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1.
Pesqui. vet. bras ; 41: e06814, 2021. graf, ilus
Article in English | LILACS, VETINDEX | ID: biblio-1279534

ABSTRACT

The protozoan Neospora caninum is known worldwide as one of the main causes of abortion in cattle. During infection, rhoptry proteins present in the apical complex of the parasite play important roles in adhesion and parasitophorous vacuole formation. The use of N. caninum ROP2 in experimental vaccines has shown promising protective results. In our study we performed cloning and expression in Escherichia coli of an antigenic portion of N. caninum ROP2. The recombinant protein (rROP2) was obtained in insoluble form, and the purified protein showed a size of approximately 18kDa. Even being a small truncate NcROP2 region, it was possible to conserve the antigenic epitopes which were recognized by bovine serum naturally infected with N. caninum. Vaccination with rROP2 on aluminum hydroxide adjuvant induced high levels of rROP2-specific IgG antibodies capable of recognizing native protein in tachyzoite lysates. In conclusion, our approaches were effective in obtaining the rROP2 protein, which induced specific mouse immune response and was also recognized by sera from N. caninum naturally infected cattle. These results suggest that it is a promising antigen for the development of neosporosis subunit vaccines as well as a suitable antigen for use in immunodiagnosis.(AU)


O protozoário Neospora caninum é conhecido mundialmente como uma das principais causas de aborto em bovinos. Durante a infecção, as proteínas rhoptry presentes no complexo apical do parasita desempenham papel importante na adesão e formação de vacúolos parasitóforos. O uso de ROP2 de N. caninum em vacinas experimentais tem mostrado resultados de proteção promissores. Em nosso estudo, realizamos a clonagem e expressão em Escherichia coli de uma porção antigênica de N. caninum ROP2. A proteína recombinante (rROP2) foi obtida na forma insolúvel, e a proteína purificada apresentou tamanho aproximado de 18kDa. Mesmo sendo uma pequena região truncada de NcROP2, foi possível conservar os epítopos antigênicos que foram reconhecidos pelo soro de bovinos naturalmente infectados com N. caninum. A vacinação com rROP2 adsorvida no adjuvante de hidróxido de alumínio induziu altos níveis de anticorpos IgG anti-rROP2, capazes de reconhecer a proteína nativa em lisados de taquizoítos. Em conclusão, nossas abordagens foram eficazes na obtenção da proteína rROP2, que induziu resposta imune específica em camundongos e também foi reconhecida por soros de bovinos naturalmente infectados com N. caninum. Estes resultados sugerem que rROP2 é um antígeno promissor para o desenvolvimento de vacinas de subunidades de neosporose, bem como um antígeno adequado para uso em imunodiagnóstico.(AU)


Subject(s)
Immunologic Tests , Immunoglobulin G , Vaccines , Neospora , Cloning, Organism
2.
Braz. arch. biol. technol ; 64: e21210292, 2021. tab, graf
Article in English | LILACS | ID: biblio-1278439

ABSTRACT

Abstract NADPH-cytochromeP450 reductase (CPR) is one of the most important components of the cytochrome P450 enzyme system. In this study, a gene encoding CPR (named EsCPR) was isolated from Eriocheir sinensis using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods. Analysis of the nucleotide sequence revealed a cDNA full-length of 3717 bp with an open reading frame of 2046 bp, a 5′-untranslated region of 42 bp, and a long 3′-untryganslated region of 1628bp, which encodes a protein of 681 amino acids with a predicted molecular weight of 30.7 kDa and an estimated pI of 4.82. The mature peptide shares amino acid of E. sinensis identity 82 % - 89 % to the CPR from Penaeus vannamei and Chionoecetes opilio. Tissues and developmental stage-dependent expression of EsCPR mRNA was investigated by real-time quantitative PCR. EsCPR mRNA was markedly expressed in the hepatopancreas and stomach. These results would provide valuable information for further study on the interactions between CPR and cytochrome P450 enzyme systems.


Subject(s)
NADPH-Ferrihemoprotein Reductase , Cloning, Organism , Brachyura , Gene Expression , Reverse Transcriptase Polymerase Chain Reaction
3.
Pesqui. vet. bras ; 40(11): 852-862, Nov. 2020. tab, ilus
Article in English | LILACS, VETINDEX | ID: biblio-1155021

