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Aeromonas aquariorum clinical isolates: antimicrobial profiles, plasmids and genetic determinants

Puah SM, Puthucheary SD, Liew FY, Chua KH

Int J Antimicrob Agents, 2013 Mar;41(3):281-4.
PMID: 23312608 DOI: 10.1016/j.ijantimicag.2012.11.012

The objective of this study was to investigate the antimicrobial resistance patterns of 47 clinical isolates of Aeromonas aquariorum and to identify the presence of plasmids and the relevant antibiotic resistance genes (ARGs). Antibiotic susceptibilities were determined by the standard disc diffusion method. The presence of plasmids and ARGs was detected by gel electrophoresis and monoplex PCR. Resistance to amoxicillin/clavulanic acid (98%), amoxicillin (91%), gentamicin (13%), trimethoprim/sulfamethoxazole (11%) and kanamycin (6%) was observed, whilst no ciprofloxacin- or amikacin-resistant strains were detected. All isolates harboured plasmids with sizes ranging from ca. 2 kb to 10 kb. PCR revealed that A. aquariorum carried three β-lactam resistance genes (bla(TEM), bla(MOX) and bla(PSE)) and two sulphonamide resistance genes (sul1 and sul2). This study provides further understanding of the phenotypic and genotypic characteristics of multiresistant A. aquariorum clinical isolates.

Matched MeSH terms: Plasmids/analysis*
Plasmid incidence rate and conjugative chloramphenicol and tetracycline resistance plasmids in Malaysian isolates of Salmonella typhi

Phipps M, Pang T, Koh CL, Puthucheary S

Microbiol. Immunol., 1991;35(2):157-61.
PMID: 1886492

Seven (6.1%) of 115 strains of Salmonella typhi isolated from Malaysian patients harbored a single large plasmid of 71 to 166 mD. Two of the seven plasmid-bearing strains were resistant to chloramphenicol (Cm) and tetracycline (Tc) and they transferred Cm and Tc resistance traits to Escherichia coli K12 at frequencies from 1.6 x 10(-7) to 1.9 x 10(-6). Agarose gel electrophoresis provided evidence that the resistance traits were cotransferred on a conjugative plasmid. The significance and importance of these results are discussed.

Matched MeSH terms: Plasmids*
Epidemiology and molecular characterization of nosocomially transmitted multidrug-resistant Klebsiella pneumoniae

Parasakthi N, Vadivelu J, Ariffin H, Iyer L, Palasubramaniam S, Arasu A

Int J Infect Dis, 2000;4(3):123-8.
PMID: 11179914

OBJECTIVES: To describe the epidemiology, antimicrobial susceptibility, genomic profiles, and control of a nosocomial outbreak of multidrug-resistant Klebsiella pneumoniae (MRKP) that occurred in the pediatric oncology unit of the University of Malaya Medical Centre in Kuala Lumpur.
MATERIALS AND METHODS: A prospective epidemiologic and microbiologic study was conducted of MRKP isolated from the blood and wound of a boy with necrotizing fasciitis after a 7-day course of ceftazidime and amikacin. In the following 2 weeks, phenotypically similar MRKP were isolated from the blood cultures of four other patients and rectal swabs of another three patients and two liquid soap samples located in the same ward.
RESULTS: Antimicrobial profiles demonstrated that all the isolates were resistant to ceftazidime, sensitive to imipenem and ciprofloxacin, and confirmed to be extended-spectrum beta-lactamase producers. Plasmids of varying molecular weights were present in all isolates. In eight of these isolates, which included four from blood, there were common large molecular weight plasmids ranging from 80 kb to 100 kb. Pulsed-field gel electrophoresis analysis using XbaI demonstrated six different DNA profiles, A to F. Profile A was shared by two blood culture isolates and were related by 91%. Profile B was found in one rectal swab isolate and one isolate from liquid soap and were related by 94%. Profile C was shared by one blood isolate and one liquid soap isolate and showed 100% relatedness. Profiles D, E, and F each were demonstrated by one blood isolate and two rectal swab isolates, respectively. These showed only 65% relatedness.
CONCLUSIONS: The MRKP strains in this outbreak were not clonal in origin. The decline of the outbreak after 4 weeks was attributed to the reemphasis of standard infection control procedures and the implementation of a program that addressed sites of environmental contamination.

