SDS-polyacrylamide Gel Electrophoresis Analysis Research Articles

Molecular characterization and immunodiagnostics of Dicrocoelium dendriticum species isolated from sheep of north-west Himalayan region.

Despite its extensive presence among grazing ruminants, dicrocoeliosis, also known as 'small liver fluke' disease, is poorly known and often underestimated by researchers and practitioners in many countries. The accurate identification and prepatent diagnosis of Dicrocoelium dendriticum infection is an essential prerequisite for its prevention and control. In the present study, the morphologically identified specimens isolated from the bile ducts of sheep (Ovis aries) were validated through molecular data. The sequence analysis of the second internal transcribed spacer (ITS-2) of our isolates showed a high degree of similarity with D. dendriticum using the BLAST function of the National Center for Biotechnology Information (NCBI). The phylogenetic analysis of our isolates showed a close relationship with previously described D. dendriticum isolates from different countries. The antigenic profiles of somatic and excretory/secretory (E/S) antigens of D. dendriticum were revealed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting using sera from sheep naturally infected with D. dendriticum. By SDS-PAGE, 16 distinct bands were revealed from crude somatic fraction. Immunoblotting analysis of these proteins with positive sera exhibited six seroreactive bands ranging from 27 to 130 kDa. Among these, the 84 and 130 kDa bands were quite specific, with high diagnostic specificity and sensitivity. The E/S fraction comprised nine distinct bands, as revealed by SDS-PAGE analysis. Immunoblotting analysis of these proteins with positive sera exhibited five antigenic bands ranging from 27 to 130 kDa. Among these, the 130 kDa band was found to be quite specific, with high diagnostic specificity and sensitivity. The present study concludes that the protein bands of 84 and 130 kDa in somatic fraction and 130 kDa in E/S fraction can be used for the immunodiagnostic purpose for this economically important parasite, which may also encourage further studies regarding their vaccine potential.

Detection and antibiotic resistance of Mycoplasma gallisepticum and Mycoplasma synoviae among chicken flocks in Egypt.

Background and Aim:Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) are the most significant pathogens of avian mycoplasmosis. This study aimed to isolate and identify MG and MS from chickens and detect the various virulence genes in the isolates. Moreover, the efficacies of different antibiotics were tested to identify suitable treatment regimens.Materials and Methods:We isolated MG and MS from 487 chicken samples of different ages located in different Governorates in Egypt using conventional isolation methods. The isolates were characterized by polymerase chain reaction (PCR) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then tested for antibiotic sensitivity by the minimum inhibitory concentration (MIC) method.Results:The prevalence of MG among the isolates was 9.85%, with the highest percentage isolated from air sacs, while the prevalence of MS among the isolates was 1.6%. Moreover, the highest levels of the prevalence of both MG and MS were during the winter and autumn sampling, while the lowest levels were in the summer and spring. Following the 16S rRNA-based detection of Mycoplasma isolates, 14 MG and 5 MS isolates were identified by different PCR-based detection methods for various virulence genes. Nine MG isolates contain the mgc2 gene, six MG isolates contain the gapA gene, and three MS isolates contain the vlhA gene. We validated a duplex PCR method for the simultaneous identification of MG and MS, based on 100% of the MG and MS isolates generating common bands at 55 and 17 kDa, respectively. The MIC method identified tiamulin and spiramycin as the antibiotics of choice for the treatment of MG and MS infections, respectively.Conclusion:For more precise diagnosis of Mycoplasma infections in chicken flocks, conventional isolation methods must be confirmed by PCR. SDS-PAGE analysis helps in epidemiological studies and vaccine preparation. The MIC method can be used to help develop therapies to control avian mycoplasmosis infections.

Relationship between semen quality and seminal plasma cholesterol, triacylglycerols and proteins in the domestic cat.

