Restriction Endonucleases BamHI Research Articles

Phosphorus-Nitrogen compounds part 74. Syntheses of (p-dimethylamino)benzyl-spiro-(N/N)-cyclotriphosphazenes: Structural characterizations, bioactivity studies, DFT calculations and reactivity parameters

Reactions of hexachlorocyclotriphosphazene (HCCP, N3P3Cl6), with equimolar amounts of N-methyl-N'-(p-dimethylaminobenzyl)-1,2-diaminoethane (1), N-ethyl-N'-(p-dimethyl-aminobenzyl)-1,2-diaminoethane (2), and N-methyl-N'-(p-dimethylaminobenzyl)-1,3-diami-nopropane (3) produced the corresponding tetrachloro-(p-dimethylaminobenzyl)spiro-(N/N)-cyclotriphosphazenes (4, 5, and 6), regioselectively. Tetrapyrrolidino (4a-6a), tetrapiperidino (4b-6b), tetramorpholinophosphazenes (4c and 6c), dichlorodimorpholinophosphazene (5c) and tetra-1,4-dioxa-8-azaspiro[4,5]decanophosphazenes (DASD) (4d-6d) were synthesized from Cl-substitution reactions of 4, 5 and 6 with excess pyrrolidine, piperidine, morpholine, and DASD, respectively. Their structures were elucidated by MS, FTIR, NMR and single crystal X-ray diffraction (for 4, 4b and 6a) techniques. Hirshfeld surface (HS) analyzes of 4, 4b, and 6a were performed to visualize intermolecular interactions in the crystals. In addition, according to the antimicrobial activity test results, compound 6b shows a better inhibitory effect on S. aureus ATCC 25923 G(+) (MİC: 78.1 µM) and C. tropicalis Y-12968 (MİC: 19.54 µM) microorganisms. The interactions of cyclotriphosphazenes with 5′-G/GATCC-3′ and 5′-A/AGCTT-3′ sequences of DNA were evaluated by examining BamHI and HindIII restriction enzyme digestion. The quantum chemical calculations of 4, 4b, and 6a were performed using DFT, and their experimental parameters were compared with the theoretical ones. Natural bond orbitals (NBOs) were determined and HOMO-LUMO energy gaps (Eg) were calculated as 3.55, 5.23, and 5.24 eV for 4, 4b, and 6a, respectively.

Generation of an Inducible Avp Knockout Mouse Line Using CRISPR/Cas9 and Cre/LoxP

Background: Arginine vasopressin (AVP) is a peptide hormone coded by the Avp gene, synthesized in the hypothalamus and secreted by the posterior pituitary. Dysregulation of AVP secretion contributes to a variety of human diseases. Previous studies of functional roles of AVP have been largely dependent on the use of Brattleboro rats, which manifest a spontaneous mutation in the Avp gene and lack circulating AVP. Despite their utility, Brattleboro rats are diffcult to breed owing largely to the fixed nature of the Avp mutation, resulting in increased neonatal death and behavioral effects in the adults. Consequently, commercial breeders have ceased production, despite a continued need. To fill this need, we have now created a mouse model with a tamoxifen-inducible deletion of AVP. Methods: We employed CRISPR/Cas9 to target the desired regions (first intron and 3' downstream) within the Avp gene. Successful insertion of the two loxP sites was confirmed by PCR using primers flanking the targeted regions. Restriction enzymes BamHI or EcoRI were used to confirm correct targeting. For additional confirmation, PCR products were cloned into the TA-cloning vector and sequenced. Mice harboring the floxed allele were mated to B6.Cg-Tg(CAG-cre/Esr1*)5Amc/J mice that globally express a tamoxifen-inducible Cre recombinase. Results: The resultant homozygous conditional knockout mice ( Cre+ Avpflx/ flx) are viable, fertile, and show no signs of polydipsia or polyuria prior to induction, indicating that the conditional alleles retain their wild-type function. Induction of Cre-mediated recombination by administration of tamoxifen (2-3 mg q.d., i.p. for 5 days) in 8-week-old mice resulted in a decrease in urine osmolality from 2076±138 to 122±6 mOsm/kgH2O on day 31 after induction (p<0.0001). Sanger sequencing demonstrated the expected 1245 bp deletion at the Avp locus. The kidney parenchyma was structurally normal with no evidence of medullary atrophy 6 weeks after induction. Immunoblotting of AQP2 in the inner medulla showed a significant decrease in AQP2 band density in Cre+ Avpflx/ flx mice to 27±14 % of values in Cre− floxed control mice (p<0.05). Conclusion: We developed an inducible AVP knockout mouse line ( Cre+ Avpflx/ flx) that will be shared with the research community and are likely to be useful for further study of regulation of water balance and polycystic kidney disease, as well as neural, vascular, and metabolic roles of vasopressin. The work was funded by the Division of Intramural Research, National Heart, Lung and Blood Institute. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