ABSTRACT

Somatic-cell nuclear transfer is a cloning technique that enables the creation of a viable embryo from a donor adult to produce a genetically identical individual. This technique opens numerous potential possibilities for medicine and animal reproduction. However, several reports have documented cloning-related issues. Embryo and fetal losses remain significantly higher than in other techniques, and there is a high incidence of dystocia and hydrops, which decreases efficiency and increases costs. Animals delivered at term often exhibit a syndrome known as macrosomia and experience difficulties in adapting to life outside the uterus, and death is a common outcome. In the present study, 41 cloned calves that died in the neonatal period were subjected to gross and histopathological examination. Most important gross lesions were found in the liver (enlargement, congestion, yellowish color), kidneys (brownish color at surface and cut, and cysts), lungs (atelectasis, parenchymal consolidation, and secretions in bronchi and bronchioles), and heart (concentric and eccentric hypertrophy, hematic cysts, persistence of ductus arteriosus). Primary microscopic findings were seen in the liver, kidneys, and lungs from neonatal calves. In the liver, 85% of the animals exhibited hepatic degeneration. The presence of a brownish pigment within the cortical tubules of the kidneys was found in approximately 90% of the samples; the presence of this pigment has not been previously reported in cloned calves. In the lungs, a large number of animals exhibiting lesions characteristic of pneumonia (55%). These changes were the pivotal causes of death, mainly due to problems in adapting to life outside the uterus and opportunistic infections in the neonatal period. Further investigation focusing on pathological anatomical changes is necessary to map these abnormalities in cloned animals.(AU)


A transferência nuclear de células somáticas ou clonagem é uma técnica que permite produzir um indivíduo geneticamente igual a um outro indivíduo adulto. Esta técnica abre inúmeras possibilidades para a medicina e para a reprodução animal. Porém, existem inúmeros relatos de problemas associados à clonagem. A taxa de perda nos períodos embrionário e fetal ainda é muito alta quando comparada a outras biotécnicas; além disso, há uma maior incidência de hidropsias e distocias, diminuindo a eficiência e aumentando o custo da técnica. Os animais que vem a termo frequentemente apresentam uma síndrome chamada de macrossomia, e apresentam dificuldades de adaptação à vida extrauterina e, por isso, o óbito é um desfecho comum. No presente trabalho realizou-se necropsia e coleta de fragmentos de órgãos para avaliação histopatológica de 41 bezerros com óbito neonatal. As lesões macroscópicas mais importantes foram encontradas no fígado (hepatomegalia, congestão e coloração amarelada), rins (coloração amarronzada na superfície e ao corte, e cistos), pulmões (atelectasia, parênquima consolidado, e secreções nos brônquios e bronquíolos), e coração (hipertrofia concêntrica e excêntrica, cistos hemáticos e persistência de ducto arterioso). As principais alterações microscópicas observadas foram presença de pigmento acastanhado no interior dos túbulos corticais renais (aproximadamente 90% dos animais), degeneração hepática (85% das amostras avaliadas) e lesões características de pneumonia (55% dos animais). A pigmentação acastanhada no interior dos túbulos corticais é uma alteração que ainda não havia sido relatada anteriormente em animais clonados. As alterações observadas nestes órgãos foram determinantes para o óbito, e devem ter ocorrido sobretudo devido a problemas na adaptação ao ambiente extrauterino e em decorrência de infecções adquiridas no período neonatal. Os achados encontrados no presente trabalho denotam a necessidade de investigação anatomopatológica detalhada de animais clonados inviáveis, na tentativa de mapear as anormalidades apresentadas por eles.(AU)


Subject(s)
Animals , Infant, Newborn , Cattle , Cattle Diseases/pathology , Cloning, Organism/veterinary , Perinatal Death/etiology
4.
Pesqui. vet. bras ; 39(7): 485-491, July 2019. tab
Article in English | LILACS, VETINDEX | ID: biblio-1040717

ABSTRACT

In order for successful extra-uterine adaptation to occur, it is necessary for the neonate to be able to establish its respiratory functions effectively, guaranteeing efficient oxygenation and good vitality. Respiratory disorders are the major cause of death during the neonatal period in cattle, and this mortality is even more significant when it comes to calves originated by in vitro fertilization (FIV) or animal cloning (CA). Blood gas analysis assesses acid-base balance changes effectively, and when associated with the neonate's clinical examination, provides subsidies for accurate diagnosis and early treatment of neonatal maladaptation. The objective of this study was to study neonates born from artificial insemination (IA) and to compare them to calves conceived by FIV and CA, regarding blood gas and clinical examination. For that, 20 AI calves, 15 FIV calves, and 15 cloned calves were evaluated immediately after calving and at 6, 12, 24 and 48 hours of life. At all experimental times, venous blood samples were collected for blood gas and clinical examination was performed. In the postpartum evaluation, Apgar score and column length and respiratory amplitude measurements were used. IVF animals showed no alterations, resembling Group IA calves. The calves from CA showed more pronounced acidosis postpartum than expected physiological acidosis mixed for neonates, with decreasing values of bicarbonate (HCO3-), and base excess (BE) and the increase in carbon dioxide pressure (PCO2) when compared to the other groups. This disorder may have reflected lower mean values of Apgar scores and increased heart and respiratory rates. Intensive follow-up of these neonates is suggested, with monitoring by clinical and hemogasometric examination for early diagnosis of this condition and treatment based on oxygen therapy and bicarbonate replacement.(AU)