The current study aimed to evaluate the relationship between specific seminal plasma components - cholesterol (CHOL), triacylglycerols (TAG) and total protein (PROT) concentrations - and semen quality in cats. A further aim was to determine the relationship between specific seminal protein bands and semen quality. Thirteen toms, 2-5 years of age, were included. Semen collection was performed by electroejaculation every 4 weeks. Fifty-eight ejaculates were assessed for motility, velocity, volume, sperm concentration, total sperm count, viability, acrosome integrity, plasma membrane integrity and sperm morphology. Samples were divided into two groups: good semen quality (GSQ) and poor semen quality (PSQ). After evaluation, seminal plasma was separated from the sperm by centrifugation and stored at -20°C. CHOL, TAG and PROT concentrations were then assessed and seminal plasma protein profile was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Seminal plasma CHOL and TAG concentrations, motility, velocity, sperm concentration, total sperm count and sperm morphology were significantly higher in GSQ cats compared with PSQ cats (P <0.01). Moreover, seminal plasma SDS-PAGE analysis showed an identifiable extra band exclusively in the GSQ group. Data obtained in this study showed that seminal plasma CHOL and TAG concentrations and specific protein bands could be used to improve semen evaluation in toms. In this sense, the 14 kDa protein band could be a valuable marker for semen quality in the cat and should be further investigated. However, more studies are necessary to determine its relationship with fertility.

Characterization, evaluation of nutritional parameters of Radix isatidis protein and its antioxidant activity in D-galactose induced ageing mice

BackgroundRadix isatidis (Isatis indigotica Fort.) is an ancient medicinal herb, which has been applied to the prevention and treatment of influenza virus since ancient times. In recent years, the antioxidant activity of Radix isatidis has been widely concerned by researchers. Our previous studies have shown that Radix isatidis protein (RIP) has good antioxidant activity in vitro. In this study, the composition of the protein was characterized and its antioxidant activity in vivo was evaluated.MethodsThe model of oxidative damage in mice was established by subcutaneous injection of D-galactose for 7 weeks. Commercially available kits were used to determine the content of protein and several oxidation indexes in different tissues of mice. The tissue samples were stained with hematoxylin and eosin (H&E) and the pathological changes were observed by optical microscope. The molecular weight of RIP was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The amino acid composition of RIP was determined by a non-derivative method developed by our research group.ResultsRIP significantly increased the activities of antioxidant enzymes such as SOD, CAT, GSH-Px and total antioxidant capability (TAOC) but decreased the MDA level in the serum, kidney and liver. H&E stained sections of liver and kidney revealed D-galactose could cause serious injury and RIP could substantially attenuate the injury. The analysis of SDS-PAGE showed that four bands with molecular weights of 19.2 kDa, 21.5 kDa, 24.8 kDa and 40.0 kDa were the main protein components of RIP.ConclusionsThe results suggested that RIP had excellent antioxidant activity, which could be explored as a health-care product to retard aging and a good source of protein nutrition for human consumption.

The use of sarcoptes scabiei crude extract as allergen: An attempt to prepare a vaccine

Scabies stands for a skin parasitic infestation as a result of mite Sarcoptes scabiei. It is a neglected international health problem; about 300 million cases develop scabies worldwide annually. Sarcoptes scabiei mite proteins are extracted, then heat-stable mite proteins concentration has been established by Bradford's method. SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) analysis was used. The activity of mite allergens was assayed through skin prick test (SPT) in scabietic patients with 1.2 μg/ml and 2.4 μg/ml. House dust mite (HDM) antigens were skin prick tested in patients with scabies and controls. The results revealed that the SDS-PAGE profile of the parasite heat-stable proteins consisted of protein bands with molecular weights ranged from less than 10 to over than 100 kDa. Skin test demonstrated that 70% and 80% of scabietic patients had a positivity against 1.2 μg/ml and 2.4 μg/ml of sarcoptic mite extracts, respectively when prick tested. HDM extract was found to be positive in 40% of scabietic patients; while controls revealed a negative result. Sarcoptic proteins contain heat-stable allergens which able to cause immediate type-1 hypersensitivity when 1.2μg/ml of mite protein is skin prick tested, and there is cross-reactivity between Sarcoptes scabiei and HDM allergens.

Purification and identification of a novel antifungal protein from Bacillus subtilis XB-1.