DNA major versus minor groove occupancy of monomeric and dimeric crystal violet derivatives. Toward structural correlations

Six monomeric (1a-1f) and five dimeric (2a-2e) derivatives of the triphenylmethane dye crystal violet (CV) have been prepared. Evaluation of the binding of these compounds to CT DNA by competitive fluorescent intercalator displacement (FID) assays, viscosity experiments, and UV and CD spectroscopy suggest that monomeric derivative 1a and dimeric derivative 2d likely associate with the major groove of DNA, while dimeric derivatives 2a and 2e likely associate with the minor groove of DNA. Additional evidence for the groove occupancy assignments of these derivatives was obtained from ITC experiments and from differential inhibition of DNA cleavage by the major groove binding restriction enzyme BamHI, as revealed by agarose gel electrophoresis. The data indicate that major groove ligands may be optimally constructed from dye units containing a sterically bulky 3,5-dimethyl-N,N-dimethylaniline group; furthermore, the groove-selectivity of olefin-tethered dimer 2d suggests that stereoelectronic interactions (n → π*) between the ligand and DNA are also an important design consideration in the crafting of major-groove binding ligands.

Novel multi-epitope vaccine against bovine brucellosis: approach from immunoinformatics to expression

Brucellosis is a zoonotic caused by the Brucella which is a well-known infectious disease agent in domestic animals and if transmitted, it can cause infection in humans. Because brucellosis is contagious, its control depends on the eradication of the animal disease in farms. There are two vaccines based on the killed and/or weakened bacteria against B. melitensis and B. abortus, but no recombinant vaccine is available for preventing the disease. The present study was designed to develop a multi-epitope vaccine against of B. melitensis and B. abortus using virB10, Omp31 and Omp16 antigens by the prediction of T lymphocytes, T cell cytotoxicity and IFN-γ epitopes. 50S L7/L12 Ribosomal protein from Mycobacterium tuberculosis was used as a bovine TLR4 and TLR9 agonist. GPGPG, AAY and KK linkers were used as a linker. Brucella construct was well-integrated in the pET-32a Shuttle vector with BamHI and HindIII restriction enzymes. The final construct contained 769 amino acids, that it was soluble protein of about ∼82 kDa after expression in the Escherichia coli SHuffle host. Modeled protein analysis based on the tertiary structure validation, molecular docking studies, molecular dynamics simulations results like RMSD, Gyration and RMSF as well as MM/PBSA analysis showed that this protein has a stable construct and is capable being in interaction with bovine TLR4 and TLR9. Analysis of the data obtained suggests that the proposed vaccine can induce the immune response by stimulating T- and B-cells, and may be used for prevention and remedial purposes, against B. melitensis and B. abortus. Communicated by Ramaswamy H. Sarma

Prevalence of JC and BK Polyomavirus Infection in Patients with Chronic Kidney Disease in the State of Pará, Brazil.