Para que ocorra adaptação extra-uterina bem sucedida é necessário que o neonato consiga estabelecer suas funções respiratórias de maneira eficaz, garantindo oxigenação eficiente e boa vitalidade. Distúrbios respiratórios são os maiores causadores de óbito durante o período neonatal em bovinos, e essa mortalidade é ainda mais expressiva quando se trata de bezerros originados por fertilização in vitro (FIV) ou clonagem animal (CA). A hemogasometria avalia alterações do equilíbrio ácido-básico de forma eficaz, e quando associada ao exame clínico do neonato, fornece subsídios para diagnóstico acurado e tratamento precoce da má adaptação neonatal. O objetivo deste trabalho foi estudar recém-nascidos bovinos originados por inseminação artificial (IA) e compará-los a bezerros concebidos por FIV e CA, no que se refere a hemogasometria e exame clínico. Para isso, foram utilizados 20 bezerros IA, 15 bezerros FIV e 15 bezerros clonados que foram avaliados imediatamente após o parto e com 6, 12, 24 e 48 horas de vida. Em todos os momentos experimentais foram colhidas amostras de sangue venoso para hemogasometria e foi realizado o exame clínico. Na avaliação pós-parto foram utilizados escore Apgar e mensurações de comprimento de coluna e amplitude respiratória. Os animais FIV não demonstraram alterações, assemelhando-se aos bezerros do Grupo IA. Os bezerros provenientes de CA apresentaram acidose pós-parto mais acentuada do que a acidose mista fisiológica esperada para neonatos, evidenciada pela diminuição dos valores de bicarbonato (HCO3-) e excesso de bases (EB) e pelo aumento de pressão parcial de dióxido de carbono (PCO2) quando comparados aos demais grupos. Esse distúrbio pode ter refletido em valores médios menores de escore Apgar e no aumento das frequências cardíaca e respiratória. Sugere-se acompanhamento intensivo desses neonatos, com monitoramento por meio do exame clínico e hemogasométrico para diagnóstico precoce dessa condição e tratamento baseado em oxigenioterapia e reposição de bicarbonato.(AU)


Subject(s)
Animals , Cattle , Apgar Score , Acidosis, Respiratory/veterinary , Blood Gas Analysis/veterinary , Maximal Voluntary Ventilation , Animals, Newborn/blood , Insemination, Artificial/veterinary , Fertilization in Vitro/veterinary , Cloning, Organism/veterinary
5.
Article in English | WPRIM | ID: wpr-763368

ABSTRACT

PURPOSE: Canine influenza virus (CIV), H3N2, carries potentiality for zoonotic transmission and genetic assortment which raises a concern on possible epidemics, and human threats in future. To manage possible threats, the development of rapid and effective methods of CIV vaccine production is required. The plant provides economical, safe, and robust production platform. We investigated whether hemagglutinin (HA) antigen from Korea-originated CIV could be produced in Nicotiana benthamiana and lettuce, Lactuca sativa by a DNA viral vector system. MATERIALS AND METHODS: We used DNA sequences of the HA gene from Korean CIV strain influenza A/canine/Korea/S3001/2015 (H3N2) for cloning into a geminiviral expression vectors to express recombinant HA (rHA) antigen in the plant. Agrobacterium-mediated infiltration was performed to introduce HA-carrying vector into host plants cells. Laboratory-grown N. benthamiana, and grocery-purchased or hydroponically-grown lettuce plant leaves were used as host plants. RESULTS: CIV rHA antigen was successfully expressed in host plant species both N. benthamiana and L. sativa by geminiviral vector. Both complex-glycosylated and basal-glycosylated form of rHA were produced in lettuce, depending on presence of endoplasmic reticulum (ER) retention signal. In terms of rHA expression level, canine HA (H3N2) showed preference to the native signal peptide than ER retention signal peptide in the tested geminiviral vector system. CONCLUSION: Grocery-purchased lettuce leaves could serve as an instant host system for the transient expression of influenza antigen at the time of emergency. The geminiviral vector was able to induce expression of complex-glycosylated and basal-glycosylated rHA in lettuce and tobacco.


