Protein Band Of Molecular Weight Research Articles

Development of Drying Methodology for Intact Whole Buffalo Liver, Its Characterization, Shelf Life, and Evaluation of Palatability as Pet Treat

Globally, large quantity of animal byproducts is generated from the slaughter of food animals, but there is lack of research articles related to drying of these byproducts and its use as pet food. Therefore, this study was conducted with the aim of utilization of intact whole buffalo liver by drying for pet treat, evaluating its shelf life and palatability. The intact liver surface was superficially sliced, and the surface was pierced. Thereafter, the livers were pretreated in 3% sugar and 4% salt solution (1 : 3 w/v) for 3 h followed by microwaving for 4 min and hot air drying at 60°C for 40 h (designated as T2L). The livers which were dried the same as T2L except surface piercing were referred as T1L, whereas the livers dried only using hot air oven were referred as control (CL). The moisture and protein contents of the dried CL and T2L were found to be 28.46% and 14.29% and 43.85% and 52.76%, respectively. Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) image of T2L revealed the presence of few low as well as high molecular weight protein bands which were absent in CL and T1L indicating a comparatively lower level of protein degradation in T2L. The shelf life of T1L and T2L samples based on microbiological and lipid oxidation analyses was found to be more than 60 days at 25 and 4°C. Palatability studies using dogs showed that all dried samples were highly palatable. Thus, it could be concluded that intact buffalo liver could be dried using surface slicing, with/without piercing followed by salt and sugar pretreatment, 4 min microwaving, and hot air drying at 60°C for 40 h. Future study should focus on the sensory properties such as aroma, texture, and flavor and sensory analysis of the dried liver by human.

Utilizing outer membrane proteins (OMPs) as a vaccine to manage motile Aeromonas septicemia (MAS) in Nile tilapia (Oreochromis niloticus) with evaluating the vaccine immunoprotective effectiveness

AbstractMotile aeromonads are an opportunistic and heterogenic bacteria that cause motile Aeromonas septicemia (MAS) which is accompanied with variety of clinical signs and lesions in fish. It has been found that their outer membrane proteins (OMPs) are highly immunogenic and may be used as a vaccine candidate. The present study was undertaken to evaluate OMP protection against Aeromonas hydrophila (A. hydrophila) strain ATCC 7966 (A5 and A8) and A. hydrophila strain DSM 30,187 (A6). Pathogenicity tests revealed that out of 12 isolated strains, seven were highly pathogenic, three were moderate, and two were avirulent. SDS-PAGE analysis of OMPs showed several protein bands (7–22) of different molecular weights ranging from 14 to 346 kDa. The common protein band was 39 kDa. It seems that pathogenicity and the number of protein bands are related in the highly pathogenic strains. Challenged A. hydrophila for Nile tilapia (O. niloticus) previously actively immunized with OMPs (A4, A5, A6, A8, and A11) showed a significant increase in relative percent survival (RPS) ranging from 50 to 100%. Immunological parameters such as immunoglobulin M (IgM), antioxidants (SOD, GSH, and CAT), and nitric oxide (NO) and expression of pro-inflammatory cytokines, interleukin 1β (IL-1β), and tumor necrosis factor-α (TNFα) showed a significant increase in OMP-immunized fish. Challenged A. hydrophila for O. niloticus previously passively immunized with OMPs (A4, A5, A6, A8, and A11) in fish-raised sera showed a significant increase in RPS (20 to 100%) in the vaccinated groups. It is concluded that OMPs from Aeromonas spp. have been identified as suitable target for protection development, especially in the case of heterogenic bacteria.