Matched MeSH terms: Plasmids/genetics
Genetic variation among Malaysian isolates of Salmonella typhi as detected by ribosomal RNA gene restriction patterns

Pang T, Altwegg M, Martinetti G, Koh CL, Puthucheary S

Microbiol. Immunol., 1992;36(5):539-43.
PMID: 1513268

Genetic variation among Malaysian isolates of Salmonella typhi was determined by analysis of ribosomal RNA gene restriction patterns. Of the 20 isolates analyzed, eight different pattern combinations were detected. The amount of variation observed was also dependent upon the restriction endonuclease used; PstI produced more different patterns than did SmaI. The results suggested that disease activity was due to a number of different clones circulating simultaneously rather than a single strain. Further implications of the data are discussed.

Matched MeSH terms: Plasmids
Resistance to extended-spectrum beta-lactams by the emergence of SHV-12 and the loss of OmpK35 in Klebsiella pneumoniae and Escherichia coli in Malaysia

Palasubramaniam S, Muniandy S, Navaratnam P

J Microbiol Immunol Infect, 2009 Apr;42(2):129-33.
PMID: 19597644

In addition to beta-lactamase production, loss of porins confers resistance to extended-spectrum beta-lactams in Klebsiella pneumoniae and Escherichia coli infection. This study describes the detection of SHV-12 extended-spectrum beta-lactamase (ESBL) subtype and the loss of OmpK35 porin in 4 strains of K. pneumoniae and E. coli.

Matched MeSH terms: Plasmids
Extended-spectrum beta-lactam resistance due to AmpC hyperproduction and CMY-2 coupled with the loss of OMPK35 in Malaysian strains of Escherichia coli and Klebsiella pneumoniae

Palasubramaniam S, Subramaniam G, Muniandy S, Parasakthi N

Microb Drug Resist, 2007;13(3):186-90.
PMID: 17949305

In this report, we describe the detection of AmpC and CMY-2 beta-lactamases with the loss of OmpK35 porin among seven sporadic strains of ceftazidime-resistant Klebsiella pneumoniae and ceftazidime-resistant Escherichia coli. Cefoxitin, which was used as a marker of resistance toward 7-alpha-methoxy-cephalosporins, exhibited high minimum inhibitory concentration (MIC) values ranging between 128 microg/ml and >256 microg/ml in all the strains. The presence of hyperproducing AmpC enzymes was indicated by the positive three-dimensional test. Isoelectric focusing (IEF) study confirmed the presence of AmpC enzymes in all the strains. The ampC gene was detected by PCR in all the strains and confirmed by DNA sequencing. Large plasmids in all the strains, ranging from 60 kb to 150 kb in size, most likely encode the ampC gene. Two E. coli strains out of the seven strains showed positive amplification of the bla(CMY-2) gene, an AmpC variant, and was confirmed by DNA sequence analyses. DNA hybridization confirmed the bla(CMY-2) gene to be plasmid-mediated in both of these strains. However, one of these two strains also mediated a chromosomal CMY gene. All the strains showed an absence of OmpK35 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS/PAGE) and was confirmed by western blot analyses using raised polyclonal anti-OmpK35 antiserum. This suggests that, apart from CMY production, absence of OmpK35 porin also contributed to cefoxitin resistance resulting in extended-spectrum beta-lactam resistance among these isolates.

