scholarly journals Serological cross-reactivity of environmental Escherichia coli strains with Shigella-specific antisera

2018 ◽  
Vol 33 (1-2) ◽  
pp. 29-33 ◽  
Author(s):  
Nafisa Azmuda ◽  
Rabeya Bilkis ◽  
Humaira Akter ◽  
Anowara Begum ◽  
Sirajul Islam Khan ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Surface Antigens ◽  
Biochemical Properties ◽  
Cross Reactivity ◽  
Freshwater Ecosystems ◽  
Shigella Species ◽  
Immunogenic Proteins

Many bacteria of clinical and environmental origin show evidence of sharing common surface antigens. The present study aimed for isolation of Escherichia coli strains that were serologically cross-reactive with Shigella species from freshwater ecosystems in Bangladesh by conventional cultural methods. Among twenty eight isolates, two isolates, termed 12(35) and 6(50) showed cross-reactivity with four polyvalent serogroup-specific Shigella antisera using slide agglutination assay. The isolates were identified and charcterized by cultural and biochemical properties and Western blot analysis. The isolates showed typical Escherichia coli cell morphology and cultural and biochemical properties and were identified as Escherichia coli by API 20E tests. Western blot analysis confirmed the isolates as cross-reactive with all the four group-specific Shigella antisera due to presence of immunogenic proteins and LPS. One of the isolates also showed cross-reactivity with multiple type-specific Shigella boydii antisera (monovalent) because of immunogenic proteins. Both the isolates were identified as nonpathogenic due to absence of virulence marker genes of diarrheagenic E. coli variants.This study revealed that a number of bacteria present in the environment could share important Shigella species surface antigens. Naturally occurring nonpathogenic environmental bacteria expressing surface antigens specific for certain types of Shigella could be a good choice for vaccine candidates against shigellosis. Bangladesh J Microbiol, Volume 33, Number 1-2, June-Dec 2016, pp 29-33

scholarly journals Characterization of Human Immunoglobulin (Ig) Isotype and IgG Subclass Response to Bartonella henselae Infection

1998 ◽  
Vol 66 (12) ◽  
pp. 5915-5920 ◽  
Author(s):  
Svena L. McGill ◽  
Russell L. Regnery ◽  
Kevin L. Karem
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Bartonella Henselae ◽  
Negative Control ◽  
Cross Reactivity ◽  
Antibody Activity ◽  
Igg Subclass ◽  
Banding Patterns ◽  
Bartonella Quintana

ABSTRACT Serologic parameters of cat scratch disease (CSD) were evaluated by Western blot analysis. Sera from patients with serologically confirmed CSD antigen were screened for immunoglobulin (Ig) isotype-specific as well as IgG subclass-specific reactivity against Bartonella henselae whole-cell antigen. Bartonella-negative control sera were used to determine baseline antibody activity. Heterogeneous B. henselae-specific IgG reactivity with numerous protein bands, ranging from >150 to <17 kDa, was observed. Though individual banding patterns were variable, one approximately 83-kDa B. henselae protein (Bh83) was immunoreactive with all CSD sera tested, suggesting it is a conserved antigen during infection. Bh83 was not recognized by reference human antisera againstRickettsia rickettsii, Chlamydia group positive, Treponema pallidum, Orientia tsutsugamushi, Fransciscella tularensis,Ehrlichia chaffeensis, Mycoplasma pneumoniae, and Escherichia coli, although other cross-reactive proteins were evident. Significantly, CSD sera failed to recognize the 83-kDa protein when tested against Bartonella quintanaantigen, though sera from B. quintana-infected patients did react to Bh83. This cross-reactivity suggests epitope conservation during infection with B. henselae or B. quintana. Western blot analysis further revealed similar banding patterns when B. henselae was reacted against the Ig isotypes IgG and IgG1 and both secretory and alpha chains of IgA. Neither IgM nor IgE reacted significantly toBartonella antigen by our Western blot analysis. Dissection of the antibody response at the IgG subclass level indicated that prominent antigen recognition was limited to IgG1. These observations provide insight into induced immunity during CSD and provide evidence for conserved epitope expression during infection withB. henselae or B. quintana.

scholarly journals Identification of Immmunogenic Salivary Proteins of Anopheles vagus based on Mass Spectrometry Analysis