Uncovering the antimicrobial activity of G-type lysozyme 2 derived from Cyprinus carpio mucus against bacterial and fungal pathogens

This study aimed to explore the antimicrobic potential of mucus samples collected from Cyprinus carpio and identify the specific antimicrobial peptides responsible for its activity. The crude extract was tested against various bacterial and fungal pathogens, and its protein content and profile were analyzed. Purification steps, including gel filtration chromatography, were employed to isolate the most active fraction (peak IV), which was further identified via liquid chromatography and mass spectroscopy. The results revealed varying degrees of antimicrobial activity of the crude extract against different bacterial and fungal strains, with Leclercia adecarboxylata, Candida glabrata, and Candida parapsilosis showing the highest susceptibility. SDS-PAGE analysis demonstrated the existence of multiple low molecular weight protein bands in the crude extract, while fraction IV obtained from gel filtration chromatography exhibited the strongest antimicrobial activity. Peak IV displayed a range of minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and minimum fungicidal concentration (MFC) values against the tested pathogens, spanning from 0.038 to 4.960 mg/mL. Further investigation identified the purified peptide derived from peak IV as G-type lysozyme 2, characterized by a molecular weight of 21 kDa. These findings shed light on the existence of a highly effective antimicrobial peptide, G-type lysozyme 2, within the mucus of Cyprinus carpio. This peptide demonstrates notable activity against diverse bacterial and fungal pathogens. The insights from this study enhance our understanding of the fish's antimicrobial defense mechanisms and hold promise for developing novel antimicrobial agents.

Serological and molecular detection of neurocysticercosis among epileptic patients in Nagpur, Maharashtra state (India).

Neurocysticercosis (NCC), one of the most important neuroparasitic diseases in humans, is caused by Cysticercus cellulosae, the metacestode stage of digenetic zoonotic cestode Taenia solium. The present study aims at the detection of anti-cysticercus antibodies in the sera of epileptic patients (n=26) visiting a tertiary care hospital in Nagpur, Maharashtra state, India, by an in-house developed indirect IgG-ELISA and enzyme-linked immunoelectro transfer blot (EITB) assay using different antigens (namely, Whole Cyst Antigen (WCA), Cystic Fluid Antigen (CFA), Scolex Antigen (SA), Excretory-Secretory Antigen (ESA) and Membrane-Body Antigen (MBA)) prepared from T. solium metacestodes to find out the status of NCC. An attempt has also been made for molecular detection of NCC from blood samples of those patients by Polymerase Chain Reaction (PCR) assay targeted at large subunit rRNA gene of T. solium. The IgG ELISA level of anti-cysticercus antibodies against WCA, CFA, SA, ESA and MBA antigens were as follows: 19.23 %, 23.07 %, 38.46 %, 30.76 % and 15.38 %. The seroreactivity to CFA, SA and ESA was found in equal proportions in patients with ring-enhancing lesions. In the EITB assay, the lower and medium molecular weight protein bands of SA and ESA were immunodominant compared to the higher WCA and CFA peptides. PCR positivity could be observed in 34.6 % (9/26) of the patients under study. It is the first report of detecting NCC among epileptic patients of the Nagpur region of Maharashtra state in India using serological and molecular tools.

Comparison of different protocols for protein extraction from murine ovarian tissue: A preliminary study

Objective: Extraction of proteins from tissues is a key step to study the protein structure and function. There is no set of universal protocols because of the enormous chemical and physical variety of both proteins and the sample sources. Hence, in this study a comparison of different methods for protein extraction from mouse ovarian tissue was carried out. Methods: Intravaginal dose of 20µl of Phosphate buffer saline was given to 7 mice for ten consecutive days followed by extraction of ovaries. For comparison, seven different protein extraction protocols from Protocol 1 to Protocol 7 were used. The supernatant was first quantified with Bradford assay to determine the total protein yield followed by SDS-PAGE analysis. Results: Highest protein yield of 1.13 mg/ml was attained after supplementing 1% v/v protease inhibitor cocktail in PBS. In total, 13 protein bands were detected by the extraction protocol 2 and 11 protein bands by the extraction protocol 1. 11 protein bands were detected in the range 180 kDa to 22 kDa following Protocol 3 and 4. Extraction with lysis buffer 1 resulted in 7 visible protein bands of molecular weight 182 kDa to 35 kDa range. Only 6 protein bands were observed in the range 182 kDa - 35 kDa following lysis buffer 2 method. For lysis buffer 3, ten visible protein bands were detected in range of 195 kDa - 13 kDa. Conclusion: Based on the protein extraction efficiency of Protocol 1, optimal combination of Phosphate buffer saline (pH 7.2) and protease inhibitor cocktail followed by hand-homogenization is suggested for protein extraction from mouse tissues.