Matched MeSH terms: Plasmids
Evaluation of the Expression of Amyloid Precursor Protein and the Ratio of Secreted Amyloid Beta 42 to Amyloid Beta 40 in SH-SY5Y Cells Stably Transfected with Wild-Type, Single-Mutant and Double-Mutant Forms of the APP Gene for the Study of Alzheimer's Disease Pathology

Pahrudin Arrozi A, Shukri SNS, Wan Ngah WZ, Mohd Yusof YA, Ahmad Damanhuri MH, Makpol S

Appl Biochem Biotechnol, 2017 Nov;183(3):853-866.
PMID: 28417423 DOI: 10.1007/s12010-017-2468-6

Neuroblastoma cell lines such as SH-SY5Y are the most frequently utilized models in neurodegenerative research, and their use has advanced the understanding of the pathology of neurodegeneration over the past few decades. In Alzheimer's disease (AD), several pathogenic mutations have been described, all of which cause elevated levels of pathological hallmarks such as amyloid-beta (Aβ). Although the genetics of Alzheimer's disease is well known, familial AD only accounts for a small number of cases in the population, with the rest being sporadic AD, which contains no known mutations. Currently, most of the in vitro models used to study AD pathogenesis only examine the level of Aβ42 as a confirmation of successful model generation and only perform comparisons between wild-type APP and single mutants of the APP gene. Recent findings have shown that the Aβ42/40 ratio in cerebrospinal fluid (CSF) is a better diagnostic indicator for AD patients than is Aβ42 alone and that more extensive Aβ formation, such as accumulation of intraneuronal Aβ, Aβ plaques, soluble oligomeric Aβ (oAβ), and insoluble fibrillar Aβ (fAβ) occurs in TgCRND8 mice expressing a double-mutant form (Swedish and Indiana) of APP, later leading to greater progressive impairment of the brain. In this study, we generated SH-SY5Y cells stably transfected separately with wild-type APP, the Swedish mutation of APP, and the Swedish and Indiana mutations of APP and evaluated the APP expression as well as the Aβ42/40 ratio in those cells. The double-mutant form of APP (Swedish/Indiana) expressed markedly high levels of APP protein and showed a high Aβ2/40 ratio compared to wild-type and single-mutant cells.

Matched MeSH terms: Plasmids/genetics
Short homologies efficiently generate detectable homologous recombination events

Osahor AN, Tan CY, Sim EU, Lee CW, Narayanan K

Anal Biochem, 2014 Oct 1;462:26-8.
PMID: 24929088 DOI: 10.1016/j.ab.2014.05.030

When recombineering bacterial artificial chromosomes (BACs), it is common practice to design the ends of the donor molecule with 50 bp of homology specifying its insertion site. We demonstrate that desired recombinants can be produced using intermolecular homologies as short as 15 bp. Although the use of shorter donor end regions decreases total recombinants by several fold, the frequency of recombinants with correctly inserted donor molecules was high enough for easy detection by simple polymerase chain reaction (PCR) screening. This observation may have important implications for the design of oligonucleotides for recombineering, including significant cost savings, especially for high-throughput projects that use large quantities of primers.

Matched MeSH terms: Plasmids/genetics
Fulltext A plasmid-encoded peptide from Staphylococcus aureus induces anti-myeloperoxidase nephritogenic autoimmunity

Ooi JD, Jiang JH, Eggenhuizen PJ, Chua LL, van Timmeren M, Loh KL, et al.

Nat Commun, 2019 07 29;10(1):3392.
PMID: 31358739 DOI: 10.1038/s41467-019-11255-0

Autoreactivity to myeloperoxidase (MPO) causes anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), with rapidly progressive glomerulonephritis. Here, we show that a Staphylococcus aureus peptide, homologous to an immunodominant MPO T-cell epitope (MPO409-428), can induce anti-MPO autoimmunity. The peptide (6PGD391-410) is part of a plasmid-encoded 6-phosphogluconate dehydrogenase found in some S. aureus strains. It induces anti-MPO T-cell autoimmunity and MPO-ANCA in mice, whereas related sequences do not. Mice immunized with 6PGD391-410, or with S. aureus containing a plasmid expressing 6PGD391-410, develop glomerulonephritis when MPO is deposited in glomeruli. The peptide induces anti-MPO autoreactivity in the context of three MHC class II allomorphs. Furthermore, we show that 6PGD391-410 is immunogenic in humans, as healthy human and AAV patient sera contain anti-6PGD and anti-6PGD391-410 antibodies. Therefore, our results support the idea that bacterial plasmids might have a function in autoimmune disease.

