Displaying publications 1 - 20 of 241 in total

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  1. Molecular cloning and characterization of an atypical butyrylcholinesterase-like protein in zebrafish
    Tan KS, Zhang Y, Liu L, Li S, Zou X, Zeng W, et al.
    Comp Biochem Physiol B Biochem Mol Biol, 2021 03 02;255:110590.
    PMID: 33662568 DOI: 10.1016/j.cbpb.2021.110590
    Cholinesterases act as bio scavengers to clear organophosphorus (OP) compounds and prodrugs. The butyrylcholinesterase (BChE) gene has been found in several types of teleost fish but this gene has yet to be identified in cyprinid fish. Indeed, BChE homologs have not been found in the zebrafish (Danio rerio) genomic database. Here, we demonstrate that BChE activity is present in zebrafish, in line with other groups' findings. Using in-gel native-PAGE enzymatic activity staining and LC-MS/MS technique, an atypical BChE-like protein was identified in zebrafish. The si:ch211-93f2.1 gene was cloned, and His-tagged recombinant protein was expressed using the Pichia yeast system. The purified protein (molecular weight ~ 180 kDa) showed BChE activity, and degraded acetylcholinesterase (ACh) at a higher rate than BCh. However, phylogram analysis shows that this novel cholinesterase shared an evolutionary origin with carboxylic esterase rather than BChE. The zebrafish BChE-like protein shares structural characteristics with cholinesterase and carboxylesterase. The 2-arachidonoylglycerol (2-AG), nicosulfuron, and triacetin exhibited a higher binding affinity to the zebrafish BChE-like protein than BCh and ACh. With the identification of BChE-like protein in zebrafish, this study could shed light on the origin of BChE and may contribute towards the development of a BChE knockout zebrafish model for sensitive drug or toxin screening.
    Matched MeSH terms: Cloning, Molecular*
  2. Versatility of subtilisin: A review on structure, characteristics, and applications
    Azrin NAM, Ali MSM, Rahman RNZRA, Oslan SN, Noor NDM
    Biotechnol Appl Biochem, 2022 Dec;69(6):2599-2616.
    PMID: 35019178 DOI: 10.1002/bab.2309
    Due to its thermostability and high pH compatibility, subtilisin is most known for its role as an additive for detergents in which it is categorized as a serine protease according to MEROPS database. Subtilisin is typically isolated from various bacterial species of the Bacillus genus such as Bacillus subtilis, B. amyloliquefaciens, B. licheniformis, and various other organisms. It is composed of 268-275 amino acid residues and is initially secreted in the precursor form, preprosubtilisin, which is composed of 29-residues signal peptide, 77-residues propeptide, and 275-residues active subtilisin. Subtilisin is known for the presence of high and low affinity calcium binding sites in its structure. Native subtilisin has general properties of thermostability, tolerance to neutral to high pH, broad specificity, and calcium-dependent stability, which contribute to the versatility of subtilisin applicability. Through protein engineering and immobilization technologies, many variants of subtilisin have been generated, which increase the applicability of subtilisin in various industries including detergent, food processing and packaging, synthesis of inhibitory peptides, therapeutic, and waste management applications.
    Matched MeSH terms: Cloning, Molecular
  3. Molecular cloning and in silico analysis of chalcone isomerase from Polygonum minus
    Shah FLA, Baharum SN, Goh HH, Leow TC, Ramzi AB, Oslan SN, et al.
    Mol Biol Rep, 2023 Jun;50(6):5283-5294.
    PMID: 37148413 DOI: 10.1007/s11033-023-08417-1
    BACKGROUND: Chalcone isomerase (CHI; EC 5.5.1.6) is one of the key enzymes in the flavonoid biosynthetic pathway that is responsible for the intramolecular cyclization of chalcones into specific 2S-flavanones.