The polyomaviruses that infect humans, JC virus (JCV) and BK virus (BKV), can establish persistent infections in the cells that make up the renal system, causing nephritis and BKV-associated nephropathy in up to 10% of renal transplant patients, and of these, 90% lose the graft and return for hemodialysis. This study aimed to determine the prevalence of polyomaviruses (PyV) in the population with chronic kidney disease (CKD), classified into three groups (conservative, dialysis, and transplanted) and a control group. Urine samples were collected from 290 individuals, including 202 patients with CKD and 88 from the control group. PyV screening was performed by PCR amplification of a fragment of the VP1 region, and the JCV and BKV species were distinguished through enzymatic digestion with the restriction endonuclease BamHI from the amplification of a TAg region. All amplification products were visualized on a 3% agarose gel. The prevalence of PyV infection was correlated with clinical-epidemiological variables using the chi-squared and Fisher's exact tests. In the group with CKD, the prevalence of PyV was 30.2%, a higher rate being observed in conservative patients (36.66%; 22/60), followed by dialysis patients (30.48%; 25/82), and transplanted patients (20%; 12/60). In the control group, the prevalence was 46.59% (41/88). The differentiation between species revealed that JCV was present in 77.8% and BKV in 22.2% of the group with CKD. The prevalence of infection was higher in male patients (59.32%), whose most common pathology was systemic arterial hypertension (35.59%). In the group of transplanted patients, there was a statistically significant association between infection and the use of the immunosuppressant azathioprine (p = 0.015). The prevalence of PyV infection was higher in the control group than in the group with CKD, being predominant in males and in patients with systemic arterial hypertension.

A quantification method of somatic mutations in normal tissues and their accumulation in pediatric patients with chemotherapy

Somatic mutations are accumulated in normal human tissues with aging and exposure to carcinogens. If we can accurately count any passenger mutations in any single DNA molecule, since their quantity is much larger than driver mutations, we can sensitively detect mutation accumulation in polyclonal normal tissues. Duplex sequencing, which tags both DNA strands in one DNA molecule, enables accurate count of such mutations, but requires a very large number of sequencing reads for each single sample of human-genome size. Here, we reduced the genome size to 1/90 using the BamHI restriction enzyme and established a cost-effective pipeline. The enzymatically cleaved and optimal sequencing (EcoSeq) method was able to count somatic mutations in a single DNA molecule with a sensitivity of as low as 3 × 10-8 per base pair (bp), as assessed by measuring artificially prepared mutations. Taking advantages of EcoSeq, we analyzed normal peripheral blood cells of pediatric sarcoma patients who received chemotherapy (n = 10) and those who did not (n = 10). The former had a mutation frequency of 31.2 ± 13.4 × 10-8 per base pair while the latter had 9.0 ± 4.5 × 10-8 per base pair (P < 0.001). The increase in mutation frequency was confirmed by analysis of the same patients before and after chemotherapy, and increased mutation frequencies persisted 46 to 64 mo after chemotherapy, indicating that the mutation accumulation constitutes a risk of secondary leukemia. EcoSeq has the potential to reveal accumulation of somatic mutations and exposure to environmental factors in any DNA samples and will contribute to cancer risk estimation.

Rolling circle amplification of begomoviral DNA from a single nucleus isolated by laser dissection microscopy

Laser dissection microscopy (LDM) is a method for isolating organelles, a specific cell or cells/tissue of interest from microscopic regions with the help of a laser. Here we describe a LDM-based isolation of begomovirus infected Nicotiana benthamiana epidermal cells and nuclei, in combination with a fast method to prepare non-fixed leaf epidermal samples for LDM. The bipartite Abutilon mosaic virus (AbMV) was used in which the coat protein gene of DNA A was deleted and replaced by the open reading frame (ORF) coding for the green fluorescent protein (GFP, accession: U87624), agro-infiltrated together with DNA B, to visualize infected cells. GFP expressing epidermal cells or nuclei were isolated by LDM with the MMi Cellcut system and viral circular DNA was amplified by rolling circle amplification (RCA). Subsequently, the RCA product was incubated with the restriction enzymes BamHI and PstI and restriction fragments were separated on an agarose gel to prove presence of the viral genome. It was shown that even a single-isolated nucleus harbored enough material to produce a sufficient restriction fragment pattern to identify a begomovirus infected cell/nucleus.