Matched MeSH terms: Plasmids/genetics
A transient assay to evaluate the expression of polyhydroxybutyrate genes regulated by oil palm mesocarp-specific promoter

Omidvar V, Siti Nor Akmar A, Marziah M, Maheran AA

Plant Cell Rep, 2008 Sep;27(9):1451-9.
PMID: 18563415 DOI: 10.1007/s00299-008-0565-2

The promoter of the oil palm metallothionein-like gene (MT3-A) demonstrated mesocarp-specific activity in functional analysis using transient expression assay of reporter gene in bombarded oil palm tissue slices. In order to investigate the tissue-specific expression of polyhydroxybutyrate (PHB) biosynthetic pathway genes, a multi-gene construct carrying PHB genes fused to the oil palm MT3-A promoter was co-transferred with a construct carrying GFP reporter gene using microprojectile bombardment targeting the mesocarp and leaf tissues of the oil palm. Transcriptional analysis using RT-PCR revealed successful transcription of all the three phbA, phbB, and phbC genes in transiently transformed mesocarp but not in transiently transformed leaf tissues. Furthermore, all the three expected sizes of PHB-encoded protein products were only detected in transiently transformed mesocarp tissues on a silver stained polyacrylamide gel. Western blot analysis using polyclonal antibody specific for phbB product confirmed successful translation of phbB mRNA transcript into protein product. This study provided valuable information, supporting the future engineering of PHB-producing transgenic palms.

Matched MeSH terms: Plasmids
Fulltext Lactococcus lactis M4, a potential host for the expression of heterologous proteins

Noreen N, Hooi WY, Baradaran A, Rosfarizan M, Sieo CC, Rosli MI, et al.

Microb Cell Fact, 2011;10:28.
PMID: 21518457 DOI: 10.1186/1475-2859-10-28

Many plasmid-harbouring strains of Lactococcus lactis have been isolated from milk and other sources. Plasmids of Lactococcus have been shown to harbour antibiotic resistance genes and those that express some important proteins. The generally regarded as safe (GRAS) status of L. lactis also makes it an attractive host for the production of proteins that are beneficial in numerous applications such as the production of biopharmaceutical and nutraceutical. In the present work, strains of L. lactis were isolated from cow's milk, plasmids were isolated and characterised and one of the strains was identified as a potential new lactococcal host for the expression of heterologous proteins.

Matched MeSH terms: Plasmids/metabolism; Plasmids/chemistry
Inadvertent expression of dengue 2 virus NS3-EGFP fusion protein in Escherlchla coll using the pEGFP-N 1 mammalian expression vector

Norazizah S, AbuBakar S

JUMMEC, 1999;4:41-46.

Dengue 2 New Guinea C (NGC) virus NS3 protein, a potentially important virulence factor was cloned to the N-terminus of the Aeqirorea victoria enhanced green fluorescent protein (EGFP) using the pEGFP-N1 mammalian expression vector. During amplification of the recombinant plasmid in E. coli, transformants expressing the EGFP were detected in vivo when viewed using fluorescence microscopy. This inadvertent expression of the recombinant fusion protein was confirmed further by detection of the T7.Tag peptide cloned to the aluino terminal of the fusion protein using T 7.Tag specific monoclonal antibody. These findings represent perhaps the first reported expression of the T7.Tag-NS3-EGFP fusion protein using the pEGFP-N1 mammalian expression vector in E. coli. KEYWORDS: Dengue, NS3, pEGFP-N1, fusion protein.