    METHODS AND RESULTS: In this study, the open reading frame (ORF) of CHI was successfully isolated from the cDNA of Polygonum minus at 711-bp long, encoding for 236 amino acid residues, with a predicted molecular weight of 25.4 kDa. Multiple sequence alignment and phylogenetic analysis revealed that the conserved residues (Thr50, Tyr108, Asn115, and Ser192) in the cleft of CHI enzyme group active site are present in PmCHI protein sequence and classified as type I. PmCHI comprises more hydrophobic residues without a signal peptide and transmembrane helices. The three-dimensional (3D) structure of PmCHI predicted through homology modeling was validated by Ramachandran plot and Verify3D, with values within the acceptable range of a good model. PmCHI was cloned into pET-28b(+) plasmid, expressed in Escherichia coli BL21(DE3) at 16 °C and partially purified.

    CONCLUSION: These findings contribute to a deeper understanding of the PmCHI protein and its potential for further characterization of its functional properties in the flavonoid biosynthetic pathway.

    Matched MeSH terms: Cloning, Molecular
  4. Molecular progress on the mapping and cloning of functional genes for blast disease in rice (Oryza sativa L.): current status and future considerations
    Ashkani S, Rafii MY, Shabanimofrad M, Ghasemzadeh A, Ravanfar SA, Latif MA
    Crit Rev Biotechnol, 2016;36(2):353-67.
    PMID: 25394538 DOI: 10.3109/07388551.2014.961403
    Rice blast disease, which is caused by the fungal pathogen Magnaporthe oryzae, is a recurring problem in all rice-growing regions of the world. The use of resistance (R) genes in rice improvement breeding programmes has been considered to be one of the best options for crop protection and blast management. Alternatively, quantitative resistance conferred by quantitative trait loci (QTLs) is also a valuable resource for the improvement of rice disease resistance. In the past, intensive efforts have been made to identify major R-genes as well as QTLs for blast disease using molecular techniques. A review of bibliographic references shows over 100 blast resistance genes and a larger number of QTLs (∼500) that were mapped to the rice genome. Of the blast resistance genes, identified in different genotypes of rice, ∼22 have been cloned and characterized at the molecular level. In this review, we have summarized the reported rice blast resistance genes and QTLs for utilization in future molecular breeding programmes to introgress high-degree resistance or to pyramid R-genes in commercial cultivars that are susceptible to M. oryzae. The goal of this review is to provide an overview of the significant studies in order to update our understanding of the molecular progress on rice and M. oryzae. This information will assist rice breeders to improve the resistance to rice blast using marker-assisted selection which continues to be a priority for rice-breeding programmes.
    Matched MeSH terms: Cloning, Molecular*
  5. Cloning and sequencing of its region of two isolates of Phellinus spp obtained from diseased roots of setang (Azadirachta excelsa [Jack] Jacobs)
    Mohd Rosli Haron, Mohd Farid Ahmad, Lee, Su See, Norwati Muhammad
    Buletin Persatuan Genetik Malaysia, 2005;11(1):0-0.
    MyJurnal
    Two isolates of brown root disease fungi were obtained from diseased roots of sentang (Azadirachta excelsa). Morphological characters from macroscopic and microscopic studies suggested that both isolates were from the same genus namely Phellinus noxius and Phellinus sp. Cloning and sequencing of ITS region were conducted to investigate further the variation between the two species at
    molecular level. PCR-amplified ITS regions were cloned in pCR2.1 and sequenced. DNA sequences sized 723bp and 710bp were obtained for Phellinus noxius and Phellinus sp respectively. Comparison between the two sequences showed 98% similarity where three nucleotide substitutions and three insertion/deletion regions were found sized 8bp, 2bp and 3bp respectively.
    Matched MeSH terms: Cloning, Molecular
  6. Fulltext Cloning and expression of a subfamily 1.4 lipase from Bacillus licheniformis IBRL-CHS2
    Reddy, Nidyaletchmy Subba, Rashidah Abdul Rahim, Darah Ibrahim, Kumar, K. Sudesh
    Trop Life Sci Res, 2016;27(11):145-150.
    MyJurnal
    We report on the cloning of the lipase gene from Bacillus licheniformis IBRLCHS2
    and the expression of the recombinant lipase. DNA sequencing analysis of the
    cloned lipase gene showed that it shares 99% identity with the lipase gene from
    B. licheniformis ATCC 14580 and belongs to subfamily 1.4 of true lipases based on amino
    acid sequence alignment of various Bacillus lipases. The 612 bp lipase gene was then
    cloned into the pET-15b(+) expression vector and the construct was transformed into
    E. coli BL21 (DE3) for bulk expression of the lipase. Expression was analysed by SDSPAGE
    where the lipase was found to have a molecular weight of about 23 kDa.