Studying of cyp2c19*2, *3 and *17 polymorphism in Vietnamese patients with coronary artery disease

Coronary artery disease is the most common type of cardiovascular disease, due to the accumulation of atherosclerotic plaque inside the arterial wall which leads to block blood supply to the heart muscle. A number of clinical trials have demonstrated that Clopidogrel is able to inhibit platelet aggregation in patients with acute coronary syndromes, reduce mortality and cardiovascular events. However, the antiplatelet effectiveness of Clopidogrel significantly depends on CYP2C19 genotypes. Therefore, the aim of this study was to identify the CYP2C19*2, *3 and allele frequencies in Vietnamese coronary artery patients by using PCR-RFLP method. Total genomic DNA were extracted from peripheral blood of 96 patients diagnosed with coronary artery disease. Thereafter, single nucleotide polymorphism sites in the CYP2C19 gene were identified by PCR with specific primers. The amplified products were then digested by restriction enzymes SmaI, BamHI, and MnlI, respectively. The results showed that the proportion of heterozygous individuals for CYP2C19*2 (c. 681G&gt;A, rs4244285), CYP2C19*3 (c. 636G&gt;A, rs4986893), and CYP2C19*17 (g. -3402C&gt;T, rs11188072) accounted for 39.58%, 6.25%, and 2.08%, respectively. Among 96 subjects, 41.67% of patients were predicted for intermediate metabolic phenotype CYP2C19*1/*2 (37.50%) and CYP2C19*1/*3 (4.17%). Approximately 10.42% of total patients represented poor metabolizers in which 8.34% had two copies of the same allele *2/*2 and 2.08% had *2/*3 genotype. Particularly, two individuals (2.08%) detected with CYP2C19*1/*17 genotype were able to increase CYP2C19 activity (ultrarapid metabolizers). The results of this study generate a foundation for introducing individualized antiplatelet therapy in Vietnam based on genetic testing.

Molecular cloning and characterizing of Bacillus subtilis cellulase collected from central-northern Iran forests

In the large-scale commercial bioconversion of complex lignocellulosic materials to biofuels, a cost-effective purification and production of cellulase is a huge concern. In this regard, genetically engineered strains have shown great merits for producing active cellulase. As an attempt to improve cellulase production; herein, bacterial strains with cellulolytic activity were collected from forest soils located across Mazandaran Province, in central-northern Iran. To verify cellulolytic activity, carboxymethyl cellulose (CMC) in aka cellulose gum medium was used. Afterward, in order to characterize the bacteria with most cellulolytic activity, 16S rDNA of bacterial colonies were amplified and then phylogenetic analysis was conducted. Cellulase gene was isolated from the most active bacterial strain, Bacillus subtilis B2 and then it was cloned into the bacterial expression vector pET-26b using HindIII and BamHI restriction endonuclease sites. The generated plasmid containing cellulase and expression promoter system was transformed into the BL21 (DE3) competent Escherichia coli. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis was performed to verify presence of the protein. The sequencing of cloned gene revealed 77% identity with endoglucanase of Bacillus subtilis subsp. Moreover, the molecular weight of cloned cellulase was 48 k Da as revealed by SDS-PAGE. Significance statementHerein, a newly isolated bacterial strain with high potential of cellulase production is introduced that benefits commercial bioconversion of complex lignocellulosic materials to biofuels.