Subject(s)
Base Sequence , Clone Cells , Cloning, Organism , DNA , Emergencies , Endoplasmic Reticulum , Hemagglutinins , Humans , Influenza, Human , Lettuce , Orthomyxoviridae , Plant Leaves , Plants , Protein Sorting Signals , Tobacco
6.
Article in English | WPRIM | ID: wpr-759593

ABSTRACT

BACKGROUND: The recent expansion of knowledge about various ABO alleles has led to the need for a comprehensive measure to cover the numerous polymorphisms dispersed in the ABO gene. A few studies have examined the diversity of the O allele compared to A or B subgroup alleles, resulting in antigenic changes. This study investigated the relationship between the serologic and molecular genetic characteristics of the O alleles in the Korean population. METHODS: One hundred and five samples from healthy blood group O subjects were selected randomly. The isoagglutinin titer was measured using a tube agglutination and gel microcolumn assay. The ABO alleles were analyzed by sequencing exons 6 and 7 of the ABO gene. When the origin of a heterozygous nucleotide sequence was ambiguous, it was separated into a single allele using mono-allele amplification or cloning. RESULTS: The median IgM isoagglutinin titer was eight. In contrast, the median IgG anti-A and anti-B isoagglutinin titers were 64 and 32, respectively. The IgG isoagglutinin titer showed a significant increase with age (P<0.0001). Six O alleles were observed in 105 blood group O populations by sequencing. The O01 and O02 alleles were common (0.57, 0.36). Three rare O alleles (O04, O05, and O06) and one novel non-deletional O allele were found. CONCLUSION: The distribution of isoagglutinin titers of blood group O and the genetic frequency of O alleles in this study would form the basis of the development and interpretation of ABO genotyping and serologic workup in the Korean population.


Subject(s)
Agglutination , Alleles , Base Sequence , Clone Cells , Cloning, Organism , Exons , Immunoglobulin G , Immunoglobulin M , Molecular Biology , Sequence Analysis
7.
Article in English | WPRIM | ID: wpr-719116

ABSTRACT

My professional journey to understand the glucose homeostasis began in the 1990s, starting from cloning of the promoter region of glucose transporter type 2 (GLUT2) gene that led us to establish research foundation of my group. When I was a graduate student, I simply thought that hyperglycemia, a typical clinical manifestation of type 2 diabetes mellitus (T2DM), could be caused by a defect in the glucose transport system in the body. Thus, if a molecular mechanism controlling glucose transport system could be understood, treatment of T2DM could be possible. In the early 70s, hyperglycemia was thought to develop primarily due to a defect in the muscle and adipose tissue; thus, muscle/adipose tissue type glucose transporter (GLUT4) became a major research interest in the diabetology. However, glucose utilization occurs not only in muscle/adipose tissue but also in liver and brain. Thus, I was interested in the hepatic glucose transport system, where glucose storage and release are the most actively occurring.


Subject(s)
Adipogenesis , Adipose Tissue , Animals , Brain , Clone Cells , Cloning, Organism , Diabetes Mellitus, Type 2 , Glucokinase , Gluconeogenesis , Glucose Transport Proteins, Facilitative , Glucose Transporter Type 2 , Glucose , Glycolysis , Homeostasis , Humans , Hyperglycemia , Liver , Promoter Regions, Genetic , Rats , Transcription Factors
8.
Laboratory Medicine Online ; : 167-170, 2018.
Article in Korean | WPRIM | ID: wpr-717394

ABSTRACT

MPL mutation is an important molecular marker in myeloproliferative neoplasms (MPN). Although MPL W515 is a hot spot for missense mutations in MPN, MPL S505 mutations have been reported in both familial and non-familial MPN. A 72-year-old male visited the hospital, complaining mainly of dizziness and epistaxis. Leukocytosis, anemia, thrombocytopenia, tear drop cells, nucleated RBCs, and myeloblasts were observed in both complete blood cell counts and peripheral blood smears. Bone marrow aspiration failed due to dilution with peripheral blood. BM biopsy indicated hypercellular marrow, megakaryocytic proliferation with atypia, and grade 3 reticulin fibrosis. Conventional cytogenetics results were as follows: 46,XY,del(13)(q12q22)[19]/46,XY[1]. Molecular studies did not detect JAK2 V617F, BCR/ABL translocation, JAK2 exon 12, and CALR exon 9 mutations. The MPL S505N mutation was verified by colony PCR and Sanger sequencing following gene cloning. Based on the above findings, a diagnosis of overt primary myelofibrosis (PMF) was indicated. Mutation studies of buccal and T cells were not conducted. Further, family members were not subjected to mutation studies. Therefore, we were unable to determine whether this mutation was familial or non-familial. Six months after the first visit to the hospital, the patient died due to pneumonia and sepsis. Thrombotic symptoms or major bleeding events did not develop during the survival period following diagnosis of PMF. To the best of our knowledge, this may be the first reported case of PMF with the MPL S505N mutation in Korea.


