Displaying publications 1 - 20 of 39 in total

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  1. Fulltext Antiviral cationic peptides as a strategy for innovation in global health therapeutics for dengue virus: high yield production of the biologically active recombinant plectasin peptide
    Rothan HA, Mohamed Z, Suhaeb AM, Rahman NA, Yusof R
    OMICS, 2013 Nov;17(11):560-7.
    PMID: 24044366 DOI: 10.1089/omi.2013.0056
    Dengue virus infects millions of people worldwide, and there is no vaccine or anti-dengue therapeutic available. Antimicrobial peptides have been shown to possess effective antiviral activity against various viruses. One of the main limitations of developing these peptides as potent antiviral drugs is the high cost of production. In this study, high yield production of biologically active plectasin peptide was inexpensively achieved by producing tandem plectasin peptides as inclusion bodies in E. coli. Antiviral activity of the recombinant peptide towards dengue serotype-2 NS2B-NS3 protease (DENV2 NS2B-NS3pro) was assessed as a target to inhibit dengue virus replication in Vero cells. Single units of recombinant plectasin were collected after applying consecutive steps of refolding, cleaving by Factor Xa, and nickel column purification to obtain recombinant proteins of high purity. The maximal nontoxic dose (MNTD) of the recombinant peptide against Vero cells was 20 μM (100 μg/mL). The reaction velocity of DENV2 NS2B-NS3pro decreased significantly after increasing concentrations of recombinant plectasin were applied to the reaction mixture. Plectasin peptide noncompetitively inhibited DENV2 NS2B-NS3pro at Ki value of 5.03 ± 0.98 μM. The percentage of viral inhibition was more than 80% at the MNTD value of plectasin. In this study, biologically active recombinant plectasin which was able to inhibit dengue protease and viral replication in Vero cells was successfully produced in E. coli in a time- and cost- effective method. These findings are potentially important in the development of potent therapeutics against dengue infection.
    Matched MeSH terms: Recombinant Fusion Proteins/genetics
  2. Fulltext Cell-Based High-Throughput Screening Protocol for Discovering Antiviral Inhibitors Against SARS-COV-2 Main Protease (3CLpro)
    Rothan HA, Teoh TC
    Mol Biotechnol, 2021 Mar;63(3):240-248.
    PMID: 33464543 DOI: 10.1007/s12033-021-00299-7
    The global public health has been compromised since the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) emerged in late December 2019. There are no specific antiviral drugs available to combat SARS-CoV-2 infection. Besides the rapid dissemination of SARS-CoV-2, several variants have been identified with a potential epidemiologic and pathogenic variation. This fact has forced antiviral drug development strategies to stay innovative, including new drug discovery protocols, combining drugs, and establishing new drug classes. Thus, developing novel screening methods and direct-targeting viral enzymes could be an attractive strategy to combat SARS-CoV-2 infection. In this study, we designed, optimized, and validated a cell-based assay protocol for high-throughput screening (HTS) antiviral drug inhibitors against main viral protease (3CLpro). We applied the split-GFP complementation to develop GFP-split-3CLpro HTS system. The system consists of GFP-based reporters that become fluorescent upon cleavage by SARS-CoV-2 protease 3CLpro. We generated a stable GFP-split-3CLpro HTS system valid to screen large drug libraries for inhibitors to SARS-CoV-2 main protease in the bio-safety level 2 laboratory, providing real-time antiviral activity of the tested compounds. Using this assay, we identified a new class of viral protease inhibitors derived from quinazoline compounds that worth further in vitro and in vivo validation.
    Matched MeSH terms: Recombinant Fusion Proteins/genetics
  3. Fulltext Characterisation of Drosophila Ubx CPTI000601 and hth CPTI000378 protein trap lines
    Choo SW, Beh CY, Russell S, White R
    ScientificWorldJournal, 2014;2014:191535.