This study aimed to characterize a powerful antifungal component from bacteria. Bacillus subtilis strain XB-1, which showed maximal inhibition of Monilinia fructicola, was isolated and identified, and an antifungal protein was obtained from it. Ammonium sulfate precipitation, ion exchange chromatography, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were used to purify and identify the proteins secreted by B. subtilis XB-1. Analyses revealed that purified fraction V had the strongest antifungal effect, with the largest pathogen inhibition zone diameter of 4.15cm after 4days (P < 0.05). This fraction showed a single band with a molecular weight of approximately 43kDa in SDS-PAGE. Results from SDS-PAGE and liquid chromatography electrospray ionization tandem mass spectrometry analyses demonstrated that fraction V was likely a member of the chitosanase family. These results suggest that B. subtilis XB-1 and its antifungal protein may be useful in potential biocontrol applications.

Purification and characteristics of a new milk-clotting enzyme from Bacillus licheniformis BL312

A new milk-clotting enzyme (MCE) was produced from Bacillus licheniformis BL312. The characteristics of BL312 MCE were compared with those of Calf rennet, R. miehei MCE, and Chymopapain. BL312 MCE showed a remarkable activity of 5291 SU/mg after purification by ammonium sulfate fractionation and DEAE-Sepharose Fast Flow, which yielded 5.09-fold purification with a recovery of 39.24%. The purified BL312 MCE gave a single protein band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel, corresponding to 27 kDa. The αs-casein (αs-CN) and β-casein (β-CN) hydrolysates generated by BL312 MCE were different from those by Calf rennet, R. miehei MCE, and Chymopapain through SDS-PAGE and reversed-phase HPLC (RP-HPLC) analyses. The main cleavage sites in κ-casein (κ-CN) were the Met106-Ala107 and Asn123-Thr124 bonds for BL312 MCE, as determined by mass spectrometry analysis. BL312 MCE hydrolyzed αs-CN to a greater level than β-CN. It was stable at a wide pH range of 5.5–11.0 and temperature below 45 °C. The milk-clotting activity (MCA) reached the maximum when the substrate contained 50 mM CaCl2 at pH 5.5. The optimal temperature of MCE was 55 °C. The successful application in Monascus-fermented cheese production confirmed the usefulness of BL312 MCE in food and dairy processing.

Isoenzymes and protein polymorphism in Blaps polycresta and Trachyderma hispida (Forsskål, 1775) (Coleoptera: Tenebrionidae) as biomarkers for ceramic industrial pollution.

The expression levels of Esterase Isoenzyme and total soluble protein fractionation were studied in two coleopteran insects Blaps polycresta and Trachyderma hispida to evaluate the possible hazards from ceramic and plastic factories in the Khorshed Region, East of Alexandria, Egypt. Two insect collection sites were selected. The first site was the garden of the Faculty of Science, Moharram Bek, Alexandria University, which is considered a non-polluted site, and Khorshed district, considered as the polluted site. Percentages of heavy metals were estimated using SEM-X-ray microanalysis of soft tissues of both sexes of the two coleopteran insects. Esterase Isoenzymes were found to be overexpressed in B. Polycresta but not T. hispida. Female B. polycresta from the polluted site exhibited overexpression of the second and third loci. Furthermore, the females were found to be more affected than males, which only showed the overexpression of the second loci. T. hispida (females and males) collected from the reference site were found to have increased esterase activity compared with those sampled from the polluted site. The Snake-Skin™ Dialysis tubing technique, used for optimizing the protein extraction method, reflected the highest quantified proteins compared to other, traditional methods. SDS polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the whole-body protein reflected definite variations between T. hispida and B. polycresta in fraction number and activity at the two sites. Varied expression levels for metallothionein (MT) heavy metal resistance proteins for B. polycresta and T. hispida were also detected in the study. Based on these results, we suggest that biochemical biomarkers could infer environmental hazards, B. polycresta and T. hispida are successful biomarkers for heavy metal pollution.

Biodegradation of fenpropathrin by Rhodopseudomonas sp. strain PSB07-21 cultured under three different growth modes.