Subject(s)
Aged , Anemia , Biopsy , Blood Cell Count , Bone Marrow , Clone Cells , Cloning, Organism , Cytogenetics , Diagnosis , Dizziness , Epistaxis , Exons , Fibrosis , Granulocyte Precursor Cells , Hemorrhage , Humans , Korea , Leukocytosis , Male , Mutation, Missense , Pneumonia , Polymerase Chain Reaction , Primary Myelofibrosis , Reticulin , Sepsis , T-Lymphocytes , Tears , Thrombocytopenia
9.
Article in English | WPRIM | ID: wpr-716973

ABSTRACT

Wnt/β-catenin signaling plays a critical role in the achievement of peak bone mass, affecting the commitment of mesenchymal progenitors to the osteoblast lineage and the anabolic capacity of osteoblasts depositing bone matrix. Recent studies suggest that this evolutionarily-conserved, developmental pathway exerts its anabolic effects in part by coordinating osteoblast activity with intermediary metabolism. These findings are compatible with the cloning of the gene encoding the low-density lipoprotein related receptor-5 (LRP5) Wnt co-receptor from a diabetes-susceptibility locus and the now well-established linkage between Wnt signaling and metabolism. In this article, we provide an overview of the role of Wnt signaling in whole-body metabolism and review the literature regarding the impact of Wnt signaling on the osteoblast's utilization of three different energy sources: fatty acids, glucose, and glutamine. Special attention is devoted to the net effect of nutrient utilization and the mode of regulation by Wnt signaling. Mechanistic studies indicate that the utilization of each substrate is governed by a unique mechanism of control with β-catenin-dependent signaling regulating fatty acid β-oxidation, while glucose and glutamine utilization are β-catenin-independent and downstream of mammalian target of rapamycin complex 2 (mTORC2) and mammalian target of rapamycin complex 1 (mTORC1) activation, respectively. The emergence of these data has provided a new context for the mechanisms by which Wnt signaling influences bone development.


Subject(s)
Anabolic Agents , beta Catenin , Bone Development , Bone Matrix , Clone Cells , Cloning, Organism , Fatty Acids , Glucose , Glutamine , Lipoproteins , Metabolism , Osteoblasts , Sirolimus
10.
Article in English | WPRIM | ID: wpr-758856

ABSTRACT

Dogs serve human society in various ways by working at tasks that are based on their superior olfactory sensitivity. However, it has been reported that only about half of all trained dogs may qualify as working dogs through conventional breeding management because proper temperament and health are needed in addition to their innate scent detection ability. To overcome this low efficiency of breeding qualified working dogs, and to reduce the enormous costs of maintaining unqualified dogs, somatic cell nuclear transfer has been applied in the propagation of working dogs. Herein, we review the history of cloning working dogs and evaluate the health development, temperaments, and behavioral similarities among the cloned dogs. We also discuss concerns about dog cloning including those related to birth defects, lifespan, and cloning efficiency.


Subject(s)
Animals , Breeding , Clone Cells , Cloning, Organism , Congenital Abnormalities , Dogs , Humans , Temperament
11.
Article in English | WPRIM | ID: wpr-713307

ABSTRACT

OBJECTIVE: To determine the localization, expression, and function of Toll-like receptors (TLRs) in fallopian tube epithelial cells. METHODS: The localization of TLRs in fallopian tube epithelial cells was investigated by immunostaining. Surprisingly, the intensity of staining was not equal in the secretory and ciliated cells. After primary cell culture of fallopian tube epithelial cells, ring cloning was used to isolate colonies of ciliated epithelial cells, distinct from non-ciliated epithelial cells. The expression of TLRs 1–10 was examined by quantitative real-time polymerase chain reaction, and protein localization was confirmed by immunostaining. The function of the TLRs was determined by interleukin (IL)-6 and IL-8 production in response to TLR2, TLR3, TLR5, TLR7, and TLR9 ligands. RESULTS: Fallopian tube epithelial cells expressed TLRs 1–10 in a cell-type-specific manner. Exposing fallopian tube epithelial cells to TLR2, TLR3, TLR5, TLR7, and TLR9 agonists induced the secretion of proinflammatory cytokines such as IL-6 and IL-8. CONCLUSION: Our findings suggest that TLR expression in the fallopian tubes is cell-type-specific. According to our results, ciliated cells may play more effective role than non-ciliated cells in the innate immune defense of the fallopian tubes, and in interactions with gametes and embryos.


Subject(s)
Clone Cells , Cloning, Organism , Cytokines , Embryonic Structures , Epithelial Cells , Fallopian Tubes , Female , Germ Cells , Humans , Interleukin-6 , Interleukin-8 , Interleukins , Ligands , Primary Cell Culture , Real-Time Polymerase Chain Reaction , Toll-Like Receptors
12.
Article in English | WPRIM | ID: wpr-742291

ABSTRACT

Histomonas meleagridis is a facultative anaerobic parasite, which can cause a common poultry disease known as histomoniasis. The species and age of the birds impacts on the susceptibility, with turkey being the most susceptible species. Chickens are less susceptible to H. meleagridis than turkeys and usually serve as reservoir hosts. Here, the diagnosis of an outbreak of histomoniasis in backyard Sanhuang chickens is described. The primary diagnosis was made based on clinical symptoms, general changes at necropsy, histopathology, and the isolation and cultivation of parasites. The pathogen was further confirmed by cloning, PCR identification, and animal inoculation tests. A strain of H. meleagridis, named HM-JSYZ-C, with a higher pathogenicity level in chickens was obtained. The study lays a foundation for further investigations into H. meleagridis and histomoniasis in chickens.