Matched MeSH terms: Plasmids
Formation and characterization of pDNA-loaded alginate microspheres for oral administration in mice

Nograles N, Abdullah S, Shamsudin MN, Billa N, Rosli R

J Biosci Bioeng, 2012 Feb;113(2):133-40.
PMID: 22093752 DOI: 10.1016/j.jbiosc.2011.10.003

Alginate, a natural polysaccharide, was explored in this study as an oral delivery vehicle of a mammalian expression vector into the murine intestinal mucosa. Alginate microspheres were produced through water-in-oil (W/O) emulsification method. Average diameter sizes of microspheres were 46.88 μm±3.07 μm with significant size reduction upon utilization of 1.0% Span80. Plasmid DNA (pDNA) carrying green fluorescent protein reporter gene (GFP), pVAX-GFP, was encapsulated within microspheres at efficiencies of 72.9 to 74.4%, carrying maximum load of 6 μg pDNA. Alginate microspheres demonstrated shrinkage in pH 1.2 and swelling in pH 9.0 with pDNA release about twice the amount released in acidic environment. Oral delivery of pVAX-GFP loaded-microspheres, at 50 μg, 100 μg and 150 μg dose, was performed on BALB/c mice. Tissue biodistribution, investigated through flow cytometric analysis, demonstrated GFP positive intestinal cells (<1.0%) with 1.3-fold higher levels for the 100 μg dose; therefore suggesting feasibility of the approach for oral gene delivery and vaccination.

Matched MeSH terms: Plasmids/genetics
Synthesis, crystal structures and characterization of late first row transition metal complexes derived from benzothiazole core: anti-tuberculosis activity and special emphasis on DNA binding and cleavage property

Netalkar PP, Netalkar SP, Budagumpi S, Revankar VK

Eur J Med Chem, 2014 May 22;79:47-56.
PMID: 24721314 DOI: 10.1016/j.ejmech.2014.03.083

Air and moisture stable coordination compounds of late first row transition metals, viz. Co(II), Ni(II), Cu(II) and Zn(II), with a newly designed ligand, 2-(2-benzo[d]thiazol-2-yl)hydrazono)propan-1-ol (LH), were prepared and successfully characterized using various spectro-analytical techniques. The molecular structures of the ligand and nickel complex were unambiguously determined by single-crystal X-ray diffraction method. The [Ni(LH)2]Cl2.3H2O complex is stabilized by intermolecular CH⋯π stacking interactions between the methyl hydrogen and the C18 atom of the phenyl ring (C11-H11B⋯C18) forming 1D zig-zag chain structure. Both, the ligand and its copper complex, were electrochemically active in the working potential range, showing quasi-reversible redox system. The interactions of all the compounds with calf thymus DNA have been comprehensively investigated using electronic absorption spectroscopy, viscosity, electrochemistry and thermal denaturation studies. The cleavage reaction on pBR322 DNA has been monitored by agarose gel electrophoresis. The results showed that the ligand can bind to CT-DNA through partial intercalation, whereas the complexes bind electrostatically. Further, [Ni(LH)2]Cl2.3H2O and [CuLCl(H2O)2] complexes in the series have high binding and cleavage affinity towards pBR322 DNA. Additionally, all the compounds were screened for anti-tuberculosis activity. All the complexes revealed an MIC value of 0.8 μg/mL, which is almost 8 times active than standard used (Streptomycin, 6.25 μg/mL).

Matched MeSH terms: Plasmids
Escherichia coli bactofection using Lipofectamine

Narayanan K, Lee CW, Radu A, Sim EU

Anal Biochem, 2013 Aug 15;439(2):142-4.
PMID: 23608053 DOI: 10.1016/j.ab.2013.04.010

Successful gene delivery into mammalian cells using bactofection requires entry of the bacterial vector via cell surface integrin receptors followed by release of plasmid DNA into the cellular environment. We show, for the first time, that addition of the DNA transfection reagent Lipofectamine improves entry of invasive Escherichia coli into HeLa cells and enhances up to 2.8-fold green fluorescent protein (GFP) expression from a reporter plasmid. The addition of Lipofectamine may be applicable to other bacterial vectors to increase their DNA delivery efficiency into mammalian cells.