    Matched MeSH terms: Cloning, Molecular
  7. Fulltext Isolation and rapid identification of streptomonospora, a strictly halophilic filamentous actinomycetes from antarctic soil (Barrientos Island, Antarctic)
    Cheah Y.K., Lee, L.H., Radu, S., Wong, M.C.V.L., Andrade, H.M.
    ASM Science Journal, 2009;3(2):113-120.
    MyJurnal
    The genus Streptomonospora is a group of extremely halophilic filamentous actinomycetes that form a distinct branch in the 16S rRNA gene phylogenetic tree adjacent to the genera Nocardiopsis and Thermobifida, family Norcadiopsaceae. To date, genus Streptomonospora only contain two validly described species which are Streptomonospora salina and Streptomonospora alba. During a biodiversity study on halophilic filamentous actinomycetes from 18 co-ordinates in Barrientos Island, Antarctic, numerous actinomycetes strains were isolated. To identify whether these isolates were members of the genus Streptomonospora, a genus specific primer that allow the rapid detection of the genus Streptomonospora by means of PCR amplification was used. Furthermore molecular cloning was performed to make identical and multiple copies of the target gene. In addition, morphological characteristic identification was performed to validate isolates with positive amplification during PCR.
    Matched MeSH terms: Cloning, Molecular
  8. Fulltext High-resolution mapping of genes involved in plant stage-specific partial resistance of barley to leaf rust
    Yeo FKS, Bouchon R, Kuijken R, Loriaux A, Boyd C, Niks RE, et al.
    Molecular breeding : new strategies in plant improvement, 2017;37(4):45.
    PMID: 28356783 DOI: 10.1007/s11032-017-0624-x
    Partial resistance quantitative trait loci (QTLs) Rphq11 and rphq16 against Puccinia hordei isolate 1.2.1 were previously mapped in seedlings of the mapping populations Steptoe/Morex and Oregon Wolfe Barleys, respectively. In this study, QTL mapping was performed at adult plant stage for the two mapping populations challenged with the same rust isolate. The results suggest that Rphq11 and rphq16 are effective only at seedling stage, and not at adult plant stage. The cloning of several genes responsible for partial resistance of barley to P. hordei will allow elucidation of the molecular basis of this type of plant defence. A map-based cloning approach requires to fine-map the QTL in a narrow genetic window. In this study, Rphq11 and rphq16 were fine-mapped using an approach aiming at speeding up the development of plant material and simplifying its evaluation. The plant materials for fine-mapping were identified from early plant materials developed to produce QTL-NILs. The material was first selected to carry the targeted QTL in heterozygous condition and susceptibility alleles at other resistance QTLs in homozygous condition. This strategy took four to five generations to obtain fixed QTL recombinants (i.e., homozygous resistant at the Rphq11 or rphq16 QTL alleles, homozygous susceptible at the non-targeted QTL alleles). In less than 2 years, Rphq11 was fine-mapped into a 0.2-cM genetic interval and a 1.4-cM genetic interval for rphq16. The strongest candidate gene for Rphq11 is a phospholipid hydroperoxide glutathione peroxidase. Thus far, no candidate gene was identified for rphq16.
    Matched MeSH terms: Cloning, Molecular
  9. Fulltext Cloning and Characterization of the Autoinducer Synthase Gene from Lipid-Degrading Bacterium Cedecea neteri
    Tan KH, How KY, Tan JY, Yin WF, Chan KG
    Front Microbiol, 2017;8:72.