Subject(s)
Animals , Birds , Chickens , Clone Cells , Cloning, Organism , Diagnosis , Parasites , Polymerase Chain Reaction , Poultry Diseases , Protozoan Infections , Turkey , Turkeys , Virulence
13.
Article in English | WPRIM | ID: wpr-742257

ABSTRACT

Cystic echinococcosis (CE) caused by E. granulosus is a serious helminthic zoonosis in humans, livestock and wildlife. Xinjiang is one of high endemic province for CE in China. A total of 55 sheep and cattle livers containing echinococcal cysts were collected from slaughterhouses in Changji and Yining City, northern region of Xinjiang. PCR was employed for cloning 2 gene fragments, 12S rRNA and CO1 for analysis of phylogenetic diversity of E. granulosus. The results showed that all the samples collected were identified as G1 genotype of E. granulosus. Interestingly, YL5 and CJ75 strains were the older branches compared to those strains from France, Argentina, Australia. CO1 gene fragment showed 20 new genotype haploids and 5 new genotype haplogroups (H1-H5) by the analysis of Network 5.0 software, and the YLY17 strain was identified as the most ancestral haplotype. The major haplotypes, such as CJ75 and YL5 strains, showed identical to the isolates from Middle East. The international and domestic trade of livestock might contribute to the dispersal of different haplotypes for E. granulosus evolution.


Subject(s)
Abattoirs , Animals , Argentina , Australia , Cattle , China , Clone Cells , Cloning, Organism , Echinococcosis , Echinococcus granulosus , Echinococcus , France , Genetic Variation , Genotype , Haploidy , Haplotypes , Helminths , Humans , Liver , Livestock , Middle East , Polymerase Chain Reaction , Sheep
14.
Iranian Journal of Veterinary Research. 2017; 18 (1): 36-42
in English | IMEMR | ID: emr-189265

ABSTRACT

Rabies virus nucleoprotein [N protein] encapsidates genomic RNA of the virus and forms the viral ribonucleoprotein complex. These N proteins represent highly organized structures which activate proliferation of B cells and production antibodies against the N protein. In addition to the B cell, the rabies virus N protein has been shown to induce potent T helper cell responses resulting in a long-lasting and strong humoral immune response. Rabies virus N protein is a molecular target of choice for development of tools to diagnose acute rabies infection. We produced a recombinant immune reactive C-terminal fragment of the rabies virus N protein which contains an antigenic determinant located between positions 360-389. Synthetic gene encoding the N protein was cloned into an expression plasmid to produce the recombinant antigen in Escherichia coli cells BL21 [DE3]. SDS-PAGE showed presence of the product with expected molecular weight [44 kDa]. The recombinant fragment of the N protein efficiently recognized antibodies in sera from mice immunized with an inactivated rabies virus. Thus produced recombinant antigen of the rabies virus N protein can be used in an enzyme-linked immunosorbent assay [ELISA] for diagnosis of the rabies infection


Subject(s)
Cloning, Organism , Gene Expression , Nucleoproteins , Escherichia coli , Enzyme-Linked Immunosorbent Assay , Antigens
15.
Article in English | WPRIM | ID: wpr-50089

ABSTRACT

Toxoplasma gondii infections occur throughout the world, and efforts are needed to develop various vaccine candidates expressing recombinant protein antigens. In this study, influenza matrix protein (M1) virus-like particles (VLPs) consisting of T. gondii rhoptry antigen 4 (ROP4 protein) were generated using baculovirus (rBV) expression system. Recombinant ROP4 protein with influenza M1 were cloned and expressed in rBV. SF9 insect cells were coinfected with recombinant rBVs expressing T. gondii ROP4 and influenza M1. As the results, influenza M1 VLPs showed spherical shapes, and T. gondii ROP4 protein exhibited as spikes on VLP surface under transmission electron microscopy (TEM). The M1 VLPs resemble virions in morphology and size. We found that M1 VLPs reacted with antibody from T. gondii-infected mice by western blot and ELISA. This study demonstrated that T. gondii ROP4 protein can be expressed on the surface of influenza M1 VLPs and the M1 VLPs containing T. gondii ROP4 reacted with T. gondii-infected sera, indicating the possibility that M1 VLPs could be used as a coating antigen for diagnostic and/or vaccine candidate against T. gondii infection.