    PMID: 25389534 DOI: 10.1155/2014/191535
    In Drosophila, protein trap strategies provide powerful approaches for the generation of tagged proteins expressed under endogenous control. Here, we describe expression and functional analysis to evaluate new Ubx and hth protein trap lines generated by the Cambridge Protein Trap project. Both protein traps exhibit spatial and temporal expression patterns consistent with the reported endogenous pattern in the embryo. In imaginal discs, Ubx-YFP is expressed throughout the haltere and 3rd leg imaginal discs, while Hth-YFP is expressed in the proximal regions of haltere and wing discs but not in the pouch region. The Ubx (CPTI000601) line is semilethal as a homozygote. No T3/A1 to T2 transformations were observed in the embryonic cuticle or the developing midgut. The homozygous survivors, however, exhibit a weak haltere phenotype with a few wing-like marginal bristles on the haltere capitellum. Although hth (CPTI000378) is completely lethal as a homozygote, the hth (CPTI000378) /hth (C1) genotype is viable. Using a hth deletion (Df(3R)BSC479) we show that hth (CPTI000378) /Df(3R)BSC479 adults are phenotypically normal. No transformations were observed in hth (CPTI000378), hth (CPTI000378) /hth (C1), or hth (CPTI000378) /Df(3R)BSC479 embryonic cuticles. We have successfully characterised the Ubx-YFP and Hth-YFP protein trap lines demonstrating that the tagged proteins show appropriate expression patterns and produce at least partially functional proteins.
    Matched MeSH terms: Recombinant Fusion Proteins/genetics*
  4. Cloning and in silico characterization of two signal peptides from Pediococcus pentosaceus and their function for the secretion of heterologous protein in Lactococcus lactis
    Baradaran A, Sieo CC, Foo HL, Illias RM, Yusoff K, Rahim RA
    Biotechnol Lett, 2013 Feb;35(2):233-8.
    PMID: 23076361 DOI: 10.1007/s10529-012-1059-4
    Fifty signal peptides of Pediococcus pentosaceus were characterized by in silico analysis and, based on the physicochemical analysis, (two potential signal peptides Spk1 and Spk3 were identified). The coding sequences of SP were amplified and fused to the gene coding for green fluorescent protein (GFP) and cloned into Lactococcus lactis pNZ8048 and pMG36e vectors, respectively. Western blot analysis indicated that the GFP proteins were secreted using both heterologous SPs. ELISA showed that the secretion efficiency of GFP using Spk1 (0.64 μg/ml) was similar to using Usp45 (0.62 μg/ml) and Spk3 (0.58 μg/ml).
    Matched MeSH terms: Recombinant Fusion Proteins/genetics
  5. Cloning, expression, and characterization of recombinant Hev b 3, a Hevea brasiliensis protein associated with latex allergy in patients with spina bifida
    Wagner B, Krebitz M, Buck D, Niggemann B, Yeang HY, Han KH, et al.
    J Allergy Clin Immunol, 1999 Nov;104(5):1084-92.
    PMID: 10550757
    BACKGROUND: Two natural rubber latex proteins, Hev b 1 and Hev b 3, have been described in spina bifida (SB)-associated latex allergy.

    OBJECTIVE: The aim of this study was to clone and express Hev b 3 and to obtain the immunologic active and soluble recombinant allergen for diagnosis of SB-associated latex allergy.

    METHODS: A complementary DNA (cDNA) coding for Hev b 3 was amplified from RNA of fresh latex collected from Malaysian rubber trees (Hevea brasiliensis). PCR primers were designed according to sequences of internal peptide fragments of natural (n) Hev b 3. The 5'-end sequence was obtained by specific amplification of cDNA ends. The recombinant (r) Hev b 3 was produced in Escherichia coli as a 6xHis tagged protein. Immunoblotting and inhibition assays were performed to characterize the recombinant allergen.

    RESULTS: An Hev b 3 cDNA clone of 922 bp encoding a protein of 204 amino acid residues corresponding to a molecular weight of 22.3 kd was obtained. In immunoblots 29/35, latex-allergic patients with SB revealed IgE binding to rHev b 3, as did 4 of 15 of the latex-sensitized group. The presence of all IgE epitopes on rHev b 3 was shown by its ability to abolish all IgE binding to nHev b 3. Hev b 3 is related to Hev b 1 by a sequence identity of 47%. Cross-reactivity between these 2 latex allergens was illustrated by the large extent of inhibition of IgE binding to nHev b 1 by rHev b 3.

    CONCLUSION: rHev b 3 constitutes a suitable in vitro reagent for the diagnosis of latex allergy in patients with SB. The determination of the full sequence of Hev b 3 and the production of the recombinant allergen will allow the epitope mapping and improve diagnostic reagents for latex allergy.