This study aimed at the biodegradation of fenpropathrin by Rhodopseudomonas sp. strain PSB07-21 cultured under different growth modes. The biomass production, cell surface hydrophobicity and fenpropathrin biodegradation efficiency of the strain PSB07-21 cultured under the photoheterotrophic growth mode were better than that shown by the strain PSB07-21 cultured under the photoautotrophic or the chemotrophic growth mode. Sodium dodecyl sulfate-polyacrylamidegelelectrophoresis analysis using cell-free protein extracts showed several distinct protein bands in the gels representing the strain PSB07-21 cultured under the photoheterotrophic growth mode. The fenpropathrin enzymatic degradation was clearly affected the bacterial growth mode. Results obtained from this study should improve our knowledge regarding fenpropathrin biodegradation under field conditions. Our findings can also be used to optimize the usage of Rhodopseudomonas sp. PSB07-21 in field applications.

Influence of Processing Conditions on the Physicochemical Properties of a New-Type of Nutritional Drink-Millet Skim Milk Beverage.

In this experiment, a new type of nutritional drink—millet skim milk beverage—was developed based on combining skim milk with millet and nutritional resource utilization. The effects of NaHCO3 concentrations in soaking water (0, 0.5 g/100 mL, and 1.0 g/100 mL) and blanching time (0, 15, and 30 min) on the physicochemical properties of millet skim milk were studied. The parameter changes caused by the above treatment were evaluated via color analysis, physicochemical analysis and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Soaking in water containing NaHCO3 had a significant (p < 0.05) effect on pH, specific gravity, viscosity, and stability. The blanching treatment had a significant (p < 0.05) influence on the total solids of the samples. However, blanching only slightly affected the physical properties of the samples. In addition, soaking and blanching treatments had significant (p < 0.05) effects on the b* value of millet skim milk beverage, whereas there was no significant (p > 0.05) change in L* and a*. SDS-PAGE analysis indicated that the blanching treatment had a significant (p < 0.05) effect on band 5 and band 6 and that the soaking treatment also had a significant effect on the bands of 6 and 7 (p < 0.05). By analyzing the substantial effects, we concluded that the optimum process conditions were soaking with 0.5 g/100 mL NaHCO3 solution and blanching for 15 min.

The effect of cigarette smoking onPorphyromonas gingivalis, a crucial periodontal pathogen

Smoking is implicated as one of the most significant risk factors for periodontal disease. The objective of this study was to examine the effect of cigarette smoke extract (CSE), on cultivation of and protein production by Porphyromonas gingivalis, a major periodontal pathogen. P. gingivalis were cultivated in the presence of CSE (0%, 2.5%, 5%, and 10%) solution, and their growth was measured by optical density. In addition, expression pattern of proteins produced by P. gingivalis under CSE influence, was examined by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). According to the results, the growth of P. gingivalis was inhibited by CSE in dose-dependent mode. SDS-PAGE analyses revealed minor difference in expression pattern of proteins. Results of this study suggest that CSE imposes an inhibitory influence on growth of P. gingivalis, and is capable of modulating protein expression in P. gingivalis.

The effect of pyometra on glycosylation of proteins in the uterine tissues from female dogs