Beneficial effect of honeybee-specific lactic acid bacteria on health and activity of Apis mellifera L. colonies

BackgroundObjective of this work was to investigate the impact of isolated honeybee-specific-lactic acid bacteria (Hbs-LAB) that isolated from worker's bee intestinal tracts on bee health as well as bee colony activity parameters. Independent assays were conducted from February to July, 2021 in apiary yard of Faculty of Agriculture, Cairo University, Giza, Egypt. Colonies were allocated to 2 experimental groups each of 10 colonies, Control colonies were fed with sugar syrup, while the treated colonies were fed with sugar syrup supplemented with mixture of Hbs-LAB (Lactobacillus brevis-HBE2, Lactobacillus casei-HBE5, and Enterococcus Faecalis-HBE1, 3 gm bacterial belts (1:1:1) mixed with 1.5 L of sugar syrup.ResultsThe treated colonies recorded high significant differences in all activity parameters (worker's and drone's brood areas, amount of honey and pollen stored, and no. of combs covered with bees) than controlled colonies. In addition, total protein in workers hemolymph samples of treated colonies showed more number of protein bands and the hemolymph molecular low weight protein bands was 99.24 ± 0.3, whereas the high weight protein bands reached 183.87 ± 0.3 KDa, while for untreated colonies the hemolymph molecular low weight protein bands (KDa) were 70.99 ± 0.3 and the high weight protein bands reached 171.57 ± 0.2 KDa.ConclusionsThe Hbs-LAB proved to be a natural protocol that will positively impact the beekeepers’ economy by providing a higher yield of bee products as well as improve the protein values in worker’s hemolymph which is a superior tool for colonies resistance against several diseases that attack their hives.

Evaluation of antibacterial activity induced by Staphylococcus aureus and Ent A in the hemolymph of Spodoptera littoralis

The problem of antibiotic resistance considers one of the most dangerous challenges facing the medical field. So, it is necessary to find substitutions to conventional antibiotics. Antimicrobial peptides (AMPs) are a bio-functional derivative that have been observed as one of the important solutions to such upcoming crisis. Owing to their role as the first line of defense against bacteria, fungi, and viruses. This study was conducted to induce the immune response of Spodoptera littoralis larvae by inoculation of sub lethal doses of Staphylococcus aureus and its enterotoxin. Since Staphylococcal enterotoxin A (SEA) considers the major causative agents of Staphylococcal food poisoning, our study oriented to purify and characterize this toxin to provoke its role in yielding AMPs with broad spectrum antimicrobial activity. A great fluctuation was recorded in the biochemical properties of immunized hemolymph not only in the total protein content but also protein banding pattern. Protein bands of ∼22 kDa (attacin-like) and ∼15 kDa (lysozyme-like) were found to be common between the AMPs induced as a result of both treatments. While protein bands of molecular weight ∼70 kDa (phenoloxidase-like) and ∼14 kDa (gloverin-like) were found specific for SEA treatment. Chromatographic analysis using HPLC for the induced AMPs showed different types of amino acids appeared with differences in their quantities and velocities. These peptides exhibited noticeable antimicrobial activity against certain Gram-positive and Gram-negative bacteria. In conclusion, the antimicrobial potential of the antimicrobial peptides (AMP) induced in the larval hemolymph of S. littoralis will be a promising molecule for the development of new therapeutic alternatives.

