scholarly journals Expression and purification of four truncated 56 kDa antigens from Orientia tsutsugamushi in Escherichia coli

2018 ◽  
Vol 16 (3) ◽  
pp. 533-541
Author(s):  
Le Thi Lan Anh ◽  
Trinh Van Toan ◽  
Pham Thi Ha Giang ◽  
Bui Thi Thanh Nga ◽  
Vo Viet Cuong ◽  
...  
Keyword(s):  
Recombinant Proteins ◽  
Inclusion Bodies ◽  
Scrub Typhus ◽  
Febrile Illness ◽  
Affinity Column ◽  
Orientia Tsutsugamushi ◽  
Expression And Purification ◽  
E Coli ◽  
Clinical Presentations ◽  
Insoluble Form

Scrub typhus is an acute febrile illness caused by Orientia tsutsugamushi, transmitted to humans by the bite of the larva of trombiculid mites. Diagnosis of scrub typhus is normally based on the clinical presentations. However, it is difficult to differentiate scrub typhus from other acute febrile illnesses, such as dengue fever, malaria and leptospirosis due to similar symptoms. For differential diagnosis of scrub typhus from other acute febrile diseases, a rapid and reliable serological diagnosis is important. In order to produce an ELISA kit for detection of antibodies against O. tsutsugamushi in Vietnam, four truncated 56 kDa antigenic genes of O. tsutsugamushi including Karp (HT-09), Gilliam (HT-11), TA763 (HT-49), and Kato (YB-50) íolated from the most prevalent cases in Vietnam were cloned and expressed in E. coli Rosetta 1 cells. The recombinant proteins formed inclusion bodies when expressed in E. coli. The recombinant 56 kDa proteins in insoluble form were solubilized in 6M urea and were successfully purified by Ni2+affinity column. The purity of four recombinant proteins,HT-09, HT-11, HT-49 and YB-50,reached more than 95% and their concentrations are 12,57 mg/ml; 11,6 mg/ml; 8,98 mg/ml và 8,02 mg/ml, respectively.

Scrub typhus (Orientia tsutsugamushi), A rapidly spreading epidemic in Chennai - A standalone lab experience

2016 ◽  
Vol 5 (09) ◽  
pp. 4896
Author(s):  
Sripriya C.S.* ◽  
Shanthi B. ◽  
Arockia Doss S. ◽  
Antonie Raj I. ◽  
Mohana Priya
Keyword(s):  
Scrub Typhus ◽  
Clinical Symptoms ◽  
Clinical Manifestations ◽  
Febrile Illness ◽  
Orientia Tsutsugamushi ◽  
The Past ◽  
Igm Elisa ◽  
The Mean ◽  
Specific Symptoms ◽  
Symptoms And Signs

Scrub typhus (Orientia tsutsugamushi), is a strict intracellular bacterium which is reported to be a recent threat to parts of southern India. There is re-emergence of scrub typhus during the past few years in Chennai. Scrub typhus is an acute febrile illness which generally causes non-specific symptoms and signs. The clinical manifestations of this disease range from sub-clinical disease to organ failure to fatal disease. This study documents our laboratory experience in diagnosis of scrub typhus in patients with fever and suspected clinical symptoms of scrub typhus infection for a period of two years from April 2014 to April 2016 using immunochromatography and IgM ELISA methods. The study was conducted on 648 patients out of whom 188 patients were found to be positive for scrub typhus. Results also showed that pediatric (0 -12 years) and young adults (20 – 39 years) were more exposed to scrub typhus infection and female patients were more infected compared to male. The study also showed that the rate of infection was higher between September to February which also suggested that the infection rate is proportional to the climatic condition. Statistical analysis showed that the mean age of the patients in this study was 37.6, standard deviation was 18.97, CV % was 50.45. 

scholarly journals Scrub Typhus and Molecular Characterization of Orientia tsutsugamushi from Central Nepal

Pathogens ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 422
Author(s):  
Rajendra Gautam ◽  
Keshab Parajuli ◽  
Mythili Tadepalli ◽  
Stephen Graves ◽  
John Stenos ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Scrub Typhus ◽  
Febrile Illness ◽  
Acute Febrile Illness ◽  
Limited Information ◽  
Orientia Tsutsugamushi ◽  
Health Measures ◽  
Central Nepal ◽  
Vector Borne