2017 ◽  
Vol 18 (2) ◽  
pp. 73
Author(s):  
Dwi Esti Febriyantiningsih ◽  
Kartika Senjarini ◽  
Rike Oktarianti
Keyword(s):  
Mass Spectrometry ◽  
Salivary Gland ◽  
Western Blot ◽  
Salivary Glands ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Mass Spectrometry Analysis ◽  
Salivary Proteins ◽  
Immunogenic Proteins ◽  
Anopheles Vagus

Malaria has been prevalent for a long time in tropical developing regions causing great morbidity and mortality. Among the malaria vectors, Anopheles vagus has been known as secondary malaria vector in East Java. Salivary glands of mosquitoes perform various functions for survival of the vectors and also conducive for blood feeding, harbouring of malaria parasites, and eventual parasite transmission. The salivary gland proteomes of An. vagus have not been carried out yet. The aim of our study was to identify and characterize the immunogenic proteins of salivary glands proteins of An. vagus. A proteomic approach combining one-dimensional electrophoresis (1DE) followed by western blot analysis using human sera from healthy people living in an endemic area (Kendal); liquid chromatography mass spectrometry (LC-MS/MS) and bioinformatic analysis was adopted to provide the first direct insight into identification and characterization of salivary proteins of An. vagus. Identification of immunogenic proteins using western blot analysis has revealed three immunogenic bands which had molecular weights of 69, 75 and 232 kDa. Among those proteins analysed by LC-MS/MS, there were alpha,1-4 glucan phosphorylase, putative myosin class I heavy chain which have the highest number of total spectrum count peptide. Other proteins like vitellogenin and heat shock protein 82 (Hsp82) were also identified. The majority of proteins were scrutinized marked for their role in metabolism, cytoskeleton protein and stress response. Keywords: Anopheles vagus, salivary gland, immunogenic, proteomics

scholarly journals Development of a Kit for Rapid Immunochromatographic Detection of Sacbrood Virus Infecting Apis cerana (AcSBV) Based on Polyclonal and Monoclonal Antibodies Raised against Recombinant VP1 and VP2 Expressed in Escherichia coli

Viruses ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 2439
Author(s):  
Song Hee Lee ◽  
Tae-Kyun Oh ◽  
Sung Oh ◽  
Seongdae Kim ◽  
Han Byul Noh ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Monoclonal Antibodies ◽  
Western Blot ◽  
Blot Analysis ◽  
Rapid Detection ◽  
Western Blot Analysis ◽  
Apis Cerana ◽  
Korean Isolate ◽  
Sacbrood Virus ◽  
Positive Reactivity

A Korean isolate of the sacbrood virus infecting Apis cerana (AcSBV-Kor) is the most destructive honeybee virus, causing serious economic damage losses in Korean apiculture. To address this, here, we attempted to develop an assay for the rapid detection of AcSBV-Kor based on immunochromatographic detection of constituent viral proteins. Genes encoding VP1 and VP2 proteins of AcSBV-Kor were cloned into an expression vector (pET-28a) and expressed in Escherichia coli BL21(DE3). During purification, recombinant VP1 (rVP1) and VP2 (rVP2) proteins were found in the insoluble fraction, with a molecular size of 26.7 and 24.9 kDa, respectively. BALB/c mice immunized with the purified rVP1 and rVP2 produced polyclonal antibodies (pAbs) such as pAb-rVP1 and pAb-rVP2. Western blot analysis showed that pAb-rVP1 strongly reacted with the homologous rVP1 but weakly reacted with heterologous rVP2. However, pAb-rVP2 strongly reacted not only with the homologous rVP2 but also with the heterologous rVP1. Spleen cells of the immunized mice fused with SP2/0-Ag14 myeloma cells produced monoclonal antibodies (mAbs) such as mAb-rVP1-1 and mAb-rVP2-13. Western blot analysis indicated that pAb-rVP1, pAb-rVP2, mAb-rVP1-1, and mAb-rVP2-13 reacted with AcSBV-infected honeybees and larvae as well as the corresponding recombinant proteins. These antibodies were then used in the development of a rapid immunochromatography (IC) strip assay kit with colloidal gold coupled to pAb-rVP1 and pAb-rVP2 at the conjugate pad and mAb-rVP1-1 and mAb-rVP2-13 at the test line. One antibody pair, pAb-rVP1/mAb-VP1-1, showed positive reactivity as low as 1.38 × 103 copies, while the other pair, pAb-rVP2/mAb-VP2-13, showed positive reactivity as low as 1.38 × 104 copies. Therefore, the antibody pair pAb-rVP1/mAb-VP1-1 was selected as a final candidate for validation. To validate the detection of AcSBV, the IC strip tests were conducted with 50 positive and 50 negative samples and compared with real-time PCR tests. The results confirm that the developed IC assay is a sufficiently sensitive and specific detection method for user-friendly and rapid detection of AcSBV.