    PMID: 28197135 DOI: 10.3389/fmicb.2017.00072
    The process of intercellular communication among bacteria, termed quorum sensing (QS), is mediated by small diffusible molecules known as the autoinducers. QS allows the population to react to the change of cell density in unison, in processes such as biofilm formation, plasmid conjugation, virulence, motility and root nodulation. In Gram-negative proteobacteria, N-acyl homoserine lactone (AHL) is the common "language" to coordinate gene expression. This signaling molecule is usually synthesized by LuxI-type proteins. We have previously discovered that a rare bacterium, Cedecea neteri, exhibits AHL-type QS activity. With information generated from genome sequencing, we have identified the luxIR gene pair responsible for AHL-type QS and named it cneIR. In this study, we have cloned and expressed the 636 bp luxI homolog in an Escherichia coli host for further characterization. Our findings show that E. coli harboring cneI produced the same AHL profile as the wild type C. neteri, with the synthesis of AHL known as N-butyryl-homoserine lactone. This 25 kDa LuxI homolog shares high similarity with other AHL synthases from closely related species. This work is the first documentation of molecular cloning and characterization of luxI homolog from C. neteri.
    Matched MeSH terms: Cloning, Molecular
  10. Isolation and cloning of microRNAs from recalcitrant plant tissues with small amounts of total RNA: a step-by step approach
    Yew CW, Kumar SV
    Mol Biol Rep, 2012 Feb;39(2):1783-90.
    PMID: 21625851 DOI: 10.1007/s11033-011-0919-7
    MicroRNAs (miRNAs) are small RNAs (sRNAs) with approximately 21-24 nucleotides in length. They regulate the expression of target genes through the mechanism of RNA silencing. Conventional isolation and cloning of miRNAs methods are usually technical demanding and inefficient. These limitations include the requirement for high amounts of starting total RNA, inefficient ligation of linkers, high amount of PCR artifacts and bias in the formation of short miRNA-concatamers. Here we describe in detail a method that uses 80 μg of total RNA as the starting material. Enhancement of the ligation of sRNAs and linkers with the use of polyethylene glycol (PEG8000) was described. PCR artifacts from the amplification of reverse-transcribed sRNAs were greatly decreased by using lower concentrations of primers and reducing the number of amplification cycles. Large concatamers with up to 1 kb in size with around 20 sRNAs/concatamer were obtained by using an optimized reaction condition. This protocol provide researchers with a rapid, efficient and cost-effective method for the construction of miRNA profiles from plant tissues containing low amounts of total RNA, such as fruit flesh and senescent leaves.
    Matched MeSH terms: Cloning, Molecular/methods*
  11. Cloning and sequence characterisation of falcipain-2 from Plasmodium falciparum Gombak A strain (Malaysia)
    Sim TS, Loke P, Lee MA, Singh M, Flotow H
    Parasitol Res, 2001 Sep;87(9):683-6.
    PMID: 11570549
    In this study, the genome of the Plasmodium falciparum Gombak A strain was examined for the presence of a gene encoding falcipain-2, a cysteine protease, using homology-based polymerase chain reaction cloning. The nucleotide sequence obtained from the gene cloned (designated pFG1) is approximately 99% homologous to other falcipain-2 genes from different strains. Comparatively, it is 69% homologous to falcipain-3 genes. Direct cloning of the falcipain-2 gene and its resemblance to the reported corresponding mRNA transcript suggests the absence of introns in this gene. Sequence alignment and comparison revealed four amino acid differences at positions 15, 51, 59 and 414 in the falcipain-2 from P. falciparum Gombak A as compared to other falcipain-2 proteins from different strains.
    Matched MeSH terms: Cloning, Molecular*
  12. Resistance gene cloning from a wild crop relative by sequence capture and association genetics
    Arora S, Steuernagel B, Gaurav K, Chandramohan S, Long Y, Matny O, et al.
    Nat Biotechnol, 2019 02;37(2):139-143.
    PMID: 30718880 DOI: 10.1038/s41587-018-0007-9
    Disease resistance (R) genes from wild relatives could be used to engineer broad-spectrum resistance in domesticated crops. We combined association genetics with R gene enrichment sequencing (AgRenSeq) to exploit pan-genome variation in wild diploid wheat and rapidly clone four stem rust resistance genes. AgRenSeq enables R gene cloning in any crop that has a diverse germplasm panel.
    Matched MeSH terms: Cloning, Molecular*
  13. Cloning and functional analysis of the genes coding for 4-aminobenzenesulfonate 3,4-dioxygenase from Hydrogenophaga sp. PBC
    Gan HM, Shahir S, Yahya A
    Microbiology (Reading), 2012 Aug;158(Pt 8):1933-1941.