Scrub typhus is a vector-borne, acute febrile illness caused by Orientia tsutsugamushi. Scrub typhus continues to be an important but neglected tropical disease in Nepal. Information on this pathogen in Nepal is limited to serological surveys with little information available on molecular methods to detect O. tsutsugamushi. Limited information exists on the genetic diversity of this pathogen. A total of 282 blood samples were obtained from patients with suspected scrub typhus from central Nepal and 84 (30%) were positive for O. tsutsugamushi by 16S rRNA qPCR. Positive samples were further subjected to 56 kDa and 47 kDa molecular typing and molecularly compared to other O. tsutsugamushi strains. Phylogenetic analysis revealed that Nepalese O. tsutsugamushi strains largely cluster together and cluster away from other O. tsutsugamushi strains from Asia and elsewhere. One exception was the sample of Nepal_1, with its partial 56 kDa sequence clustering more closely with non-Nepalese O. tsutsugamushi 56 kDa sequences, potentially indicating that homologous recombination may influence the genetic diversity of strains in this region. Knowledge on the circulating strains in Nepal is important to the development of diagnostic tests and vaccines to support public health measures to control scrub typhus in this country.

scholarly journals Expression and Isolation of N-Terminal Truncated Human Recombinant Renalase in Prokaryotic Cells

2021 ◽  
Vol 4 (3) ◽  
pp. e00158
Author(s):  
V.I. Fedchenko ◽  
A.A. Kaloshin ◽  
S.A. Kaloshina ◽  
A.E. Medvedev
Keyword(s):  
Recombinant Protein ◽  
Signal Peptide ◽  
Extracellular Space ◽  
Inclusion Bodies ◽  
High Concentration ◽  
Genetic Construct ◽  
E Coli ◽  
Insoluble Form ◽  
Guanidine Chloride ◽  
Catalytic Functions

Renalase (RNLS) is a flavoproteinin which its N-terminal peptide (residues 1-17) has several important functions. In cells, it participates in the formation of the so-called Rossmanfold (residues 2-35), needed for «accommodation» of the FAD cofactor and for performing the catalytic functions of RNLS as a FAD-dependent oxidoreductase (EC 1.6.3.5). RNLS secretion into the extracellular space is accompanied by cleavage of this peptide. The resultant truncated extracellular RNLS cannot bind FAD and therefore performs various noncatalytic functions. In this work, we have performed expression the genetic construct encoding RNLS lacking its N-terminal signal peptide (tRNLS) in E. coli Rosetta (DE3) cells. The recombinant protein was accumulated in inclusion bodies in an insoluble form, which could be solubilized in the presence of a high concentration of urea or guanidine chloride. In contrast to full-length RNLS, which was effectively solubilized in the presence of 8 M urea, tRNLS was preferentially solubilized in the presence of 6 M guanidine chloride.

scholarly journals Searching and Finding the Hidden Treasure: A Retrospective Analysis of Rickettsial Disease Among Dutch International Travelers

2020 ◽  
Author(s):  
Sophia G de Vries ◽  
Louise E van Eekeren ◽  
Hans van der Linden ◽  
Benjamin J Visser ◽  
Martin P Grobusch ◽  
...  
Keyword(s):  
Retrospective Analysis ◽  
Medical Records ◽  
Scrub Typhus ◽  
Spotted Fever ◽  
Febrile Illness ◽  
Empiric Antibiotic Therapy ◽  
Spotted Fever Group ◽  
Orientia Tsutsugamushi ◽  
Rickettsial Disease ◽  
Empiric Antibiotic