Zoonotic Cryptosporidium Parasites Possess a Unique Carbohydrate-binding Protein (Malectin) that is Absent in other Apicomplexan Lineages

Objective. Malectin is a carbohydrate-binding protein that binds Glc(2)-N-glycan and is present in animals and some alveolates. This study aimed to characterize the general molecular and biochemical features of Cryptosporidium parvum malectin (CpMal). Methods. Polyclonal antibodies were raised for detecting native CpMal by western blotting and immunofluorescence assays. Recombinant CpMal and human malectin (HsMal) were produced, and their binding activities to amylose and the host cell surface were compared. Far-western blotting and far-immunofluorescence assays were used to detect potential binding partners of CpMal in the parasite. Results. Native CpMal appeared to exist in dimeric form in the parasite and was distributed in a diffuse pattern over sporozoites but was highly concentrated on the anterior and posterior sides near the nuclei. CpMal, compared with HsMal, had significantly lower affinity for binding amylose but substantially higher activity for binding host cells. Recombinant CpMal recognized three high molecular weight protein bands and labeled the sporozoite posterior end corresponding to the crystalloid body, thus suggesting the presence of its potential ligands in the parasite. Two proteins identified by proteomics should be prioritized for future validation of CpMal-binding. Conclusion. CpMal notably differs from HsMal in molecular and biochemical properties; thus, further investigation of its biochemical and biological roles is warranted.

Impact of germination on structural, functional properties and in vitro protein digestibility of sesame (Sesamum indicum L.) protein

To enhance the functional properties and utilization of sesame protein, the impact of different germination treatments (0 d, 2 d, and 4 d) on the structural and functional properties of sesame protein was investigated in this study. The results showed that germination significantly increased the content of free sulfhydryl groups and the surface hydrophobicity of sesame protein, indicating that the protein molecular structure was unfolded. The high molecular weight protein bands gradually disappeared, and the secondary structure of the sesame protein was changed after germination. Moreover, germination led to changes in the foaming and emulsifying properties of sesame protein. The in vitro protein digestibility and solubility of sesame protein were also increased after germination. These results suggested that germination treatment can improve the structural and functional properties of sesame protein and can be used as an effective approach to enhance the nutritional properties and processing value of plant seed protein.

The Inhibitory Effects of Ficin on Streptococcus mutans Biofilm Formation.

To investigate the effects of ficin on biofilm formation of conditionally cariogenic Streptococcus mutans (S. mutans). Biomass and metabolic activity of biofilm were assessed using crystal violet assay, colony-forming unit (CFU) counting, and MTT assay. Extracellular polysaccharide (EPS) synthesis was displayed by SEM imaging, bacteria/EPS staining, and anthrone method while acid production was revealed by lactic acid assay. Growth curve and live/dead bacterial staining were conducted to monitor bacterial growth state in both planktonic and biofilm form. Total protein and extracellular proteins of S. mutans biofilm were analyzed by protein/bacterial staining and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), severally. qRT-PCR was conducted to detect acid production, acid tolerance, and biofilm formation associated genes. Crystal violet assay, CFU counting, and MTT assay showed that the suppression effect of ficin on S. mutans biofilm formation was concentration dependent. 4 mg/mL ficin had significant inhibitory effect on S. mutans biofilm formation including biomass, metabolic activity, EPS synthesis, and lactic acid production (p < 0.05). The growth curves from 0 mg/mL to 4 mg/mL ficin were aligned with each other. There was no significant difference among different ficin groups in terms of live/dead bacterial staining result (p > 0.05). Protein/bacterial staining outcome indicated that ficin inhibit both total protein and biofilm formation during the biofilm development. There were more relatively small molecular weight protein bands in extracellular proteins of 4 mg/mL ficin group when compared with the control. Generally, ficin could inhibit biofilm formation and reduce cariogenic virulence of S. mutans effectively in vitro; thus, it could be a potential anticaries agent.