The pET-Rep recombinant plasmid construction and geminivirus Rep [C1] gene expression in Escherichia coli strain BL21

Geminivirus Rep [C1] gene produces a replication protein [Rep] that interacts with Retinoblastoma-related protein [RBR] when Geminivirus infect the plants. That interactions interfere with RBR and transcription factor function. So the mechanism of chili plant defense against symptoms of Pepper Yellow Leaf Curl Disease is blocked. The NPR1 gene is a transcriptional co-activator involved in the regulation defense mechanism of Systemic Acquired Resistance [SAR] in chili plants. In silico, Rep protein blocks the SAR mechanism that is regulated by the NPR1 gene. This study aimed to obtain a construction of pET-28a+ plasmid expression recombinant Rep [C1] gene and Rep protein that can be expressed in E.coli BL21. This research is useful to study the interaction of Geminivirus with transcription co-activator in chili plants. The construction was done by ligating plasmid and Rep genes which have been cut using restriction enzymes BamHI and SacI. The construction of a recombinant pET-Rep plasmid is confirmed by amplification and verification of the nucleotide sequence through sequencing techniques. Nucleotide sequence verification results proved that the Rep [C1] gene is successfully constructed into a pET-28a+ plasmid with proper position and orientation. The pET-Rep recombinant proceeds to the expression test using the E.coli BL21 host. Expression was performed using the induction method with IPTG. Proteins Rep is purified using the MagnehisTM Protein Purification System. Rep protein is visualized with SDS-PAGE. Visualization showed that Rep protein was successfully expressed in E.coli BL21 where protein size is estimated of 41,03 kDa in size.

Cloning,Sequencingand Analyzingof16S rRNA Gene from Inulin HydrolyzingBacteria

Gene of 16S rRNA is used for molecular identification of bacteria. This research aims to analyze the sequences of 16S rRNA gene from inulin hydrolyzing bacteria of UKG isolate. The 16S rRNA gene was isolated by PCR using BacF1 and UniB1 primers. The gene was cloned to pGEM-T Easy vector with E.coli TOP10F as host cell. Recombinant DNA was sequenced using T7 and SP6 primers. The size of the nucleotide base of the recombinant DNA of the sequencing result using SP6 primer was 1167 bp, whereas using T7 primer was 1218 bp. The both of the sequences overlapped 760 bp. The size of 16S rRNA gene in the sequences was found 1501 bp. Recombinant DNA that was restricted using EcoR1 showed 3 bands on agarose gel. They were 3000 bp, 800 bp, and 700 bp. The position of the EcoR1 palindrome sequence on the 16S rRNA gene was at the 832-837. The 16S rRNA gene also has been known by HindIII, BamHI, BalI, HaeIII, and SmaI restriction enzymes.

New source of cellulase production using a metagenomic technique

The cellulase enzymes with high effectiveness under conditions agreeable to the industrial processes necessities are one of the keys for the successful development of chemical and drug synthesis. The soil metagenome is an affluent source for the discovery of new natural products. The objective of the current study was to identify the isolated functional gene(s) of the cellulase enzyme by using metagenomics. The plan of work composed of collection of different soil samples, isolation of total DNA, fragmentation, cloning, and expression of the isolated gene(s) in the suitable host microorganism. The total genomic DNA was extracted using a kit (QIAGEN), and then digested by different restriction enzymes BamHI. The digested fragments ranging from ~300-5000 bp were ligated, cloned into pUC19 vector, and then transformed into Escherichia coli DH5α. The resulting clones were screened as cellulase producers using a qualitative method. The positive clones which showed hydrolysis on the plate were screened once more in Luria-Bertani (LB) medium. The plasmids were isolated and then tested using universal primer (M13), to detect the fragment size and sequence for the Polymerase Chain Reaction (PCR) products. This study establishes an effortless and professional method for cloning of recent cellulase genes through ecological metagenomes. In the outlook, the metagenomic guide approachs may be functional to the elevated selection of novel cellulase from the environment.