Subject(s)
Animals , Baculoviridae , Blotting, Western , Clone Cells , Cloning, Organism , Enzyme-Linked Immunosorbent Assay , Influenza, Human , Insecta , Mice , Microscopy, Electron, Transmission , Toxoplasma , Toxoplasmosis , Virion
16.
Article in English | WPRIM | ID: wpr-194442

ABSTRACT

Phage display technology provides a powerful tool to screen a library for a binding molecule via an enrichment process. It has been adopted as a critical technology in the development of therapeutic antibodies. However, a major drawback of phage display technology is that because the degree of the enrichment cannot be controlled during the bio-panning process, it frequently results in a limited number of clones. In this study, we applied next-generation sequencing (NGS) to screen clones from a library and determine whether a greater number of clones can be identified using NGS than using conventional methods. Three chicken immune single-chain variable fragment (scFv) libraries were subjected to bio-panning on prostate-specific antigen (PSA). Phagemid DNA prepared from the original libraries as well as from the Escherichia coli pool after each round of bio-panning was analyzed using NGS, and the heavy chain complementarity-determining region 3 (HCDR3) sequences of the scFv clones were determined. Subsequently, through two-step linker PCR and cloning, the entire scFv gene was retrieved and analyzed for its reactivity to PSA in a phage enzyme immunoassay. After four rounds of bio-panning, the conventional colony screening method was performed for comparison. The scFv clones retrieved from NGS analysis included all clones identified by the conventional colony screening method as well as many additional clones. The enrichment of the HCDR3 sequence throughout the bio-panning process was a positive predictive factor for the selection of PSA-reactive scFv clones.


Subject(s)
Antibodies , Bacteriophages , Chickens , Clone Cells , Cloning, Organism , Complementarity Determining Regions , DNA , Escherichia coli , Immunoenzyme Techniques , Mass Screening , Methods , Polymerase Chain Reaction , Prostate-Specific Antigen , Single-Chain Antibodies
17.
Article in English | WPRIM | ID: wpr-165936

ABSTRACT

Initial discovery on sphingosine 1-phosphate (S1P) as an intracellular second messenger was faced unexpectedly with roles of S1P as a first messenger, which subsequently resulted in cloning of its G protein-coupled receptors, S1P₁₋₅. The molecular identification of S1P receptors opened up a new avenue for pathophysiological research on this lipid mediator. Cellular and molecular in vitro studies and in vivo studies on gene deficient mice have elucidated cellular signaling pathways and the pathophysiological meanings of S1P receptors. Another unexpected finding that fingolimod (FTY720) modulates S1P receptors accelerated drug discovery in this field. Fingolimod was approved as a first-in-class, orally active drug for relapsing multiple sclerosis in 2010, and its applications in other disease conditions are currently under clinical trials. In addition, more selective S1P receptor modulators with better pharmacokinetic profiles and fewer side effects are under development. Some of them are being clinically tested in the contexts of multiple sclerosis and other autoimmune and inflammatory disorders, such as, psoriasis, Crohn’s disease, ulcerative colitis, polymyositis, dermatomyositis, liver failure, renal failure, acute stroke, and transplant rejection. In this review, the authors discuss the state of the art regarding the status of drug discovery efforts targeting S1P receptors and place emphasis on potential clinical applications.


Subject(s)
Acute Kidney Injury , Animals , Clone Cells , Cloning, Organism , Colitis, Ulcerative , Dermatomyositis , Drug Discovery , Fingolimod Hydrochloride , Graft Rejection , In Vitro Techniques , Liver Failure , Mice , Multiple Sclerosis , Polymyositis , Psoriasis , Receptors, Lysosphingolipid , Second Messenger Systems , Sphingosine , Stroke
18.
Acta bioeth ; 22(2): 203-211, nov. 2016.
Article in Spanish | LILACS | ID: biblio-827607

ABSTRACT

En febrero de 2015 el Reino Unido dio el primer paso para la aprobación de la transferencia mitocondrial como técnica terapéutica. Teóricamente, gracias a eso será posible para muchas mujeres engendrar descendencia libre de patologías asociadas a defectos mitocondriales. Sin embargo, esta práctica enfrenta severas dudas desde un punto de vista ético. Entre las objeciones destacan: su estrecha vinculación con la clonación humana; la alteración de los genes de la línea germinal; la modificación de la identidad del ser humano al que dará lugar; la destrucción de embriones humanos que envuelve, o el elevado riesgo que encierra para la salud del ser humano resultante. En este texto se analiza la solvencia de todas estas objeciones de forma crítica, resaltando las fortalezas de algunas de ellas. En particular, se aboga por una restricción cuidadosa del uso de esta técnica, que promueva el empleo de alternativas más respetuosas con la salud del futuro ser humano.