A novel Bacillus thuringiensis isolate toxic to cotton pink bollworm (Pectinophora gossypiella Saunders)

In this study, we report a novel indigenous Bacillus thuringiensis (Bt) isolate, T26, which showed spores and crystals under scanning electron microscope and pathogenicity against the pink bollworm (Pectinophora gossypiella Saunders) in artificial diet based bioassay. SDS-PAGE analysis of the spore-crystal mixture of the Bt isolate, T26 revealed presence of three major protein bands of approximate molecular weights of 80, 55 and 40 kDa. The draft genome assembly consists of 56 scaffolds with an entire draft genome size of 5,054,095 bp. NCBI blast analysis revealed that assembled draft genome is spread over in a chromosome (4,818,543 bp) and one plasmid (235,552 bp). NCBI Prokaryotic Genome Annotation Pipeline (PGAP) showed presence of 5033 coding gene sequences and 159 RNAs genes. None of the known lepidopteran active genes (cry1, cry2 and cry9) could be detected with PCR or with whole genome sequence analysis using Bt toxin scanner tool or CryProcessor tool. Thus, presence of protein crystals and toxicity towards cotton pink bollworm and absence of any known cry/vip/cyt type of genes in draft genome indicates it is a novel type of Bt isolate. Further investigation of this genome sequence along with protein sequencing will lead to understand the novel factors responsible for its virulence and could be a useful tool for the insect resistance management in pink bollworm.

Chromium (VI) tolerance and bioaccumulation by Candida tropicalis isolated from textile wastewater

In the present study a yeast strain isolated from industrial wastewater, identified as Candida tropicalis, showed chromium (Cr) tolerance level up to 5 mM. Yeast grown in minimal salt medium containing Cr (VI) ions for 48 h and crude enzyme extracts were tested for chromate reductase activity. Optimum temperature and pH of chromate reductase were 30 °C and pH of 7. The enzyme activity was greatly enhanced in the presence of divalent metal cations. Total protein profile revealed some protein bands were present in hexavalent chromium [Cr (VI)] treated samples but were absent in non-treated samples, especially low molecular-weight protein bands in the mass range of < 25 kDa with greater intensity in Cr (VI) treated samples. Yeast cells were able to uptake Cr (VI) between 21 and 80 mg g− 1 within 2–12-d of time, indicating yeast strain promising potential for Cr (VI) removal from the wastewater. The present study results suggest that C. tropicalis is a suitable candidate for bioremediating chromium ions from the contaminated-environment.

Developing Liquid Rhizobium Inoculants with Enhanced Long-Term Survival, Storage Stability, and Plant Growth Promotion Using Ectoine Additive.

Liquid microbial inoculants have recently received great attention due to their vital roles for sustainable agricultural practices. However, long-term conservation under ambient temperature conditions and deleterious environmental factors might negatively impact microbial cell survival and limit their efficacy in the field. Thus, developing efficient liquid formulation providing prolonged survival of rhizobia in the final product and after an application is crucial. Therefore, this study investigates the effect of various additives on the long-term survival of rhizobia stored in liquid cultures at room temperature (25°C) for 12months. Various yeast sucrose media amended with polyvinylpyrrolidone (PVP) or gum arabic as colloidal agents in combination with ectoine (as a compatible solute) and/or glycerol were evaluated. A dramatic decline in viable cell count was obtained in formulas amended only with PVP from Log 8.5 to Log 5 in the first six months and then to Log 1.5 after 12months. In contrast, rhizobia stored at PVP-based formulas amended with 10 mg L‒1ectoine exhibited almost constant survival level till the end of the storage period. The same trend was obtained using formulas based on gum arabic as a colloidal dispersing agent; however, less decline in cell count using a formula containing gum arabic alone as compared to using PVP. On the other hand, PVP based formulas exhibited higher viscosity compared with another formula. Increased viscosity till the 8th month of storage was achieved in the presence of ectoine indicating the increase of exopolymeric substances production. Electrophoretic protein pattern of rhizobial cells (stored for 12months) exhibited several low molecular weight protein bands in cells stored in PVP based formula with ectoine as compared to the other treatments. Thus, the amendment of the liquid formulation of rhizobia bioinoculant with PVP plus ectoine not only improved cell survival but also enhanced the culture viscosity and consequently ameliorate the colonization and performance of rhizobial inoculants.