Matched MeSH terms: Plasmids
Influence of iron, growth temperature and plasmids on siderophore production in Aeromonas hydrophila

Naidu AJ, Yadav M

J Med Microbiol, 1997 Oct;46(10):833-8.
PMID: 9364139

Aeromonas hydrophila strains obtained from diarrhoeal samples of human patients (19 isolates) and freshwater ponds (11 isolates) were analysed for siderophore production. Both clinical and environmental isolates showed significantly increased siderophore production under iron-limiting conditions both at 28 degrees C and at 37 degrees C. Clinical isolates consistently produced higher levels of siderophores than did the environmental isolates. The role of plasmids in moderating siderophore production was studied after curing with acridine orange. Treatment with acridine orange for 24 h removed the larger plasmids but the smaller plasmids (< 5 MDa), more common in the environmental isolates, were resistant to curing. As found in the untreated isolates, the cured clinical isolates produced higher mean levels of siderophores than the cured environmental isolates. Siderophore production in A. hydrophila was significantly influenced by iron-limiting cultural conditions and the source of isolates, but plasmid content and growth temperature at 28 degrees C or 37 degrees C had little effect on production. The basis for the greater production of siderophores in clinical isolates than in environmental isolates needs further study.

Matched MeSH terms: Plasmids
Fulltext The complete genome sequence of Ensifer meliloti strain CCMM B554 (FSM-MA), a highly effective nitrogen-fixing microsymbiont of Medicago truncatula Gaertn

Nagymihály M, Vásarhelyi BM, Barrière Q, Chong TM, Bálint B, Bihari P, et al.

Stand Genomic Sci, 2017;12:75.
PMID: 29255570 DOI: 10.1186/s40793-017-0298-3

Strain CCMM B554, also known as FSM-MA, is a soil dwelling and nodule forming, nitrogen-fixing bacterium isolated from the nodules of the legume Medicago arborea L. in the Maamora Forest, Morocco. The strain forms effective nitrogen fixing nodules on species of the Medicago, Melilotus and Trigonella genera and is exceptional because it is a highly effective symbiotic partner of the two most widely used accessions, A17 and R108, of the model legume Medicago truncatula Gaertn. Based on 16S rRNA gene sequence, multilocus sequence and average nucleotide identity analyses, FSM-MA is identified as a new Ensifer meliloti strain. The genome is 6,70 Mbp and is comprised of the chromosome (3,64 Mbp) harboring 3574 predicted genes and two megaplasmids, pSymA (1,42 Mbp) and pSymB (1,64 Mbp) with respectively 1481 and 1595 predicted genes. The average GC content of the genome is 61.93%. The FSM-MA genome structure is highly similar and co-linear to other E. meliloti strains in the chromosome and the pSymB megaplasmid while, in contrast, it shows high variability in the pSymA plasmid. The large number of strain-specific sequences in pSymA as well as strain-specific genes on pSymB involved in the biosynthesis of the lipopolysaccharide and capsular polysaccharide surface polysaccharides may encode novel symbiotic functions explaining the high symbiotic performance of FSM-MA.

Matched MeSH terms: Plasmids
Fulltext Molecular cloning of full-length haemagglutinin (H5HA) gene of highly pathogenic avian influenza virus (HPAI) H5N1

Mustapha Bala Abubakar, Aini Ideris, AbdulRahman Omar, Mohd Hair Bejo

Buletin Persatuan Genetik Malaysia, 2009;15(1):20-23.
MyJurnal

Avian Influenza viruses belonging to the Orthomyxoviridae family are enveloped viruses with segmented negative sense RNA genome surrounded by a helical symmetry capsid. Influenza viruses, especially the highly pathogenic avian influenza virus (HPAI) such as H5 or H7 subtype are the most important pathogens for the poultry industry in recent times. The haemagglutinin protein and neuraminidase, serves as the target for the immune response of the host. Due to recurrent genetic reassortments between avian and human influenza viruses, global pandemics may emerge and the naive human immunity could not withstand pressure by the novel hybrid virus. The emergence of genetic engineering technology provided the industry with new methods of manufacturing diagnostics tools and vaccines. After extraction of RNA from the cell culture of strain influenza A/Chicken/Malaysia/2004(H5N1) of AIV, the viral RNA was converted to cDNA by a specific primer. The cDNA was amplified by the polymerase chain reaction (PCR) and analyzed
by agarose gel electrophoresis. The intact PCR product of full length haemagglutinin gene was cloned in TO POTM TA Cloning vector. The full-length HA-encoding gene of H5N1 AIV was subcloned into a pPICZA vector. After successful ligation, the constructed plasmid was transformed into E.coli.Top10, Plasmid DNA from transformed bacteria was extracted in white colony and positive clones were confirmed by restriction digestion with Sacl and Not1 restriction enzymes, colony PCR screening and nucleotide sequencing. Construction of a recombinant pPICZA/H5HA plasmid containing the full length haemagglutinin gene was achieved as a first step
towards the expression in Pichia pastoris.

