E. coli provided protection against a hvIBDV challenge in specificpathogen-free (SPF) chickens. Six out of seven chickens that were injected three times with crude VP2 protein developed significant antibody titer against IBDV. However, only four out of the seven chickens survived the hvIBDV challenge. Despite showing low antibody titer profiles, all chickens immunized with the heat-inactivated whole virus also survived the challenged with hvIBDV. However, all of these chickens had bursal atrophy and mild to moderate depletion of lymphocytes. Thus, antibodies raised against IBDV VP2 protein expressed in E. coli and denatured IBDV proteins induced some degree of protection against mortality but not against bursal damage following challenge with hvIBDV.

Matched MeSH terms: Recombinant Proteins/genetics; Recombinant Proteins/immunology*
Construction and characterization of recombinant single-chain variable fragment antibodies against Toxoplasma gondii MIC2 protein

Hoe LN, Wan KL, Nathan S

Parasitology, 2005 Dec;131(Pt 6):759-68.
PMID: 16336729

The protozoan parasite Toxoplasma gondii produces a family of microneme proteins that are thought to play diverse roles in aiding the parasite's intracellular existence. Among these, TgMIC2 has a putative function in parasite adhesion to the host cell to initiate the invasion process. The invasion process may be localized and inhibited by monoclonal antibodies against the protein(s) involved. Here we report on the construction of a phage-displayed single-chain variable fragment (scFv) library from mice immunized with whole T. gondii parasites. The library was subsequently panned against recombinant TgMIC2 (rpTgMIC2) and 2 different groups of antibody clones were obtained, based on fingerprinting and sequencing data. The expressed recombinant scFv antibody was able to recognize rpTgMIC2 in a Western blot detection experiment. These results show that the phage display technology allows quick and effective production of monoclonal antibodies against parasite antigens. By panning the scFv-displayed library, we should be able to obtain a plethora of multi-functional scFv antibodies towards T. gondii proteins.

Matched MeSH terms: Recombinant Proteins/genetics; Recombinant Proteins/immunology
Rational discovery of dengue type 2 non-competitive inhibitors

Heh CH, Othman R, Buckle MJ, Sharifuddin Y, Yusof R, Rahman NA

Chem Biol Drug Des, 2013 Jul;82(1):1-11.
PMID: 23421589 DOI: 10.1111/cbdd.12122

Various works have been carried out in developing therapeutics against dengue. However, to date, no effective vaccine or anti-dengue agent has yet been discovered. The development of protease inhibitors is considered as a promising option, but most previous works have involved competitive inhibition. In this study, we focused on rational discovery of potential anti-dengue agents based on non-competitive inhibition of DEN-2 NS2B/NS3 protease. A homology model of the DEN-2 NS2B/NS3 protease (using West Nile Virus NS2B/NS3 protease complex, 2FP7, as the template) was used as the target, and pinostrobin, a flavanone, was used as the standard ligand. Virtual screening was performed involving a total of 13 341 small compounds, with the backbone structures of chalcone, flavanone, and flavone, available in the ZINC database. Ranking of the resulting compounds yielded compounds with higher binding affinities compared with the standard ligand. Inhibition assay of the selected top-ranking compounds against DEN-2 NS2B/NS3 proteolytic activity resulted in significantly better inhibition compared with the standard and correlated well with in silico results. In conclusion, via this rational discovery technique, better inhibitors were identified. This method can be used in further work to discover lead compounds for anti-dengue agents.

Matched MeSH terms: Recombinant Proteins/biosynthesis; Recombinant Proteins/genetics

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