Potential Antioxidant and Angiotensin I-converting Enzyme Inhibitory Activity in Crust of Dry-aged Beef

Antioxidant activity, angiotensin I-converting enzyme (ACE) inhibitory activity, and protein profile of crust (the dried surface of dry-aged beef) were evaluated compared to unaged, wet-, and dry-aged beef. Antioxidant activity was determined using 2,2-diphenyl-1-picrylhydrazyl and 2,2′-azino-di-(3-ethylbenzthiazoline sulfonate) radical scavenging activities, ferric reducing antioxidant power, and ferrous ion chelating activity. The crust samples showed the greatest (P < 0.05) ACE inhibitory and antioxidant activity resulting from the three different mechanisms of action (radical scavenging, non-radical redox potential activity, and metal chelating) among the treatments. Small molecular weight protein bands and small peptides (<3 kDa) indicating potent bioactivity were evident in the myofibrillar protein profile of crust samples. The lowest (P < 0.05) ACE inhibitory activity was observed in unaged beef. The results indicate that crust could be utilized in various areas as a functional ingredient possessed antioxidant and ACE inhibitory activity instead of being discarded. In addition, dry aging can use for generation of functional ingredient from beef as the regime.

Oxidative and biochemical responses as indicators of chemical stress in Clarias gariepinus (Burchell, 1822) juvenile exposed to glyphosate

A sub-lethal toxicity experiment was undertaken to study the response of some enzyme activities, tissue proteins and muscular stress proteins in Clarias gariepinus juveniles exposed to glyphosate herbicide. The juveniles were exposed to 0.19 mgL−1, 0.28 mgL−1and 0.57 mgL−1 of the test herbicide, which represented 1/9th, 1/6th and 1/3rd of the LC50, respectively in 24 h static renewal assay. The liver and muscle tissues of the exposed juveniles were sampled on day 1 and 14 during assay. The results showed that catalase, TBARS, ALP and ALT increased significantly (p < 0.05) with concentrations and time. Protein bands of different molecular weight ranging from 15 kDa to 250 kDa were expressed at different treatments. The results showed that glyphosate herbicides induce oxidative stress in fishes even at sub-lethal doses.

Evaluation of Genetic Diversity of Seven Species of Solanum in Nigeria Using SDS-PAGE

Seed protein profile of 12 accessions belonging to 7 species of the genus Solanum of the family solanaceae were investigated through poly acrylamide gel electrophoresis. The accessions were selected based on their overlapping morphological associations observed on the field. Occurrence of two protein bands of molecular weights 100 kda and 20 kda in all accession showed interspecific relationships and could be used as generic bands among the genus Solanum. All morphologically similar accessions manifested similarities in their protein bands except S. melongena. Dendrogram based on upgma also revealed the generic status and intraspecific relationships of the accession of S. aethiopicum and S. macrocarpon.

Biotic potential of mucus extracts of giant mudskipper Periophthalmodon schlosseri (Pallas, 1770) from Pichavaram, southeast coast of India

BackgroundThe epidermal mucus of fish contains various bioactive compounds, which play a crucial role in the animal defense mechanisms. However, the mucus from giant mudskipper is not well characterized for its biochemical and pharmacological effects. This study for the first time analyzed the mucus extracts of this species for antimicrobial (against human pathogen) and hemolytic activities. Fourier-transform infrared spectrum analysis (FT-IR) and SDS-PAGE of mucus extracts were also carried out.ResultsThe protein content in Periophthalmodon schlosseri (Gobiidae: Oxudercine) mucus extracts was 0.66 ± 0.04 mg/ml. The mucus extracts of P. schlosseri showed antibacterial activity against all eight human pathogenic bacteria and four fungal strains tested in this study. The hemolytic activities against chicken and human blood cells were observed with maximum activity for dichloromethane extracts. The FT-IR spectrum of mucus extract of P. schlosseri showed the presence of functional groups, evident protein bands of various molecular weights ranging from 14 kDa to 97 kDa.ConclusionThe obtained results suggested that the mucus of this species has proteinaceous compounds responsible for antimicrobial and hemolytic activities.