Cloning, expression, purification and interaction evaluation of 30 amino acids in C terminus of Clostridium perfringens toxin with its receptor Claudin-4

Oral vaccine is a strategy being the most interested about treatments of gastrointestinal infections because of many great benefits outweigh conventional injection vaccines. In order to resolve the dispersion of antigens in gastrointestinal surfaces, the immunological tolerance and also be capable to stimulate immune responses effectively, M cells are targeted for antigens delivery. A number of researches reported that 30 amino acids in C terminus of Clostridium perfringens toxin (CPE30) have a high affinity to Claudin-4 receptor presenting on M cells. It is highly indispensable to produce a resource for assessing of CPE30 binding ability so cpe30 gene was cloned into the pET-gfp plasmid by two restriction enzymes BamHI and NdeI on the E. coli DH5α strain. The expression and confirmation of the fusion protein CPE30-GFP which was induced by IPTG in E. coli BL21 (DE3) strain and assessed by SDS-PAGE and Western blot with 6xHis Taq antibody demonstrated that there was the over expression of CPE30 GFP fusion protein in the cytoplasm, mainly in the soluble form. Finally, CPE30-GFP was purified which the purity was approximately 92.3%. In vitro protein interaction measurement using silicon nanowire field-effect transistors (SiNW FETs) showed that CPE30-GFP had a good binding affinity with its receptor Claudin-4 (R4). This result laid the groundwork for the CPE30 interaction study with the M cell in vivo.

Precise Construction and Tuning of an Aerolysin Single-Biomolecule Interface for Single-Molecule Sensing

Precise Construction and Tuning of an Aerolysin Single-Biomolecule Interface for Single-Molecule Sensing

Predicting Diffusion Coefficients of DNA-Protein Complexes by Convex Hull Modelling

The recent development of single-molecule diffusometry establishes the diffusion coefficient as a new observable in single-molecule experiments to visualize biomolecular interactions. To decipher the rich information available from these experiments, accurate modeling of the hydrodynamic properties of biomolecular complexes is needed. Currently available tools for predicting diffusion coefficients of biomolecules, such as Hydropro and HullRad, rely on the existence of atomic coordinates for the molecule of interest. Composite molecular complexes that lack detailed Protein Data Bank entries, but can be modeled structurally from their constituent parts (i.e. a complex with multiple proteins bound to a single DNA) are frequently encountered in experiments but cannot yet be properly modeled for hydrodynamic properties. We present a simple program to predict the diffusion coefficients of composite DNA-protein complexes. Our algorithm extends the computational framework introduced by Fleming et al. in the HullRad software to construct a convex hull of the molecular complex and then uses an ellipsoidal approximation to compute hydrodynamic parameters. In order to construct the convex hull of the whole complex, the algorithm incorporates a priori knowledge of the complex such as the crystal structures of the constituent biomolecules, binding site positions, geometry and mechanics of DNA duplexes. The program is computationally efficient, integrates naturally with PyMOL, and outputs the 3D convex hull for visualization. We validate the program by accurately predicting hydrodynamic parameters of known crystal structures of DNA-protein complexes. We then predict diffusion coefficients of the restriction enzyme BamHI bound to double strand DNA of different lengths and compare these predictions with single-molecule diffusometry measurements. We envision that this program will aid researchers in using diffusion coefficient to identify and characterize relevant biological structures at the nanometer scale.

Bacterial Expression and Characterization of Recombinant β-Xylosidase from the Thermophilic Xylanolytic Bacterium Bacillus sp.

With the passage of time, energy sources are decreasing day by day. In order to meet the world's demand, much attention is being paid to the study of enzymes with xylanolytic activity as a potential means of generating energy. A thermophilic xylanolytic bacterium, Bacillus sp., was isolated from naturally decaying material by enrichment culture and serial dilution methods. The bacterium was grown in MH medium at 50°C and pH 7 for 10 h. The xylanolytic Bacillus sp. produced clear yellow haloes around the colonies in the presence of p-nitrophenyl beta-D-xylopyranoside (pNPX) as a substrate. After condition optimization, it was found that the organism produced the higher level of xylosidase activity after 14 h in the presence of arabinose as a carbon source and ammonium sulfate as a nitrogen source in the pH 7 medium of at 55°C. The maximum β-xylosidase activity after optimizing the culture condition was 5.0 U/mL. Later this thermophilic Bacillus sp. was used as a donor in cloning of the β-xylosidase gene. A genomic library of Bacillus sp. was prepared by digesting the genomic DNA of the Bacillus with the restriction endonuclease BamHI, ligating the fragments in the pUC18 cloning vector and then transforming the competent E. coli DH5α cells with the resultant chimeric plasmid. The β-xylosidase gene was identified by screening the transformants in duplicates on LB agar plates overlaid with pNPX as a substrate. Commercial production of β-xylosidase to be used as a methanol-producing enzyme can help to overcome fuel shortages.