    Matched MeSH terms: Recombinant Fusion Proteins/genetics
  6. Cloning, expression, and purification of recombinant protein from a single synthetic multivalent construct of Mycobacterium tuberculosis
    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/genetics
  7. Fulltext Delivery of chimeric hepatitis B core particles into liver cells
    Lee KW, Tey BT, Ho KL, Tan WS
    J Appl Microbiol, 2012 Jan;112(1):119-31.
    PMID: 21992228 DOI: 10.1111/j.1365-2672.2011.05176.x
    To display a liver-specific ligand on the hepatitis B virus core particles for cell-targeting delivery.
    Matched MeSH terms: Recombinant Fusion Proteins/genetics
  8. Dengue virus type 2 NS3 protease and NS2B-NS3 protease precursor induce apoptosis
    Shafee N, AbuBakar S
    J Gen Virol, 2003 Aug;84(Pt 8):2191-2195.
    PMID: 12867651 DOI: 10.1099/vir.0.19022-0
    Apoptosis was detected in Vero cell cultures expressing transfected dengue virus type 2 (DENV-2) genes. Approximately 17.5 and 51.5 % of cells expressing NS3 serine protease and NS2B-NS3(185) serine protease precursor protein [NS2B-NS3(185)(pro)] genes, respectively, were apoptotic. The percentage of apoptotic cells was significantly higher in cell cultures expressing NS2B-NS3(185)(pro). NS2B-NS3(185)(pro) was detected as NS2B-NS3(185)(pro)-EGFP fusion protein in cytoplasmic vesicular structures in the apoptotic cells. Site-directed mutagenesis which replaced His(51) with Ala within the protease catalytic triad significantly reduced the ability of the expressed NS3 and NS2B-NS3(185)(pro) to induce apoptosis. Results from the present study showed that DENV-2-encoded NS3 serine protease induces apoptosis, which is enhanced in cells expressing its precursor, NS2B-NS3(185)(pro). These findings suggest the importance of NS2B as a cofactor to NS3 protease-induced apoptosis.
    Matched MeSH terms: Recombinant Fusion Proteins/genetics
  9. Fulltext Dengue virus type 2 envelope protein displayed as recombinant phage attachment protein reveals potential cell binding sites
    Abd-Jamil J, Cheah CY, AbuBakar S
    Protein Eng. Des. Sel., 2008 Oct;21(10):605-11.
    PMID: 18669522 DOI: 10.1093/protein/gzn041
    A method to map the specific site on dengue virus envelope protein (E) that interacts with cells and a neutralizing antibody is developed using serially truncated dengue virus type 2 (DENV-2) E displayed on M13 phages as recombinant E-g3p fusion proteins. Recombinant phages displaying the truncated E consisting of amino acids 297-423 (EB2) and amino acids 379-423 (EB4) were neutralized by DENV-2 patient sera and the DENV-2 E-specific 3H5-1 monoclonal antibodies suggesting that the phages retained the DENV-2 E antigenic properties. The EB4 followed by EB2 recombinant phages bound the most to human monocytes (THP-1), African green monkey kidney (Vero) cells, mosquito (C6/36) cells, ScFv specific against E and C6/36 cell proteins. Two potential cell attachment sites were mapped to loop I (amino acids 297 to 312) and loop II (amino acids 379-385) of the DENV-2 E using the phage-displayed truncated DENV-2 E fragments and by the analysis of the E structure. Loop II was present only in EB4 recombinant phages. There was no competition for binding to C6/36 cell proteins between EB2 and EB4 phages. Loop I and loop II are similar to the sub-complex specific and type-specific neutralizing monoclonal antibody binding sites, respectively. Hence, it is proposed that binding and entry of DENV involves the interaction of loop I to cell surface glycosaminoglycan-motif and a subsequent highly specific interaction involving loop II with other cell proteins. The phage displayed truncated DENV-2 E is a powerful and useful method for the direct determination of DENV-2 E cell binding sites.
    Matched MeSH terms: Recombinant Fusion Proteins/genetics
  10. Design and validation of small interfering RNA on respiratory syncytial virus M2-2 gene: A potential approach in RNA interference on viral replication
    Chin VK, Atika Aziz NA, Hudu SA, Harmal NS, Syahrilnizam A, Jalilian FA, et al.