Urea can inhibit efficient reduction and alkylation of protein dimers in solution demonstrated by the beta subunit of alpha glucosidase II

Protein reduction and alkylation is routinely used for analysis of protein dimers and protein complexes in cell fractions using two dimensional gel electrophoresis and mass spectrometry. To resolve the heterogeneity of a high molecular weight protein band that is highlighted by an antibody to the beta subunit of alpha glucosidase II (GIIβ), we performed reduction and alkylation of cytosolic proteins extracted from mouse brain. The presence of urea in the reduction/alkylation buffer inhibited the chemical processes. It is thus recommended that protein reduction/alkylation be performed both in the presence and absence of urea for the separation of mono-/hetero-mers.

MORPHOLOGICAL AND BIOCHEMICAL DIVERSITY AND RESPONSE OF EGYPTIAN FABA BEAN TO HEAT AND DROUGHT STRESSES

Four Egyptian faba bean (Vicia faba L.) varieties (Giza843, Giza716, “Misr1” and “Sakha1”) have been evaluated under heat and drought stresses using seventeen morphological traits and total protein content. Analysis of variance of the morphological trait revealed highly significant differences among treatments and genotypes for all studied traits and the interaction between genotypes and treatment was also significant. Both varieties Giza716 and Giza843 gave the highest means for most of the morphological traits. The control treatment surpass all the other treatments in the most of the morphological traits. The lowest significant means were obtained from the heat with drought treatment while the effect of heat on the morphological traits was less than the effect of drought stress. According to the cluster analysis of both morphological and biochemical data, the varieties Giza716 and Giza843 were clustered together on one group where the varieties “Misr1” and “Sakha1” were clustered in another group. A protein band of molecular weight of about 76 kDa was noticed in the protein pattern of the variety Giza716 (heat with drought treatment) and of about 100 kDa was obtained in the protein pattern of the variety Giza843 (heat with drought treatment). The morphological and biochemical parameters along with susceptibility test revealed that Giza716 and Giza843 appeared to be tolerant for drought and heat. However “Misr1” and “Sakha1” varieties appeared to be susceptible.

Identification of immunogenic proteins and evaluation of recombinant PDHA1 and GAPDH as potential vaccine candidates against Streptococcus iniae infection in flounder (Paralichthys olivaceus).

Streptococcus iniae is a major Gram-positive pathogen that causes invasive disease in fish worldwide. In this study, in order to identify immunogenic proteins for developing highly effective vaccine against S. iniae, whole-cell lysate proteins of S. iniae were analyzed by western blotting using flounder anti-S. iniae antibodies, and two positive protein bands of molecular weight 37 kDa and 40 kDa were screened, which were identified as pyruvate dehydrogenase E1 subunit alpha (PDHA1), BMP family ABC transporter substrate-binding protein (BMP) and L-lactate dehydrogenase (LDH), as well as ornithine carbamoyltransferase (OCT), lactate oxidas (LOx) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by mass spectrometry. Subsequently, the six recombinant proteins were produced and used to immunize healthy flounder, and the relative percent survival (RPS) value was 72.73%, 27.27%, 36.36%, 9.09%, 36.36% and 63.64% respectively after intraperitoneal challenge with live S. iniae, revealing that rPDHA1 and rGAPDH produced higher relative percent survival than formalin-killed S. iniae (36.36%). To further investigate the protective efficacy of rPDHA1 and rGAPDH, the proliferation of surface membrane immunoglobulin-positive (sIg+) lymphocytes in peripheral blood leucocytes, the total serum IgM, specific IgM against S. iniae and RPS were detected. The results showed that rPDHA1, rGAPDH and formalin-killed S. iniae significantly induced the proliferation of sIg+ lymphocytes, the production of total serum IgM and specific IgM as compared with the control group, and rGAPDH and rPDHA1 provide higher RPS (62.5% and 75%, respectively) again. These results demonstrated that rPDHA1 and rGAPDH are promising vaccine candidates against S. iniae infection in flounder.