The Production of the Recombinant Protein of Anthrax Bacteriolysine PlyPH and In Vitro Study of the Lytic Properties of the Protein Coded for them Against Bacillus anhracis Microbial Cells

The causative agent of anthrax - Bacillus anthracis, due to the prevalence of its natural foci in Russia, high virulence for humans and most mammals, the unique resistance of spore forms to environmental factors and repeated use in terrorist acts, is an extremely dangerous biological agent. Therefore, the search for new effective drugs for the diagnosis and treatment of anthrax, including diseases caused by antibiotic-resistant strains of B. anthracis is necessary. The use of lytic enzymes of species-specific bacteriophages is a new trend in the diagnosis, prevention and treatment of infectious diseases. The goal of this work is the cloning of the anthrax bacteriolysin PlyPH gene as part of the pTrcHis2C vector in Escherichia coli and the in vitro study of the lytic properties of the protein encoded by it against B. anthracis microbial cells. According to the complete sequencing of the B. anthracis genomes of the Ames, Stern 34F2 and JB17 strains, a prophage was found in their chromosomal DNA, which lost part of the structural genes necessary for its replication, but retained a gene with a high degree of homology with the bacteriolysin γ phage gene. For amplification and subsequent cloning of the PlyPH gene, we developed primers containing EcoRI and BamHI restriction enzyme recognition sites. Amplification of the PlyPH gene in a polymerase chain reaction (PCR) with a developed pair of primers was performed using the Stern 34F2 strain of the anthrax microbe as a template. Based on the obtained amplification products and the pTrcHis2C vector, we constructed a recombinant plasmid containing the bacteriolysin synthesis PlyPH gene and stably functioning in the cells of the recombinant E. coli strain. In the course of research, it has been established that microbial cells of the E. coli recombinant TOP10 strain provide for the production of the bacteriolysin of the anthrax prophage, PlyPH , which has the ability to in vitro lyse the vegetative cells of the STI-1 vaccine strain of B. anthracis

Production of MPT-64 recombinant protein from virulent strain of Mycobacterium bovis.

Tuberculosis (TB) is a zoonotic infectious disease common to humans and animals which has been caused by a rod shaped, acid fast bacterium, called Mycobacterium bovis. The rapid and sensitive detection is a great challenge for TB diagnosis. The virulent strains of Mycobacterium tuberculosis complex (MTBC) have 16 different regions of difference (RD) in their genome which encode some important antigens. The major protein of M. bovis 64 (MPT-64) is one of the main immune-stimulating antigens which are encode by RD-2 region. The aim of the present study was cloning, expression and purification of MPT-64 as a protein antigen of M. bovis in a prokaryotic system for the usage in the future diagnostic studies. In this experimental study, the mpt-64 gene with 687 bp has been proliferated from M. bovis whole genome by polymerase chain reaction (PCR) method. The PCR product has been digested by BamHI and HindIII restriction enzymes and cloned into pQE-30 plasmid. The recombinant protein has been expressed in the Escherichia coli M15 with induction by isopropyl-β-D-thiogalactopyranoside (IPTG). The expressed protein was analyzed on SDS-PAGE, and purified with Nitrilotriacetic acid (Ni-NTA) column. Finally, its biological properties were confirmed in Western blotting method using specific antibodies. Data showed the successful cloning of mpt-64 gene (as a 687 bp segment) in expression vector. The MPT-64 recombinant protein was ideally expressed and purified as a 24 kDa protein. The result of this study indicated that MPT-64 recombinant protein (24 kDa) has been successfully expressed and purified in a prokaryotic system, so this protein could be used for differential diagnosis of pathogenic and non-pathogenic Mycobacterium, in suspected BTB cases.