The main aim of this study was to investigate the effect of pyometra on glycosylation of proteins in the uterine tissues from female dogs, using western blotting with selected lectins (Sambucus nigra agglutinin - SNA and Maackia amurensis agglutinin – MAL II). In addition protein pattern of examined tissues was also evaluated. The study was performed on 10 female dogs undergoing ovariohysterectomy because of pyometra and 10 clinically healthy female dogs, undergoing elective spaying (ovariohysterectomy). Uterine tissue samples of 1 cm2 were taken from the middle region of each uterine horn in both group of animals immediately after ovariohysterectomy. Tissue samples were homogenized and analysed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting with SNA and MAL II. SDS-PAGE analysis showed differences between pyometra samples and controls in the amount of obtained protein fractions and the protein content in the individual fractions. Five protein (with a molecular weight of 193.78 kDa, 103.18 kDa, 77.67 kDa, 70.39 kDa, and 53.00 kDa) were found only in the pyometra samples. The remaining fractions differed in intensity of staining, which indicated differ abundance of a given protein. The results of western blotting with SNA and MAL II demonstrated that the pattern obtained from densitometric analysis differs between adequate healthy and pyometra samples with regard to the amount of protein fraction obtained as well as the intensity of staining of particular fraction. The pyometra tissues contained seven SNA-binding proteins (with a molecular weight 189.94 kDa, 165.51 kDa, 100.94 kDa, 59.42 KDa, 41.32 kDa, 35.16 kDa, and 32.6 kDa) that were not in the healthy tissues. Of the nine remaining fractions, six showed significantly higher (P < 0.05) intensity of staining in the healthy uterine tissues. In turn, the MAL II-binding protein with a molecular weight 75.85 kDa, 51.12 kDa, and 49.98 kDa were found only in the pyometra samples. Of the 28 remaining fractions, ten demonstrated significantly higher (P < 0.05), and five fractions had significantly lower (P < 0.05) intensity of staining in the pyometra tissues. The results obtained indicate that proteins in uterine tissues from female dogs with pyometra are differently glycosylated compared to normal uterine tissues. These findings provide the basis for further studies of the possible role of glycosylation in the pathogenesis of canine pyometra.

Biosynthesis of selenium nanoparticles mediated by fungus Mariannaea sp. HJ and their characterization

The biosynthesis of selenium nanoparticles (SeNPs) by Mariannaea sp. HJ was reported for the first time. Different cultural conditions, including SeO2 concentrations and pH were studied, 2 mM SeO2 and pH 10 were favorable for the removal of Se(IV), and extracellular SeNPs were observed at alkaline pH values. UV–vis spectroscopy, transmission electron microscopy (TEM), scanning electron microscopy (SEM), X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were used to characterize SeNPs. The results showed that removal of Se(IV) ions occurred during the late exponential phase of strain HJ. TEM and SEM images revealed that spherical nanoparticles were deposited on the cell wall and in the cytoplasmic region of strain HJ with a uniform size distribution. XRD pattern suggested the standard hexagonal phase of selenium crystals. FTIR, XPS and selective passivation experiments implicated that lipid and amide groups were involved in the formation process and Se(-I) or Se(-II) species were demonstrated to be functioned in this process. Based on the SDS-PAGE analysis, the up-regulated proteins of MW ∼91.6 and ∼37.4–57.2 kDa might promote SeNP formation, and a number of proteins adsorbed on the surface of nanoparticles, which may act as a natural stabilizing agent.

Identification of Hemin-binding Protein of Oral Streptococci via Electrophoresis in SDS Polyacrylamide Gel

Background and Objectives: It has been reported that hemin binding proteins are involved in the mechanism of obtaining iron in some bacteria. Oral streptococci in the dental plaque are assumed to acquire iron through hemin or hemin compounds. The aim of this study was to identify the presence of a protein (hemin binding protein) involved in the hemin binding mechanism of oral streptococci.&#x0D; Methodology: In this study, we investigated the presence of proteins involved in hemin binding of oral streptococci through sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis using hemin-agarose beads.&#x0D; Results: As a result of SDS-PAGE analysis, similar or different sizes of hemin binding protein bands were observed depending on the strains belonging to streptococci. The molecular weight of hemin binding protein in Streptococcus gordonii, Streptococcus rattus, Streptococcus sobrinus, Streptococcus sanguis and Streptococcus oralis were approximately 95 kDa, 43 kDa, 43 kDa, 39 kDa, and 39 kDa, respectively.&#x0D; Conclusion: In this study, the presence of hemin binding protein in streptococci was confirmed and the proteins involved in hemin binding in different species of oral streptococci may be different.

The difference in biofilm molecular weight in Streptococcus mutans and Aggregatibacter actinomycetemcomitans induced by sucrose and soy protein (glycine soja).