    PMID: 22609751 DOI: 10.1099/mic.0.059550-0
    The gene coding for the oxygenase component, sadA, of 4-aminobenzenesulfonate (4-ABS) 3,4-dioxygenase in Hydrogenophaga sp. PBC was previously identified via transposon mutagenesis. Expression of wild-type sadA in trans restored the ability of the sadA mutant to grow on 4-ABS. The inclusion of sadB and sadD, coding for a putative glutamine-synthetase-like protein and a plant-type ferredoxin, respectively, further improved the efficiency of 4-ABS degradation. Transcription analysis using the gfp promoter probe plasmid showed that sadABD was expressed during growth on 4-ABS and 4-sulfocatechol. Heterologous expression of sadABD in Escherichia coli led to the biotransformation of 4-ABS to a metabolite which shared a similar retention time and UV/vis profile with 4-sulfocatechol. The putative reductase gene sadC was isolated via degenerate PCR and expression of sadC and sadABD in E. coli led to maximal 4-ABS biotransformation. In E. coli, the deletion of sadB completely eliminated dioxygenase activity while the deletion of sadC or sadD led to a decrease in dioxygenase activity. Phylogenetic analysis of SadB showed that it is closely related to the glutamine-synthetase-like proteins involved in the aniline degradation pathway. This is the first discovery, to our knowledge, of the functional genetic components for 4-ABS aromatic ring hydroxylation in the bacterial domain.
    Matched MeSH terms: Cloning, Molecular*
  14. Strategies for the Construction of Cassava Brown Streak Disease Viral Infectious Clones
    Duff-Farrier CRA, Mbanzibwa DR, Nanyiti S, Bunawan H, Pablo-Rodriguez JL, Tomlinson KR, et al.
    Mol Biotechnol, 2019 Feb;61(2):93-101.
    PMID: 30484144 DOI: 10.1007/s12033-018-0139-7
    Cassava brown streak disease (CBSD) has major impacts on yield and quality of the tuberous roots of cassava in Eastern and Central Arica. At least two Potyviridae species cause the disease: Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV). Cloned viral genome sequences known as infectious clones (ICs) have been important in the study of other viruses, both as a means of standardising infectious material and characterising viral gene function. IC construction is often technically challenging for Potyviridae due to sequence instability in E. coli. Here, we evaluate three methods for the construction of infectious clones for CBSD. Whilst a simple IC for in vitro transcription was made for UCBSV isolate 'Kikombe', such an approach failed to deliver full-length clones for CBSV isolates 'Nampula' or 'Tanza', necessitating more complex approaches for their construction. The ICs successfully generated symptomatic infection in the model host N. benthamiana and in the natural host cassava. This shows that whilst generating ICs for CBSV is still a technical challenge, a structured approach, evaluating both in vitro and in planta transcription systems should successfully deliver ICs, allowing further study into the symptomology and virulence factors in this important disease complex.
    Matched MeSH terms: Cloning, Molecular/methods*
  15. Magnetic Nanoparticle-Based Semi-Automated Panning for High-Throughput Antibody Selection
    Ch'ng ACW, Hamidon NHB, Konthur Z, Lim TS
    Methods Mol Biol, 2018;1701:301-319.
    PMID: 29116512 DOI: 10.1007/978-1-4939-7447-4_16
    The application of recombinant human antibodies is growing rapidly mainly in the field of diagnostics and therapeutics. To identify antibodies against a specific antigen, panning selection is carried out using different display technologies. Phage display technology remains the preferred platform due to its robustness and efficiency in biopanning experiments. There are both manual and semi-automated panning selections using polystyrene plastic, magnetic beads, and nitrocellulose as the immobilizing solid surface. Magnetic nanoparticles allow for improved antigen binding due to their large surface area. The Kingfisher Flex magnetic particle processing system was originally designed to aid in RNA, DNA, and protein extraction using magnetic beads. However, the system can be programmed for antibody phage display panning. The automation allows for a reduction in human error and improves reproducibility in between selections with the preprogrammed movements. The system requires minimum human intervention to operate; however, human intervention is needed for post-panning steps like phage rescue. In addition, polyclonal and monoclonal ELISA can be performed using the semi-automated platform to evaluate the selected antibody clones. This chapter will summarize the suggested protocol from the panning stage till the monoclonal ELISA evaluation. Other than this, important notes on the possible optimization and troubleshooting are also included at the end of this chapter.