Molecular characterization of Babesia microti seroreactive antigen 5-1-1 and development of rapid detection methods for anti-B. microti antibodies in serum

Babesiosis has become a new global threat impacting human health, and most human babesiosis cases are caused by Babesia microti. Until now few antigens of B. microti have been described which can be used for the diagnosis of human babesiosis. In the present study, we report on the bioinformatic analysis, cloning and expression of the sequence encoding the B. microti seroreactive antigen 5-1-1 to investigate its potential incorporation in serologic diagnostic tools for babesiosis. Bioinformatic analysis and recombinant gene expression were performed to molecularly characterize seroreactive antigen 5-1-1. Enhanced chemiluminescence (ECL)-Western blot methods were used to detect specific antibodies in infected mice. Immunofluorescence antibody assays (IFA) were performed to detect the localization of BmSA5-1-1 in B. microti parasites. ELISA and immunochromatographic (ICT) tests were developed using recombinant BmSA5-1-1 to evaluate its potential use in rapid detection methods for B. microti antibodies and for the diagnosis of babesiosis. A recombinant expression plasmid was constructed by inserting the target gene fragment in the pET28a vector after double digestion with BamHI and XhoI restriction enzymes. The recombinant BmSA5-1-1 protein was expressed in Escherichia coli (rBmSA5-1-1) and purified by means of Ni-nitrilotriacetic acid (NTA) agarose columns. Polyclonal antibodies were generated against rBmSA5-1-1. Based on indirect immunofluorescence assay results, BmSA5-1-1 appeared to localize on the surface of B. microti. ELISA tests using the rBmSA5-1-1 antigen detected specific antibodies from infected mice as early as 4 days post-infection. Our results indicate that the two methods we developed can detect specific antibodies in mice at different stages of infection with sensitivities of 100% (rBmSA5-1-1 ELISA) and 90% (ICT). The specificity of the two methods was 100%. Sera of patients suffering from other closely related parasitic diseases, such as malaria and toxoplasmosis, produced negative results. In conclusion, seroreactive antigen 5-1-1, a member of the BMN1 protein family, is expressed on the outer surface of B. microti and is a promising candidate antigen for the early diagnosis of babesiosis. rBmSA5-1-1 ELISA and ICT methods show good potential for detecting specific antibodies in mice at different stages of infection.

Antibiotic resistance and distribution of SodCI, sopE, sefA genes among Salmonella enteric serotype Enteritidis isolates from poultry

Present work aimed to examine the antibiotic resistance of the Salmonella enterica serotype Enteritidis (SE) isolates from poultry, to study the plasmid-mediated ampicillin resistance and to detect and determine the distribution of sodCI, sopE and sefA genes. Thirty-five SE isolates from one-day chicks, layers and broilers were studied for susceptibility/resistance to sixteen antimicrobial agents; 23 (65.7%) of them showed resistance to ampicillin, 5 (14.3%) to ampicillin and tetracycline, 4 (11.45%) to tetracycline and 1 (2.9%) isolate showed multi-drug resistance. Ampicillin (AmpR) and ampicillin/tetracycline (AmpRTeR) resistance was easily transferred by conjugation, and all isolates except two possessed a common band. The molecular mass of the plasmid carrying ampicillin resistance was approximately determined at 41kb after DNA digestion with BamHI, HindIII, EcoRI, EcoRV and PstI restriction enzymes and ligation of EcoRI fragments to pET29c. For the detection of TEM-1 or/and TEM-2 ß-lactamases, two pairs of primers were used in a polymerase chain reaction (PCR). The PCR products showed the presence of blaTEM-1 gene in all isolates. The presence of SodCI, sopE, sefA genes was also examined by PCR. Twenty-two (62,8%) isolates carried the sodCI gene, thirty-four (97,2%) isolates carried the sopE gene and all isolates carried the sefA fimbrial locus.