    J Virol Methods, 2016 10;236:117-125.
    PMID: 27432115 DOI: 10.1016/j.jviromet.2016.07.012
    Human respiratory syncytial virus (RSV) is the leading cause of severe lower respiratory tract infection in infants and young children globally and is a significant pathogen of the elderly and immunocompromised. The M2-2 protein of respiratory syncytial virus (RSV) is particularly important in regulation of viral RNA transcription and replication that could be a potential anti-viral candidate against RSV infection. In this study, we designed and validated siRNAs that specifically target the RSV M2-2 gene. Four siRNAs targeting different regions of the M2-2 gene were designed using web tool. In-vitro evaluation of silencing effect was performed by using RSV infected Vero cell line. Viral M2-2 linked GFP recombinant plasmid was co-transfected with non-targeted siRNA, Pooled siRNA, siRNA 1, siRNA 2, siRNA 3 and siRNA 4 using synthetic cationic polymer. The silencing effect of M2-2 gene at the protein level was measured both qualitatively and quantitatively by using fluorescence microscopy and flow cytometry. Meanwhile, the silencing effect at the mRNA level was assessed by using RT-qPCR. This study showed that all four designed siRNAs can effectively and efficiently silence M2-2 gene. siRNA 2 showed the highest (98%) silencing effect on protein level and siRNA 4 with 83.1% at the mRNA level. The viral assay showed no significant cytopathic effects observed after 6days post-infection with siRNAs. In conclusion, this study showed the effectiveness of siRNA in silencing M2-2 gene both at the protein and mRNA level which could potentially be used as a novel therapeutic agent in the treatment of RSV infection. However, further study is warranted to investigate the silencing effect of M2-2 protein and inhibition of RSV infection.
    Matched MeSH terms: Recombinant Fusion Proteins/genetics
  11. Fulltext Development and evaluation of a sensitive and specific assay for diagnosis of human toxocariasis by use of three recombinant antigens (TES-26, TES-30USM, and TES-120)
    Mohamad S, Azmi NC, Noordin R
    J Clin Microbiol, 2009 Jun;47(6):1712-7.
    PMID: 19369434 DOI: 10.1128/JCM.00001-09
    Diagnosis of human toxocariasis currently relies on serologic tests that use Toxocara excretory-secretory (TES) antigen to detect immunoglobulin G (IgG) antibodies to the larvae. In general, however, these assays do not have adequate specificity for use in countries in which other soil-transmitted helminths are endemic. The use of recombinant antigens in these assays, however, is promising for improving the specificity of the diagnosis of toxocariasis. Toward this goal, we developed an IgG4 enzyme-linked immunosorbent assay (ELISA) involving three recombinant antigens: rTES-30USM (previously produced), rTES-26, and rTES-120. The latter two antigens were produced by reverse transcription-PCR cloning; subcloned into glutathione S-transferase (GST)-tagged and His-tagged prokaryotic expression vectors, respectively; and expressed in Escherichia coli. The recombinant proteins were subsequently purified by affinity chromatography using GST and His-Trap resins. The diagnostic potential of each purified recombinant antigen was tested with various immunoglobulin classes (IgG, IgM, and IgE) and IgG subclasses. The IgG4 ELISA was determined to have the highest specificity and was further evaluated using a panel of serum samples. The rTES-26 IgG4 ELISA showed 80.0% (24/30 samples positive) sensitivity, and both the rTES-30USM IgG4 ELISA and rTES-120 IgG4 ELISA had 93.0% (28/30) sensitivity. Combined use of rTES-120 and rTES-30 IgG4 ELISA for the diagnosis of toxocariasis provided 100% sensitivity. The specificities of rTES-26, rTES-30USM, and rTES-120 antigens were 96.2%, 93.9%, and 92.0%, respectively. These results indicate that the development of a diagnostic test using the three recombinant antigens will allow for more-accurate detection of toxocariasis.
    Matched MeSH terms: Recombinant Fusion Proteins/genetics
  12. Development of monoclonal antibodies against recombinant LipL21 protein of pathogenic Leptospira through phage display technology
    Mohd Ali MR, Sum JS, Aminuddin Baki NN, Choong YS, Nor Amdan NA, Amran F, et al.