    Matched MeSH terms: Cloning, Molecular/methods*
  16. Mass Spectrometry Immuno Assay (MSIA™) Streptavidin Disposable Automation Research Tips (D.A.R.T's®) Antibody Phage Display Biopanning
    Chin CF, Choong YS, Lim TS
    Methods Mol Biol, 2018;1701:285-299.
    PMID: 29116511 DOI: 10.1007/978-1-4939-7447-4_15
    Antibody phage display has been widely established as the method of choice to generate monoclonal antibodies with various efficacies post hybridoma technology. This technique is a popular method which takes precedence over ease of methodology, time- and cost-savings with comparable outcomes to conventional methods. Phage display technology manipulates the genome of M13 bacteriophage to display large diverse collection of antibodies that is capable of binding to various targets (nucleic acids, peptides, proteins, and carbohydrates). This subsequently leads to the discovery of target-related antibody binders. There have been several different approaches adapted for antibody phage display over the years. This chapter focuses on the semi-automated phage display antibody biopanning method utilizing the MSIA™ streptavidin D.A.R.T's® system. The system employs the use of electronic multichannel pipettes with predefined programs to carry out the panning process. The method should also be adaptable to larger liquid handling instrumentations for higher throughput.
    Matched MeSH terms: Cloning, Molecular/methods*
  17. Cloning, expression and purification of squalene synthase from Candida tropicalis in Pichia pastoris
    Lee PY, Yong VC, Rosli R, Gam LH, Chong PP
    Protein Expr Purif, 2014 Feb;94:15-21.
    PMID: 24184232 DOI: 10.1016/j.pep.2013.10.012
    Squalene synthase (SS) is the key precursor and first committed enzyme of the sterol biosynthesis pathway. In a previous work, SS has been identified as one of the immunogenic proteins that could be a potential diagnostic candidate for the pathogenic fungus Candida tropicalis. In this study, SS from C. tropicalis was cloned and expressed as recombinant protein in Pichia pastoris to investigate its reactivity with serum antibodies. ERG9 gene that encodes for SS was amplified by PCR and cloned in-frame into pPICZB expression vector. The recombinant construct was then transformed into P. pastoris GS115 host strain. Expression of the recombinant protein was confirmed by SDS-PAGE and Western blot analysis using anti-His tag probe. Optimal protein production was achieved by cultivating the culture with 1.0% methanol for 72h. The recombinant protein was purified to approximately 97% pure in a single step immobilized metal affinity chromatography with a yield of 70.3%. Besides, the purified protein exhibited specific reactivity with immune sera on Western blot. This is the first report on heterologous expression of antigenic SS from C. tropicalis in P. pastoris which can be exploited for large-scale production and further research. The results also suggested that the protein might be of great value as antigen candidate for serodiagnosis of Candida infection.
    Matched MeSH terms: Cloning, Molecular*
  18. Secretory expression of thermostable T1 lipase through bacteriocin release protein
    Rahman RN, Leow TC, Basri M, Salleh AB
    Protein Expr Purif, 2005 Apr;40(2):411-6.
    PMID: 15766884
    The extracellular production of T1 lipase was performed by co-expression of pJL3 vector encoding bacteriocin release protein in prokaryotic system. Secretory expression was optimized by considering several parameters, including host strains, inducer (IPTG) concentration, media, induction at A(600 nm), temperature, and time of induction. Among the host strains tested, Origami B excreted out 18,100 U/ml of lipase activity into culture medium when induced with 50 microM IPTG for 12 h. The Origami B harboring recombinant plasmid pGEX/T1S and pJL3 vector was chosen for further study. IPTG at 0.05 mM, YT medium, induction at A(600 nm) of 1.25, 30 degrees C, and 32 h of induction time were best condition for T1 lipase secretion with Origami B as a host.