Biofilms consist of microbial cells and extracellular polymeric substance (EPS). Streptococcus mutans and Aggregatibacter actinomycetemcomtans are bacteria that can form biofilms and generate EPS. Biofilm formation can be induced by specific substances such as sucrose and protein. To identify the molecular weight that determines biofilm protein profile expression of S. mutans and A. actinomycetemcomitans induced by sucrose (carbohydrate) and soy protein (glycine soja). Experimental laboratory study. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to determine the molecular weight. Nil. The results of analysis of protein SDS-PAGE showed the presence of 28 protein bands on A. actinomycetemcomitans biofilm in the media trypticase soy broth (TSB), 20 protein bands on biofilms of S. mutans in the media TSB, 29 protein bands on biofilm A. actinomycetemcomitans in the media brain heart infusion (BHI) + sucrose 2%, and 13 protein bands on biofilms of S. mutans in the media BHI + sucrose 2%. There are differences in biofilm protein profile expression that determine the molecular weight of S. mutans biofilm and A. actinomycetemcomitans induced by sucrose (carbohydrate) and soy protein (glycine soja).

Nanoparticle-biomolecular corona: A new approach for the early detection of non-small-cell lung cancer.

Lung cancer (LC) is the most common type of cancer and the second cause of death worldwide in men and women after cardiovascular diseases. Non-small-cell lung cancer (NSCLC) is the most frequent type of LC occurring in 85% of cases. Developing new methods for early detection of NSCLC could substantially increase the chances of survival and, therefore, is an urgent task for current research. Nowadays, explosion in nanotechnology offers unprecedented opportunities for therapeutics and diagnosis applications. In this context, exploiting the bio-nano-interactions between nanoparticles (NPs) and biological fluids is an emerging field of research. Upon contact with biofluids, NPs are covered by a biomolecular coating referred to as "biomolecular corona" (BC). In this study, we exploitedBC for discriminating between NSCLC patients and healthy volunteers. Blood samples from 10 NSCLC patients and 5 subjects without malignancy were allowed to interact with negatively charged lipid NPs, leading to the formation of a BC at the NP surface. After isolation, BCs were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). We found that the BCs of NSCLC patients was significantly different from that of healthy individuals. Statistical analysis of SDS-PAGE results allowed discriminating between NSCLC cancer patients and healthy subjects with 80% specificity, 80% sensitivity and a total discriminate correctness rate of 80%. While the results of the present investigation cannot be conclusive due to the small size of the data set, we have shown that exploitation of the BC is a promising approach for the early diagnosis of NSCLC.

Cloning and Expression of Immunogenic Regions of EMA-1 Gene of Theileria equi From Infected Horses.

Diversity among the pathogenic strains of Theileria equi (T. equi), a major agent of equine piroplasmosis, can affect the appropriate detection of parasite and host immunization. Production of recombinant surface proteins from an infected horse in natural endemic area provides a reliable tool for immunodiagnosis of parasite. Regarding this, the present study was targeted toward the cloning, expression, and purification of the immunogenic regions of equine merozoite antigen 1 (EMA-1 gene), as one of the most important immunodominant surface proteins in T. equi, from naturally infected horses in Iran. The immunogenic region of EMA-1 gene was amplified using the blood of infected horses. EMA-1 gene was cloned into pET26b vector. Then, recombinant plasmids (pET 26b-EMA-1) were transformed into competent E. coli BL21 (DE3) cells. Cloning was confirmed by polymerase chain reaction (PCR), restriction enzyme assays, and DNA sequence analysis. The recombinant protein was expressed using isopropyl &amp;beta;-D-1-thiogalactopyranoside as an inducer, purified using nickle-nitrilotriacetic acid column, and then confirmed by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and dot blot analysis utilizing Anti-His Tag antibody. Furthermore, the immunoreactivity of recombinant protein against the serum of the infected horses was evaluated using dot blot analysis. The PCR product analysis showed a 750-bp band belonging to immunogenic regions of EMA-1 gene. Sequence analysis revealed that cloned EMA-1 and protein had 94% and 97% homology to EMA-1 sequences submitted to GenBank from different countries, respectively. Restriction enzyme and sequence analyses confirmed the subcloning and correction of the orientation of inserted gene. The SDS-PAGE analysis confirmed the expression of EMA-1 protein with a 28-kDa band. The results of the dot blot analysis revealed that the horse serum containing antibody against T. equi could react with the purified recombinant protein. Purified EMA-1 protein can be used as a reliable tool for the future development of diagnostic tests or vaccines.