Abstract Background Rickettsial disease (RD) is a prevalent and underestimated cause of febrile illness worldwide, especially in the absence of an inoculation eschar. We attempted to quantify this underestimation at our clinic, by investigating past cases of febrile illness in travelers who had tested negative for leptospirosis, a disease that can initially present similarly to non-eschar RD, and which we routinely consider when other important causes of unspecified febrile illness have tested negative. Methods We performed a retrospective analysis in febrile returned travelers from Asia, Africa, or the Americas between 2010 and 2017, who had tested negative for leptospirosis. Serologic immunofluorescence assays were performed for Orientia tsutsugamushi (scrub typhus), typhus group, and spotted fever group RD. We performed a medical records review of all patients who tested positive. In case of a fitting medical history, cases were deemed either confirmed (based on convalescent serology) or suspected (based on single serology). Results Among 97 patients, convalescent serology was available in 16 (16.5%) patients, and a single serology in 81 (83.5%) patients. RD was the likely diagnosis in 8 of 16 (50.0%) patients with convalescent serology, and in 8 of 81 (9.9%) with single serology. Of the 16 confirmed/suspected cases, 11 (69%) had been missed and 7 (44%) had not received adequate empiric antibiotic therapy. Conclusions This study shows that non-eschar RD is an important and poorly recognized cause of illness in travelers, even in a specialized travel clinic. A lower threshold to test and treat for RD is warranted in returning travelers with febrile illness.

scholarly journals Confirmation of local transmission of Orientia tsutsugamushi during scrub typhus outbreaks in Nepal

2019 ◽  
Author(s):  
Meghnath Dhimal ◽  
Shyam Prakhas Dumre ◽  
Guna Niddhi Sharma ◽  
Pratik Khanal ◽  
Kamal Ranabhat ◽  
...  
Keyword(s):  
Preventive Measures ◽  
Scrub Typhus ◽  
Case Fatality ◽  
Febrile Illness ◽  
Orientia Tsutsugamushi ◽  
2015 Gorkha Earthquake ◽  
Causative Agents ◽  
The Government ◽  
Local Transmission ◽  
Immediate Response

ABSTRACTBackgroundScrub typhus is a severely ignored tropical disease and a leading cause of undifferentiated febrile illness worldwide caused by infection of an obligate intracellular bacteria Orientia tsutsugamushi. It has been rapidly expanding in South Asian countries, although clear epidemiological information is not available from Nepal. After the 2015 earthquake in Nepal, a sudden upsurge in scrub typhus cases was reported. The objective of this study was to investigate scrub typhus and its causative agents in human, rodent and chigger mites to better understand the ongoing transmission ecology.MethodsScrub typhus cases with confirmed diagnosis throughout the country were included in the analysis. Studies were concentrated in the Chitwan district, the site of a major outbreak in 2016. Additional country-wide data from 2015 to 2017 was made available from the government database to analyse the disease distribution using geographical mapping.ResultsDuring 2015-2017, 1,239 scrub typhus cases were confirmed with the largest outbreak occurring in 2016 with 831 (67.1%) cases. The remainder 267 cases were reported in 2017. The case fatality rate was 5.7% in 2015 and declined to 1.1% in 2017. Nationwide outbreak of scrub typhus was identified as the cases were found from 52 of the 75 districts of Nepal. A seasonal trend was observed with a peak during August and September (p = 0.01). In addition to the human cases, the presence of O. tsutsugamushi was also confirmed in rodents and chigger mites from the outbreak areas of southern Nepal.ConclusionThe detection of O. tsutsugamushi in human, rodent, and chigger mites from outbreak locations and wide-spread reports of scrub typhus throughout the country over two years confirms the ongoing transmission of O. tsutsugamushi with a firmly established ecology in Nepal. The country’s health system needs to be strengthened for systematic surveillance, early outbreaks detection, and immediate response actions including treatment and preventive measures.Author SummaryScrub typhus is a disease caused by a bacteria called Orientia tsutsugamushi and transmitted to people through bites of infected chiggers (larval mites). After the 2015 Gorkha earthquake in Nepal, a sudden upsurge in scrub typhus cases was reported with repeated outbreaks from different parts of the country. This study has documted epidemiology of scrub typhus and its causative agents in human, rodent and chigger mites confimring the local transmission O. tsutsugamushi with a firmly established ecology in Nepal. The local transmission of the diseases from most parts of the country demands strengthening for systematic surveillance, early outbreaks detection, and immediate response actions including treatment and preventive measures.