    Matched MeSH terms: Cloning, Molecular/methods*
  19. The mannose-binding lectin (MBL) gene cloned from Exopalaemon carinicauda plays a key role in resisting infection by Vibrio parahaemolyticus
    Shi T, Gao J, Xu W, Liu X, Yan B, Azra MN, et al.
    Comp Biochem Physiol B Biochem Mol Biol, 2024;274:111001.
    PMID: 38908544 DOI: 10.1016/j.cbpb.2024.111001
    Mannose-binding lectin (MBL) is a vital member of the lectin family, crucial for mediating functions within the complement lectin pathway. In this study, following the cloning of the mannose-binding lectin (MBL) gene in the ridgetail white prawn, Exopalaemon carinicauda, we examined its expression patterns across various tissues and its role in combating challenges posed by Vibrio parahaemolyticus. The results revealed that the MBL gene spans 1342 bp, featuring an open reading frame of 972 bp. It encodes a protein comprising 323 amino acids, with a predicted relative molecular weight of 36 kDa and a theoretical isoelectric point of 6.18. The gene exhibited expression across various tissues including the eyestalk, heart, gill, hepatopancreas, stomach, intestine, ventral nerve cord, muscle, and hemolymph, with the highest expression detected in the hepatopancreas. Upon challenge with V. parahaemolyticus, RT-PCR analysis revealed a trend of MBL expression in hepatopancreatic tissues, characterized by an initial increase followed by a subsequent decrease, peaking at 24 h post-infection. Employing RNA interference to disrupt MBL gene expression resulted in a significant increase in mortality rates among individuals challenged with V. parahaemolyticus. Furthermore, we successfully generated the Pet32a-MBL recombinant protein through the construction of a prokaryotic expression vector for conducting in vitro bacterial inhibition assays, which demonstrated the inhibitory effect of the recombinant protein on V. parahaemolyticus, laying a foundation for further exploration into its immune mechanism in response to V. parahaemolyticus challenges.
    Matched MeSH terms: Cloning, Molecular*
  20. Molecular cloning, homology modeling and site-directed mutagenesis of vanadium-dependent bromoperoxidase (GcVBPO1) from Gracilaria changii (Rhodophyta)
    Baharum H, Chu WC, Teo SS, Ng KY, Rahim RA, Ho CL
    Phytochemistry, 2013 Aug;92:49-59.
    PMID: 23684235 DOI: 10.1016/j.phytochem.2013.04.014
    Vanadium-dependent haloperoxidases belong to a class of vanadium enzymes that may have potential industrial and pharmaceutical applications due to their high stability. In this study, the 5'-flanking genomic sequence and complete reading frame encoding vanadium-dependent bromoperoxidase (GcVBPO1) was cloned from the red seaweed, Fracilaria changii, and the recombinant protein was biochemically characterized. The deduced amino acid sequence of GcVBPO1 is 1818 nucleotides in length, sharing 49% identity with the vanadium-dependent bromoperoxidases from Corralina officinalis and Cor. pilulifera, respectively. The amino acid residues associated with the binding site of vanadate cofactor were found to be conserved. The Km value of recombinant GcVBPO1 for Br(-) was 4.69 mM, while its Vmax was 10.61 μkat mg(-1) at pH 7. Substitution of Arg(379) with His(379) in the recombinant protein caused a lower affinity for Br(-), while substitution of Arg(379) with Phe(379) not only increased its affinity for Br(-) but also enabled the mutant enzyme to oxidize Cl(-). The mutant Arg(379)Phe was also found to have a lower affinity for I(-), as compared to the wild-type GcVBPO1 and mutant Arg(379)His. In addition, the Arg(379)Phe mutant has a slightly higher affinity for H2O2 compared to the wild-type GcVBPO1. Multiple cis-acting regulatory elements associated with light response, hormone signaling, and meristem expression were detected at the 5'-flanking genomic sequence of GcVBPO1. The transcript abundance of GcVBPO1 was relatively higher in seaweed samples treated with 50 parts per thousand (ppt) artificial seawater (ASW) compared to those treated in 10 and 30 ppt ASW, in support of its role in the abiotic stress response of seaweed.
    Matched MeSH terms: Cloning, Molecular
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