    Int J Biol Macromol, 2021 Jan 31;168:289-300.
    PMID: 33310091 DOI: 10.1016/j.ijbiomac.2020.12.062
    Leptospirosis is a potentially fatal zoonosis that is caused by spirochete Leptospira. The signs and symptoms of leptospirosis are usually varied, allowing it to be mistaken for other causes of acute febrile syndromes. Thus, early diagnosis and identification of a specific agent in clinical samples is crucial for effective treatment. This study was aimed to develop specific monoclonal antibodies against LipL21 antigen for future use in leptospirosis rapid and accurate immunoassay. A recombinant LipL21 (rLipL21) antigen was optimized for expression and evaluated for immunogenicity. Then, a naïve phage antibody library was utilized to identify single chain fragment variable (scFv) clones against the rLipL21 antigen. A total of 47 clones were analysed through monoclonal phage ELISA. However, after taking into consideration the background OD405 values, only 4 clones were sent for sequencing to determine human germline sequences. The sequence analysis showed that all 4 clones are identical. The in silico analysis of scFv-lip-1 complex indicated that the charged residues of scFv CDRs are responsible for the recognition with rLipL21 epitopes. The generated monoclonal antibody against rLipL21 will be evaluated as a detection reagent for the diagnosis of human leptospirosis in a future study.
    Matched MeSH terms: Recombinant Fusion Proteins/genetics
  13. Effect of signal peptides on the secretion of β-cyclodextrin glucanotransferase in Lactococcus lactis NZ9000
    Subramaniam M, Baradaran A, Rosli MI, Rosfarizan M, Khatijah Y, Raha AR
    J. Mol. Microbiol. Biotechnol., 2012;22(6):361-72.
    PMID: 23295307 DOI: 10.1159/000343921
    Cyclodextrin glucanotransferase (CGTase) is an extracellular enzyme which catalyzes the formation of cyclodextrin from starch. The production of CGTase using lactic acid bacterium is an attractive alternative and safer strategy to produce CGTase. In this study, we report the construction of genetically modified Lactococcus lactis strains harboring plasmids that secrete the Bacillus sp. G1 β-CGTase, with the aid of the signal peptides (SPs) SPK1, USP45 and native SP (NSP). Three constructed vectors, pNZ:NSP:CGT, pNZ:USP:CGT and pNZ:SPK1:CGT, were developed in this study. Each vector harbored a different SP fused to the CGTase. The formation of halo zones on starch plates indicated the production and secretion of β-CGTase by the recombinants. The expression of this enzyme is shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and zymogram analysis. A band size of ∼75 kDa corresponding to β-CGTase is identified in the intracellular and the extracellular environments of the host after medium modification. The replacement of glucose by starch in the medium was shown to induce β-CGTase production in L. lactis. Although β-CGTase production is comparatively low in NZ:SPK1:CGT, the SP SPK1 was shown to have higher secretion efficiency compared to the other SPs used in this study.
    Matched MeSH terms: Recombinant Fusion Proteins/genetics
  14. Evaluation of different promoters driving the GFP reporter gene and selected target tissues for particle bombardment of Dendrobium Sonia 17
    Tee CS, Marziah M, Tan CS, Abdullah MP
    Plant Cell Rep, 2003 Jan;21(5):452-8.
    PMID: 12789448
    Three different morphological callus types, identified as type A, B and C, and tips of in vitro inflorescences were used as target tissues for genetic transformation. Five different DNA plasmids carrying a synthetic green fluorescent protein (gfp) gene driven by different promoters, CaMV 35S, HBT, and Ubi1 were tested for the genetic transformation of Dendrobium Sonia 17. 35S-sgfp-TYG-nos (p35S) with the CaMV 35S promoter showed the highest GFP transient expression rate, while the HBT and Ubi1 promoters showed a relatively lower expression rate in all of the target tissues tested. The highest number of GFP-expressing cells was observed on day 2 post-bombardment, and the number declined gradually over the course of the next 2 weeks. Type A and B callus were found to be the best potential target tissues for genetic transformation.
    Matched MeSH terms: Recombinant Fusion Proteins/genetics
  15. Fulltext Expression of the Streptococcus pneumoniae yoeB chromosomal toxin gene causes cell death in the model plant Arabidopsis thaliana
    Bakar FA, Yeo CC, Harikrishna JA
    BMC Biotechnol, 2015;15:26.