Expression, solubilization, refolding and final purification of recombinant proteins as expressed in the form of "classical inclusion bodies" in E. coli

2020 ◽  
Vol 27 ◽  
Author(s):  
Mohammad Sadegh Hashemzadeh ◽  
Mozafar Mohammadi ◽  
Hadi Esmaeili Gouvarchin Ghaleh ◽  
Mojtaba Sharti ◽  
Ali Choopani ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Proteins ◽  
Inclusion Bodies ◽  
Native Conformation ◽  
Native Form ◽  
Over Expression ◽  
E Coli ◽  
Mild Conditions ◽  
Final Purification

: Escherichia coli has been most widely used for production of the recombinant proteins. Over-expression of the recombinant proteins is the mainspring of the inclusion bodies formation. The refolding of these proteins into bioactive forms is cumbersome and partly time-consuming. In the present study, we reviewed and discussed most issues regarding the recovery of "classical inclusion bodies" by focusing on our previous experiences. Performing proper methods of expression, solubilization, refolding and final purification of these proteins, would make it possible to recover higher amounts of pro-teins into the native form with appropriate conformation. Generally, providing mild conditions and proper refolding buffers, would lead to recover more than 40% of inclusion bodies into bioactive and native conformation.

scholarly journals Scrub Typhus in Children

2018 ◽  
Vol 38 (1) ◽  
pp. 59-62
Author(s):  
Madiha Zainab ◽  
Atul Kumar Gupta ◽  
Suparna Guha
Keyword(s):  
Acute Kidney Injury ◽  
Scrub Typhus ◽  
Kidney Injury ◽  
Febrile Illness ◽  
Acute Febrile Illness ◽  
Common Complication ◽  
Orientia Tsutsugamushi ◽  
Pain Abdomen ◽  
One Year ◽  
High Index Of Suspicion

Introduction: Scrub typhus is an acute febrile illness caused by infection with rickettsial bacilli Orientia tsutsugamushi. This was a retrospective observational study to study the clinical profile of paediatric scrub typhus, its associated complications and response to treatmentMaterial and Methods: Record files of all patients diagnosed with positive Weil felix (OXK>1:80) and Scrub IgM positive over a period of one year were analysed. Total of 10 cases were diagnosed as scrub with median age of presentation 4.1 years.Results: Fever was present in all followed by pain abdomen (50%), rash. Anaemia (90%), lymphadenopathy (70%) hepatomegaly (100%), Leukopenia was present in those cases with fever <1 week while leucocytosis was found thereafter. Most common complication were hepatitis (100%) shock (50%), acute kidney injury (AKI) 30%, DIC in 20% cases. Secondary HLH was found in 20% and pancarditis in one case. All the cases showed dramatic response to doxycycline.Conclusion: So a high index of suspicion is required to diagnose scrub and early initiation of treatment is essential to prevent mortality from the disease.

scholarly journals Characterization and Analysis of Sip1Aa Protein Expressed by cry1Ac Promoter

2021 ◽  
Author(s):  
Jing Wang ◽  
Mingyue Ding ◽  
Lin Wang ◽  
Jun Cui ◽  
Haitao Li ◽  
...  
Keyword(s):  
Recombinant Proteins ◽  
Insecticidal Activity ◽  
Inclusion Bodies ◽  
Functional Verification ◽  
T7 Promoter ◽  
E Coli ◽  
Significant Difference ◽  
New Ideas ◽  
Pcr Technology ◽  
New Research

Abstract In this study, based on the pUC19 vector and using overlapping PCR technology, the researchers constructed an expression vector of the Sip1Aa protein guided by a cry1Ac promoter. The expression situation, insecticidal activity and solubility were researched, and the expression of the Sip1Aa protein, as guided by a T7 promoter, was compared. Additionally, the fermentation conditions were explored on a preliminary basis and a histidine label was added to the recombinant plasmid via reverse PCR for subsequent purification of recombinant proteins. The results showed that both the cry1Ac and T7 promoters could guide Sip1Aa to express as a soluble protein of 37.6 kDa, and there was no significant difference in insecticidal activity against Colaphellus bowringi Baly, with LC50 values of 1.637 mg/mL and 1.683 μg/mL, respectively. The soluble component of the Sip1Aa protein, when guided by the cry1Ac promoter, was significantly higher than when it was guided by the T7 promoter. The expression of the cry1Ac guider-promoted Sip1Aa protein was more suitable at 37℃ and 16 h. The recombinant protein was purified after an exogenous histidine sequence was added. This provides a new research method and idea to solve the problem of the Sip1Aa protein usually producing a large number of inclusion bodies when expressed in E. coli, and provides new ideas for the study of sip gene rapid expression, functional verification and insecticidal mechanisms.