scholarly journals Detection of immunogenic protein from salivary gland of Aedes albopictus

2021 ◽  
Vol 40 (3) ◽  
pp. 234-242
Author(s):  
Rike Oktarianti ◽  
Rochmatul Nuryu Khasanah ◽  
Syubbanul Wathon ◽  
Kartika Senjarini
Keyword(s):  
Salivary Gland ◽  
Western Blot ◽  
Salivary Glands ◽  
Aedes Albopictus ◽  
Blot Analysis ◽  
Gel Electrophoresis ◽  
Western Blot Analysis ◽  
Sodium Dodecyl ◽  
Immunogenic Proteins ◽  
Healthy Persons

BackgroundDengue virus is transmitted by several species of Aedes mosquitoes, with Aedes albopictus as secondary vector. During blood feeding, these vectors inject saliva into the vertebrate hosts. The saliva contains anticoagulant, anti-inflammatory and immunogenic factors. The objective of this research was to detect immunogenic proteins from Ae.albopictus salivary glands reacting with sera of people living in dengue endemic areas. MethodsThe identification of immunogenic proteins of Ae. albopictus salivary gland used one-dimensional gel electrophoresis (sodium dodecyl sulfate polyacrylamide gel electrophoresis), and western blot analysis, respectively. To determine the immunogenic nature of the candidate proteins, the antigens from the salivary gland of Ae. albopictus were reacted with sera from healthy persons, dengue hemorrhagic fever (DHF) patients, and neonates, each of the groups comprising 10 samples. ResultsThe protein profiles of Ae. albopictus salivary glands showed 13 bands with molecular weights from 16 kDa up to 97 kDa, i.e. 16, 17, 26, 28, 31, 32, 45, 55, 60, 67, 73, 76, and 97 kDa. According to western blot analysis result, the 31 kDa proteins were recognized in all endemic population sera, both in DHF patients and healthy persons. In contrast, protein bands of 47 and 67 kDa were only recognized by the sera of DHF patients. ConclusionThree immunogenic proteins of 31, 47 and 67 kDa were detected from Ae. albopictus salivary glands. These immunogenic proteins may be developed as candidate biomarkers for bite exposure to Ae. albopictus and as vector-based DHF vaccines.

scholarly journals Purification and characterization of recombinant tissue kallikrein from Escherichia coli and yeast

1991 ◽  
Vol 276 (1) ◽  
pp. 63-71 ◽  
Author(s):  
J Wang ◽  
J Chao ◽  
L Chao
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Shuttle Vector ◽  
Affinity Column ◽  
Tissue Kallikrein ◽  
E Coli ◽  
Rat Tissue

A full-length rat tissue kallikrein cDNA was constructed by oligonucleotide engineering through an extension of RSK 1105, a partial cDNA clone containing 534 bp of the 3′ end of tissue kallikrein, followed by site-directed mutagenesis to remove the vector sequence from within the chimaeric coding sequence. The cDNA has been cloned both into the plasmid pET3b under the control of the T7 promoter/polymerase system, and into the shuttle vector PYE directed by the alpha-factor promoter. Expression in Escherichia coli was detected by direct radioimmunoassay, and recombinant kallikrein of 36 kDa was identified by Western-blot analysis using both polyclonal and monoclonal antibodies to rat tissue kallikrein, and by autoradiography of 14C-labelled L-amino acid-labelled-protein synthesis in the presence of rifampicin. Expression in yeast was also detected by direct radioimmunoassay, and recombinant kallikrein was identified by Western-blot analysis with a molecular mass of 39 kDa. The recombinant kallikrein from yeast, however, remained mostly inactive. Kallikrein was purified to apparent homogeneity from E. coli by DEAE-Sepharose CL-6B and aprotinin-affinity column chromatography and confirmed by the N-terminal ten-amino-acid sequence, which matched the deduced sequence from the cDNA. Both E. coli and yeast recombinant kallikreins have Tos-Arg-OMe-esterolytic and kininogenase activities similar to those of purified tissue kallikrein. Comparisons were made between recombinant kallikreins and rat tissue kallikrein with respect to size, charge, substrate specificity, susceptibility to inhibitors and immunological properties. Our results open the way for the study of kallikrein structure-function relationships through protein engineering.