    PMID: 25887501 DOI: 10.1186/s12896-015-0138-8
    Bacterial toxin-antitoxin systems usually comprise of a pair of genes encoding a stable toxin and its cognate labile antitoxin and are located in the chromosome or in plasmids of several bacterial species. Chromosomally-encoded toxin-antitoxin systems are involved in bacterial stress responses and activation of the toxins usually leads to cell death or dormancy. Overexpression of the chromosomally-encoded YoeB toxin from the yefM-yoeB toxin-antitoxin locus of the Gram-positive bacterium Streptococcus pneumoniae has been shown to cause cell death in S. pneumoniae as well as E. coli.
    Matched MeSH terms: Recombinant Fusion Proteins/genetics
  16. FISH analysis using PPAR γ-specific probes for detection of PAX8-PPAR γ translocation in follicular thyroid neoplasms
    Sharifah NA, Zakaria Z, Chia WK
    Methods Mol Biol, 2013;952:187-96.
    PMID: 23100233 DOI: 10.1007/978-1-62703-155-4_13
    Fluorescence in situ hybridization (FISH) is increasingly gaining importance in clinical diagnostics settings. Due to the ability of the technique to detect chromosomal abnormalities in samples with low cellularity or containing a mixed population of cells even at a single-cell level, it has become more popular in cancer research and diagnosis. Here, we describe the FISH technique for detection of PAX8-PPARγ translocation in follicular thyroid neoplasms, and the optimal protocol for the detection of this fusion gene using in archival formalin-fixed paraffin-embedded (FFPE) thyroid tissue sections.
    Matched MeSH terms: Recombinant Fusion Proteins/genetics
  17. Fulltext Fusion of protegrin-1 and plectasin to MAP30 shows significant inhibition activity against dengue virus replication
    Rothan HA, Bahrani H, Mohamed Z, Abd Rahman N, Yusof R
    PLoS One, 2014;9(4):e94561.
    PMID: 24722532 DOI: 10.1371/journal.pone.0094561
    Dengue virus (DENV) broadly disseminates in tropical and sub-tropical countries and there are no vaccine or anti-dengue drugs available. DENV outbreaks cause serious economic burden due to infection complications that requires special medical care and hospitalization. This study presents a new strategy for inexpensive production of anti-DENV peptide-fusion protein to prevent and/or treat DENV infection. Antiviral cationic peptides protegrin-1 (PG1) and plectasin (PLSN) were fused with MAP30 protein to produce recombinant antiviral peptide-fusion protein (PG1-MAP30-PLSN) as inclusion bodies in E. coli. High yield production of PG1-MAP30-PLSN protein was achieved by solubilization of inclusion bodies in alkaline buffer followed by the application of appropriate refolding techniques. Antiviral PG1-MAP30-PLSN protein considerably inhibited DENV protease (NS2B-NS3pro) with half-maximal inhibitory concentration (IC50) 0.5±0.1 μM. The real-time proliferation assay (RTCA) and the end-point proliferation assay (MTT assay) showed that the maximal-nontoxic dose of the peptide-fusion protein against Vero cells is approximately 0.67±0.2 μM. The cell-based assays showed considerable inhibition of the peptide-fusion protein against binding and proliferating stages of DENV2 into the target cells. The peptide-fusion protein protected DENV2-challeged mice with 100% of survival at the dose of 50 mg/kg. In conclusion, producing recombinant antiviral peptide-fusion protein by combining short antiviral peptide with a central protein owning similar activity could be useful to minimize the overall cost of short peptide production and take advantage of its synergistic antiviral activities.
    Matched MeSH terms: Recombinant Fusion Proteins/genetics
  18. Fulltext Gene expression profiles in primary duodenal chick cells following transfection with avian influenza virus H5 DNA plasmid encapsulated in silver nanoparticles
    Jazayeri SD, Ideris A, Shameli K, Moeini H, Omar AR
    Int J Nanomedicine, 2013;8:781-90.