scholarly journals Detection of Antibodies against Orientia tsutsugamushi Sca Proteins in Scrub Typhus Patients and Genetic Variation ofscaGenes of Different Strains

2012 ◽  
Vol 19 (9) ◽  
pp. 1442-1451 ◽  
Author(s):  
Na-Young Ha ◽  
Yuri Kim ◽  
Ji-Hye Choi ◽  
Myung-Sik Choi ◽  
Ik-Sang Kim ◽  
...  
Keyword(s):  
Scrub Typhus ◽  
Pcr Amplification ◽  
Febrile Illness ◽  
Immunofluorescence Assay ◽  
Antibody Responses ◽  
Diagnostic Methods ◽  
Orientia Tsutsugamushi ◽  
Content Type ◽  
Asian Pacific Region ◽  
Different Strains

ABSTRACTScrub typhus, caused byOrientia tsutsugamushiinfection, is one of the main causes of acute febrile illness in the Asian-Pacific region. Although early diagnosis and immediate antibiotic treatment are critical for reducing disease severity and mortality, current diagnostic methods using serological and molecular approaches have some limitations in sensitivity and applicability in clinical laboratories. In this study, we identified and characterizedO. tsutsugamushisurface cell antigen (sca) family genes encoding autotransporter proteins in order to test them as novel diagnostic targets. We evaluated antibody responses against the Sca proteins in scrub typhus patient sera and examined the genetic diversity of these genes in different strains after PCR amplification. Specific antibody responses against ScaA and ScaC were observed in patients with high indirect immunofluorescence assay titers (≥1:640), whereas specific responses against ScaB and ScaE were relatively low. Genetic analysis using genomic DNAs revealed thescagenes to be quite variable among the different strains. In contrast toscaA,scaC, andscaD, which were detected in all of the tested strains,scaBandscaEwere amplified differentially from the different strains, suggesting a differential presence of the genes in the genomes. Among the members of the gene family, the sequence ofscaCis the most highly conserved between the different strains, and the size ofscaDis the most variable due to the presence of different numbers of internal repeat sequences. These results suggest that thescagenes ofO. tsutsugamushimay be valuable targets for use in combination with classical assay methods for scrub typhus diagnosis.

scholarly journals Expression, Purification, and Functional Analysis of Novel RelE Operon fromX. nematophila

2014 ◽  
Vol 2014 ◽  
pp. 1-7 ◽  
Author(s):  
Jitendra Singh Rathore ◽  
Lalit Kumar Gautam
Keyword(s):  
Escherichia Coli ◽  
Functional Analysis ◽  
Recombinant Proteins ◽  
Bacterial Toxin ◽  
Type Ii ◽  
Xenorhabdus Nematophila ◽  
Expression And Purification ◽  
Toxicity Assay ◽  
Neutralization Activity ◽  
E Coli

Bacterial toxin-antitoxin (TA) complexes induce programmed cell death and also function to relieve cell from stress by various response mechanisms.Escherichia coliRelB-RelE TA complex consists of a RelE toxin functionally counteracted by RelB antitoxin. In the present study, a novel homolog of RelE toxin designated as Xn-relE toxin fromXenorhabdus nematophilapossessing its own antitoxin designated as Xn-relEAT has been identified. Expression and purification of recombinant proteins under native conditions with GST and Ni-NTA chromatography prove the existence of novel TA module. The expression of recombinant Xn-relE under tightly regulated ara promoter inE. coliTop 10 cells confirms its toxic nature in endogenous toxicity assay. The neutralization activity in endogenous toxicity assay by Xn-relEAT antitoxin confirms its antidote nature when studying the whole TA operon under ara regulated promoter. This study promotes newly discovered TA module to be regarded as important as other proteins of type II toxin-antitoxin system.

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