Matched MeSH terms: Plasmids
Recombinant Enterovirus 71 Viral Protein 1 Fused to a Truncated Newcastle Disease Virus NP (NPt) Carrier Protein

Mustafa S, Abd-Aziz N, Saw WT, Liew SY, Yusoff K, Shafee N

Vaccines (Basel), 2020 Dec 07;8(4).
PMID: 33297428 DOI: 10.3390/vaccines8040742

Enterovirus 71 (EV71) is the major causative agent in hand, foot, and mouth disease (HFMD), and it mainly infects children worldwide. Despite the risk, there is no effective vaccine available for this disease. Hence, a recombinant protein construct of truncated nucleocapsid protein viral protein 1 (NPt-VP1198-297), which is capable of inducing neutralizing antibody against EV71, was evaluated in a mouse model. Truncated nucleocapsid protein Newcastle disease virus that was used as immunological carrier fused to VP1 of EV71 as antigen. The recombinant plasmid carrying corresponding genes was constructed by recombinant DNA technology and the corresponding protein was produced in Escherichia coli expression system. The recombinant NPt-VP1198-297 protein had elicited neutralizing antibodies against EV71 with the titer of 1:16, and this result is higher than the titer that is elicited by VP1 protein alone (1:8). It was shown that NPt containing immunogenic epitope(s) of VP1 was capable of inducing a greater functional immune response when compared to full-length VP1 protein alone. It was capable to carry larger polypeptide compared to full-length NP protein. The current study also proved that NPt-VP1198-297 protein can be abundantly produced in recombinant protein form by E. coli expression system. The findings from this study support the importance of neutralizing antibodies in EV71 infection and highlight the potential of the recombinant NPt-VP1198-297 protein as EV71 vaccine.

Matched MeSH terms: Plasmids
Fulltext Rapid Generation of a Recombinant Genotype VIII Newcastle Disease Virus (NDV) Using Full-Length Synthetic cDNA

Murulitharan K, Yusoff K, Omar AR, Peeters BPH, Molouki A

Curr Microbiol, 2021 Apr;78(4):1458-1465.
PMID: 33660046 DOI: 10.1007/s00284-021-02421-z

Rescue of (-)ssRNA viruses involves the sequential assembly and cloning of the full-length cDNA, which is often a challenging and time-consuming process. The objective of this study was to develop a novel method to rapidly clone the full-length cDNA of a very virulent NDV by only one assembly step. A completely synthetic 15 kb cDNA of a Malaysian genotype VIII NDV known as strain AF2240-I with additional flanking BsmBI sites was synthesised. However, to completely follow the rule-of-six, the additional G residues that are traditionally added after the T7 promoter transcription initiation site were not synthesised. The synthetic fragment was then cloned into low-copy number transcription vector pOLTV5-phiX between the T7 promoter and HDV Rz sequences through digestion with BbsI. The construct was co-transfected with helper plasmids into BSRT7/5 cells. A recombinant NDV called rAF was successfully rescued using transfection supernatant harvested as early as 16 h post-transfection. Virus from each passage showed an intracerebral pathogenicity index (ICPI) and a mean death time (MDT) similar to the parent strain AF2240-I. Moreover, rAF possessed an introduced mutation which was maintained for several passages. The entire rescue using the one-step assembly procedure was completed within a few weeks, which is extremely fast compared to previously used methods.

Matched MeSH terms: Plasmids

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