scholarly journals Overproduction and Purification of Soluble Recombinant Human Granulocyte Colony Stimulating Factor in Escherichia coli Using Thioredoxin as Fusion

2017 ◽  
Vol 21 (1) ◽  
pp. 1
Author(s):  
Dian Fitria Agustiyanti ◽  
Debbie Sofie Retnoningrum ◽  
Heni Rachmawati ◽  
Asrul Muhamad Fuad
Keyword(s):  
Mass Spectrometry ◽  
Escherichia Coli ◽  
Fusion Protein ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Soluble Form ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Stimulating Factor

Recombinant human Granulocyte Colony Stimulating Factor (G-CSF) has been produced in a soluble form in Escherichia coli BL21 (DE3) as a fusion protein. The open reading frame of G-CSF was synthetically constructed in previous work and was codon optimized for best expression in E. coli. In this research, the gene was fused to thioredoxin (Trx) at the N-terminal in pET32 vector. The purpose of this research was to optimize the overproduction and purification processes to obtain high yield recombinant protein in soluble form, and to characterize the Trx-G-CSF fusion protein. Overproduction was performed using IPTG induction method for 3 and 6 hours. The protein was purified by Ni-NTA affinity chromatography and separated using gradient concentration of imidazole. The purified protein was then characterized by SDS-PAGE and Western Blot analysis. Further, enterokinase was used to separate G-CSF from the fusion protein. The purified form of G-CSF was subsequently characterized using Western Blot and mass spectrometry using MALDI-TOF. The results showed that the fusion protein was successfully produced in soluble part as much as 48.25% were obtained after 3 hours of induction. The yield of  fusion protein was 67.37%  from total protein (229.65  mg protein/L culture). The Western Blot analysis showed the G-CSF band at around 18.6 kDa. Mass spectrometry with MALDI-TOF/ TOF revealed that 25.86% of amino acid residue was recognized as part of human G-CSF sequence. 

scholarly journals Characterization of Four Viral Species Belonging to the Family Potyviridae Isolated from Ranunculus asiaticus

2006 ◽  
Vol 96 (6) ◽  
pp. 560-566 ◽  
Author(s):  
M. Turina ◽  
M. Ciuffo ◽  
R. Lenzi ◽  
L. Rostagno ◽  
L. Mela ◽  
...  
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Aphid Transmission ◽  
Cross Reactivity ◽  
Polyclonal Antiserum ◽  
Enzyme Linked Immunosorbent Assay ◽  
Coated Plate ◽  
Ranunculus Asiaticus ◽  
Das Elisa

Four different viral species were isolated from diseased Ranunculus asiaticus plants growing in Imperia Province (Italian Riviera-Liguria Region). Infected plants exhibited mosaic symptoms and growth abnormalities. The viruses were mechanically inoculated to a range of herbaceous hosts and differentiated biologically. Long flexuous particles were present in leaf dip extracts observed by electron microscopy. A general protocol for the amplification of potyvirus genome fragments through reverse transcription-polymerase chain reaction generated products that were cloned and sequenced. Sequence and phylogenetic analysis suggested that three of these isolates could be considered new viral species belonging to the genus Potyvirus. The fourth isolate is a new member of the genus Macluravirus. Purified virus was used as antigen to produce a specific polyclonal antiserum in rabbit; serological features were established through double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), antigen coated plate (ACP)-ELISA, and western blot analysis. DAS-ELISA was highly specific for each virus isolate, whereas some cross-reactivity was shown in ACP-ELISA and western blot analysis. Aphid transmission by Myzus persicae was demonstrated in a controlled environment for each of the four viral isolates, whereas no transmission through seed was observed.