In February 2015 the United Kingdom took the first step towards the adoption of mitochondrial transfer as a therapeutic technique. Theoretically, it will make it possible for many women to get rid of pathologies associated with mitochondrial defects. However, this practice has been subjected to severe doubts from an ethical standpoint. Among these objections, we could highlight the following: its close association with human cloning; the alteration of the germline genes; the modification in the identity of the human being involved; the destruction of human embryos; or the high risk to the health of the human being. In this text we will analyze these objections critically, highlighting the strength of all of them. As a result, we will call for a careful restriction of the use of this technique, and the promotion of the use of alternative options much more respectful of the human future.


Em fevereiro de 2015 o Reino Unido deu o primeiro passo para a aprovação da transferência mitocondrial como técnica terapêutica. Teoricamente, graças a isso será possível a muitas mulheres engendrar descendência livre de patologias associadas a defeitos mitocondriais. No entanto, esta prática enfrenta severas dúvidas a partir de um ponto de vista ético. Entre as objeções destacam: sua estreita vinculação com a clonagem humana; a alteração dos genes da linha germinal; a modificação da identidade do ser humano ao qual dará lugar; a destruição de embriões humanos que envolve, ou o elevado risco que encerra para a saúde do ser humano resultante. Neste texto se analisa a solvência de todas estas objeções de forma crítica, ressaltando as fortalezas de algumas delas. Em particular, se advoga por uma restrição cuidadosa do uso desta técnica, que se promova o emprego de alternativas mais respeitosas com a saúde do futuro ser humano.


Subject(s)
Humans , Cloning, Organism/ethics , Genetic Therapy/ethics , Mitochondrial Replacement Therapy/ethics , Mitochondria/transplantation , Nuclear Transfer Techniques/ethics , Bioethics
19.
Pakistan Journal of Pharmaceutical Sciences. 2016; 29 (1 Supp.): 287-293
in English | IMEMR | ID: emr-177606

ABSTRACT

A full length about 105 kb gene cluster containing 35 open reading frames involved in the biosynthesis of lobophorins was cloned and sequenced from a fosmid genomic library of Streptomyces olivaceus strain FXJ7.023. The cluster was identified by genome wide annotation and analysis of secondary metabolite biosynthesis gene clusters by anti SMASH and knockout of loading module-contained region of polyketide skeleton synthesis gene [the starter of lobS1]. Gene cluster comparative analysis suggested that the cluster encoded the complete genes for lobophorin polyketide assembly, modification, substrate catalysis, regulation, transportation and resistance, and shows great identity to the newest reported lobophorin biosynthetic gene cluster from Streptomyces sp. SCSIO 01127, but with a significant gene rearrangement in the PKS modules


Subject(s)
Multigene Family , Streptomyces , Cloning, Organism , Aquatic Organisms
20.
J. venom. anim. toxins incl. trop. dis ; 22: 28, 2016. tab, graf, ilus
Article in English | LILACS, VETINDEX | ID: biblio-954789

ABSTRACT

Background: Wasp venom is a complex mixture containing proteins, enzymes and small molecules, including some of the most dangerous allergens. The greater banded wasp (Vespa tropica) is well-known for its lethal venom, whose one of the major components is a hyaluronidase (HAase). It is believed that the high protein proportion and activity of this enzyme is responsible for the venom potency. Methods: In the present study, cDNA cloning, sequencing and 3D-structure of Vespa tropica venom HAase were described. Anti-native HAase antibody was used for neutralization assay. Results: Two isoforms, VesT2a and VesT2b, were classified as members of the glycosidase hydrolase 56 family with high similarity (42-97 %) to the allergen venom HAase. VesT2a gene contained 1486 nucleotide residues encoding 357 amino acids whereas the VesT2b isoform consisted of 1411 residues encoding 356 amino acids. The mature VesT2a and VesT2b are similar in mass and pI after prediction. They are 39119.73 Da/pI 8.91 and 39571.5 Da/pI 9.38, respectively. Two catalytic residues in VesT2a, Asp107 and Glu109 were substituted in VesT2b by Asn, thus impeding enzymatic activity. The 3D-structure of the VesT2s isoform consisted of a central core (α/β)7 barrel and two disulfide bridges. The five putative glycosylation sites (Asn79, Asn99, Asn127, Asn187 and Asn325) of VesT2a and the three glycosylation sites (Asn1, Asn66 and Asn81) in VesT2b were predicted. An allergenic property significantly depends on the number of putative N-glycosylation sites. The anti-native HAase serum specifically recognized to venom HAase was able to neutralize toxicity of V. tropica venom. The ratio of venom antiserum was 1:12. Conclusions: The wasp venom allergy is known to cause life-threatening and fatal IgE-mediated anaphylactic reactions in allergic individuals. Structural analysis was a helpful tool for prediction of allergenic properties including their cross reactivity among the vespid HAase.(AU)


Subject(s)
Animals , Wasp Venoms , Wasps , Cloning, Organism , Glycoside Hydrolases , Hyaluronoglucosaminidase
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