Validation of molecular heterogeneity of Fluorescent Pseudomonas spp. and correlation with their potential biocontrol traits against fusarium wilt disease

Fluorescent Pseudomonas (FPs) are major rhizospheric bacteria with a variety of plant growth promotion attributes having potential field applications. The genomic and outer membrane protein (OMP) diversity were valued of 11 shortlisted FPs exhibiting potential biocontrol activity. The taxonomic diversity of isolates was studied using genomic fingerprinting assays—random amplification of polymorphic DNA (RAPD), enterobacterial repetitive intergenic consensus (ERIC), BOX, repetitive extragenic palindromic (REP) and (GTG5), and OMP analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Genomic diversity parameters were calculated: total allelic number, polymorphic information content, expected heterozygosity, dominance, and the Shannon and Simpson indices. All isolates were used to prime tomato seeds and plants were challenge-inoculated with Fusarium oxysporum and the disease incidence was assessed under greenhouse condition. Cluster analyses of RAPD and combined Rep analyses classified isolates into two major clusters. SDS-PAGE analysis of OMP resulted in bands in the range 29–66 kDa. Furthermore, the isolates treated with tomato seeds followed by challenge-inoculation with F. oxysporum under greenhouse conditions showed that the three FPs, M80 (SUB1688209 Seq1 KX570929), M96 (SUB1688209 Seq1 KX570930) and T109 (SUB1688209 Seq1 KX570931) were able to induce systemic resistance. The study examined the possible correlation between strain-specific molecular diversity of FPs and provided an account of their potential biological control activity against F. oxysporum.

English

To assay the efficiency of graphite nanoparticles (GtNPs) in sterilizing tissues and their role in enhancing genetic diversity, sweet potato is considered an important crop; hence its explants were used. In this experiment, GtNPs of 200, 400 and 800 ppm concentrations were used for sterilization of MS callus induction and regeneration media in Abees cultivar. The results showed that GtNPs had a good potential for removing bacterial contaminants without having side effects on the explant viability during the sterilization of sweet potato tissue in all their concentrations. Also, the percentage of callus induction increased from 98.67% in control to 100% in all GtNPs concentrations. The number of shoots per callus was enhanced at 400 ppm concentration. RAPD molecular markers and SDS-PAGE analysis were used to assess the genetic diversity of the sweet potato selected plants obtained from somaclonal variations in combination with GtNPs. Five decamer random amplified polymorphic DNA (RAPD) primers generated a total of 96 DNA fragments from the selected variants and their parent. Out of them, 82 polymorphic bands appeared with 85.42% polymorphism. The levels of DNA and protein patterns polymorphism within each treatment varied. RAPD and protein markers revealed that the concentration of 800 ppm showed the lowest similarity average among the ten selected variants and their parent. The obtained results indicated that somaclonal variation with GtNPs can be combined to increase the induced mutations frequency. Key words: Graphite nanoparticles, somaclonal variation, random amplified polymorphic DNA (RAPD), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

Protein profile of FAdV-4 based on SDS-PAGE and Western blot isolated from chickens in India.

Fowl adenovirus-4 (FAdV-4) causes hydropericardium syndrome (HPS) in poultry worldwide. An understanding of viral structural protein composition is important for developing novel immunodiagnostics and immunoprophylactics. Here we report isolation, culture, molecular and protein profile of FAdV-4 isolates recovered from HPS outbreaks in chicken in the states of Himachal Pradesh and Tamil Nadu in India. We performed a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting-based protein profiling of FAdV-4 isolates against a reference FAdV-1 or Chicken Embryo Lethal Orphan (CELO) virus. SDS-PAGE analysis showed that seven protein bands in FAdV-4 isolates were similar to CELO expect an additional band of 110 kDa in CELO virus. On Western blotting, two protein fractions of 43 kDa and 78 kDa size were observed in FAdV-4 isolates. Overall, results show that FAdV-4 isolates recovered from different regions of the country had similar protein profile and possibly a common source of origin.