    PMID: 23459681 DOI: 10.2147/IJN.S39074
    In order to develop a systemically administered safe and effective nonviral gene delivery system against avian influenza virus (AIV) that induced cytokine expression, the hemagglutinin (H5) gene of AIV, A/Ck/Malaysia/5858/04 (H5N1) and green fluorescent protein were cloned into a coexpression vector pIRES (pIREGFP-H5) and formulated using green synthesis of silver nanoparticles (AgNPs) with poly(ethylene glycol) and transfected into primary duodenal cells taken from 18-day-old specific-pathogen-free chick embryos. The AgNPs were prepared using moderated temperature and characterized for particle size, surface charge, ultraviolet-visible spectra, DNA loading, and stability. AgNPs and AgNP-pIREGFP-H5 were prepared in the size range of 13.9 nm and 25 nm with a positive charge of +78 ± 0.6 mV and +40 ± 6.2 mV, respectively. AgNPs with a positive surface charge could encapsulate pIREGFP-H5 efficiently. The ultraviolet-visible spectra for AgNP-pIREGFP-H5 treated with DNase I showed that the AgNPs were able to encapsulate pIREGFP-H5 efficiently. Polymerase chain reaction showed that AgNP-pIREGFP-H5 entered into primary duodenal cells rapidly, as early as one hour after transfection. Green fluorescent protein expression was observed after 36 hours, peaked at 48 hours, and remained stable for up to 60 hours. In addition, green fluorescent protein expression generally increased with increasing DNA concentration and time. Cells were transfected using Lipocurax in vitro transfection reagent as a positive control. A multiplex quantitative mRNA gene expression assay in the transfected primary duodenal cells via the transfection reagent and AgNPs with pIREGFP-H5 revealed expression of interleukin (IL)-18, IL-15, and IL-12β.
    Matched MeSH terms: Recombinant Fusion Proteins/genetics
  19. Glycosylation is not necessary for recognition of the fusion glycoprotein domain of the human respiratory syncytial virus by a polyclonal antibody
    Lim SH, Jahanshiri F, Jalilian FA, Rahim RA, Sekawi Z, Yusoff K
    Acta Virol., 2010;54(3):181-7.
    PMID: 20822310
    Human respiratory syncytial virus (HRSV) is a leading pathogen causing lower respiratory tract infections in infants and young children worldwide. In line with the development of an effective vaccine against HRSV, a domain of the fusion (F) glycoprotein of HRSV was produced and its immunogenicity and antigenic properties, namely the effect of deficient glycosylation was examined. A His-tagged recombinant F (rF) protein was expressed in Escherichia coli, solubilized with 8 mol/l urea, purified by the Ni-NTA affinity chromatography and used for the raising of a polyclonal antibody in rabbits. The non-glycosylated rF protein proved to be a strong immunogen that induced a polyclonal antibody that was able to recognize also the glycosylated F1 subunit of native HRSV. The other way around, a polyclonal antibody prepared against the native HRSV was able to react with the rF protein. These results indicated that glycosylation was not necessary for the F domain aa 212-574 in order to be recognized by the specific polyclonal antibody.
    Matched MeSH terms: Recombinant Fusion Proteins/genetics
  20. High level expression of thermostable lipase from Geobacillus sp. strain T1
    Leow TC, Rahman RN, Basri M, Salleh AB
    Biosci Biotechnol Biochem, 2004 Jan;68(1):96-103.
    PMID: 14745170
    A thermostable extracellular lipase of Geobacillus sp. strain T1 was cloned in a prokaryotic system. Sequence analysis revealed an open reading frame of 1,251 bp in length which codes for a polypeptide of 416 amino acid residues. The polypeptide was composed of a signal peptide (28 amino acids) and a mature protein of 388 amino acids. Instead of Gly, Ala was substituted as the first residue of the conserved pentapeptide Gly-X-Ser-X-Gly. Successful gene expression was obtained with pBAD, pRSET, pET, and pGEX as under the control of araBAD, T7, T7 lac, and tac promoters, respectively. Among them, pGEX had a specific activity of 30.19 Umg(-1) which corresponds to 2927.15 Ug(-1) of wet cells after optimization. The recombinant lipase had an optimum temperature and pH of 65 degrees C and pH 9, respectively. It was stable up to 65 degrees C at pH 7 and active over a wide pH range (pH 6-11). This study presents a rapid cloning and overexpression, aimed at improving the enzyme yield for successful industrial application.
    Matched MeSH terms: Recombinant Fusion Proteins/genetics
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