Radiosensitivity of glioma to Gamma Knife treatment enhanced in vitro and in vivo by RNA interfering Ku70 that is mediated by a recombinant adenovirus

2010 ◽  
Vol 113 (Special_Supplement) ◽  
pp. 228-235 ◽  
Author(s):  
Qiang Jia ◽  
Yanhe Li ◽  
Desheng Xu ◽  
Zhenjiang Li ◽  
Zhiyuan Zhang ◽  
...  
Keyword(s):  
Western Blot ◽  
Gamma Knife ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Glioma Cells ◽  
C6 Glioma ◽  
C6 Glioma Cells ◽  
C6 Cells

Object The authors sought to evaluate modification of the radiation response of C6 glioma cells in vitro and in vivo by inhibiting the expression of Ku70. To do so they investigated the effect of gene transfer involving a recombinant replication-defective adenovirus containing Ku70 short hairpin RNA (Ad-Ku70shRNA) combined with Gamma Knife treatment (GKT). Methods First, Ad-Ku70shRNA was transfected into C6 glioma cells and the expression of Ku70 was measured using Western blot analysis. In vitro, phenotypical changes in C6 cells, including proliferation, cell cycle modification, invasion ability, and apoptosis were evaluated using the MTT (3′(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) assay, Western blot analysis, and cell flow cytometry. In vivo, parental C6 cells transfected with Ad-Ku70shRNA were implanted stereotactically into the right caudate nucleus in Sprague-Dawley rats. After GKS, apoptosis was analyzed using the TUNEL (terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling) method. The inhibitory effects on growth and invasion that were induced by expression of proliferating cell nuclear antigen and matrix metalloproteinase–9 were determined using immunohistochemical analyses. Results The expression of Ku70 was clearly inhibited in C6 cells after transfection with Ad-Ku70shRNA. In vitro following transfection, the C6 cells showed improved responses to GKT, including suppression of proliferation and invasion as well as an increased apoptosis index. In vivo following transfection of Ad-Ku70shRNA, the therapeutic efficacy of GKT in rats with C6 gliomas was greatly enhanced and survival times in these animals were prolonged. Conclusions Our data support the potential for downregulation of Ku70 expression in enhancing the radiosensitivity of gliomas. The findings of our study indicate that targeted gene therapy–mediated inactivation of Ku70 may represent a promising strategy in improving the radioresponsiveness of gliomas to GKT.

Synthesis and Characterization of Quinoline-3-Carboxamide Derivatives as Inhibitors of the ATM Kinase

2020 ◽  
Vol 20 (23) ◽  
pp. 2070-2079
Author(s):  
Srimadhavi Ravi ◽  
Sugata Barui ◽  
Sivapriya Kirubakaran ◽  
Parul Duhan ◽  
Kaushik Bhowmik
Keyword(s):  
Western Blot ◽  
Cancer Cells ◽  
Cell Line ◽  
Cell Lines ◽  
Cancer Cell ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Cancer Cell Lines ◽  
Atm Kinase ◽  
Cytotoxicity Studies

Background: The importance of inhibiting the kinases of the DDR pathway for radiosensitizing cancer cells is well established. Cancer cells exploit these kinases for their survival, which leads to the development of resistance towards DNA damaging therapeutics. Objective: In this article, the focus is on targeting the key mediator of the DDR pathway, the ATM kinase. A new set of quinoline-3-carboxamides, as potential inhibitors of ATM, is reported. Methods: Quinoline-3-carboxamide derivatives were synthesized and cytotoxicity assay was performed to analyze the effect of molecules on different cancer cell lines like HCT116, MDA-MB-468, and MDA-MB-231. Results: Three of the synthesized compounds showed promising cytotoxicity towards a selected set of cancer cell lines. Western Blot analysis was also performed by pre-treating the cells with quercetin, a known ATM upregulator, by causing DNA double-strand breaks. SAR studies suggested the importance of the electron-donating nature of the R group for the molecule to be toxic. Finally, Western-Blot analysis confirmed the down-regulation of ATM in the cells. Additionally, the PTEN negative cell line, MDA-MB-468, was more sensitive towards the compounds in comparison with the PTEN positive cell line, MDA-MB-231. Cytotoxicity studies against 293T cells showed that the compounds were at least three times less toxic when compared with HCT116. Conclusion: In conclusion, these experiments will lay the groundwork for the evolution of potent and selective ATM inhibitors for the radio- and chemo-sensitization of cancer cells.

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