Eukaryotic expression and antigen epitope prediction of the LRRC15 protein in excretory secretory antigens of Taenia solium cysticercus / 中国血吸虫病防治杂志

OBJECTIVE@#To conduct eukaryotic expression of the leucine-rich repeat containing 15 (LRRC15), a differentially expressed protein in excretory secretory antigens of Taenia solium cysticercus, and predict its antigen epitope.@*METHODS@#The molecular weight, stability, amino acid sequence composition, isoelectric point and T lymphocyte epitope of the LRRC15 protein were predicted using the bioinformatics online softwares ExPASy-PortParam and Protean. The full-length splicing primers were designed using PCR-based accurate synthesis, and the LRRC15 gene was synthesized. The recombinant pcDNA3.4-LRRC15 plasmid was constructed and transfected into HEK293 cells to express the LRRC15 protein. In addition, the LRRC15 protein was characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting.@*RESULTS@#The recombinant pcDNA3.4-LRRC15 plasmid was successfully constructed, which expressed the target LRRC15 protein with an approximately molecular weight of 70 kDa. Bioinformatics prediction with the ExPASy-PortParam software showed that LRRC15 was a hydrophilic protein, which was consisted of 644 amino acids and had a molecular weight of 69.89 kDa and an isoelectric point of 5.6. The molecular formula of the LRRC15 protein was C3073H4942N846O953S28 and had an instability coefficient is 50.3, indicating that LRRC15 was an instable protein. Bioinformatics prediction with the Protean software showed that the dominant T-cell antigen epitopes were located in 292 to 295, 353 to 361, 521 to 526 and 555 to 564 amino acids of the LRRC15 protein, and the T-cell antigen epitopes with a high hydrophilicity, good flexibility, high surface accessibility and high antigenicity index were found in 122 to 131, 216 to 233, 249 to 254, 333 to 343, 358 to 361, 368 to 372, 384 to 386, 407 to 412, 445 to 450, 469 to 481, 553 to 564, 588 to 594, 607 to 617 and 624 to 639 amino acids. Following transfection of the recombinant pcDNA3.4-LRRC15 plasmid into HEK293 cells, SDS-PAGE and Western blotting identified LRRC15 proteins in cell secretory culture media, cell lysis supernatants and sediments. The LRRC15-His fusion protein was purified from the cell culture medium, and SDS-PAGE identified a remarkable band at approximately 70 kDa, while Western blotting successfully recognized the band of the recombinant LRRC15 protein.@*CONCLUSIONS@#The eukaryotic expression and antigen epitope prediction of the LRRC15 protein in the excretory secretory antigens of T. solium cysticercus have been successfully performed, which provides insights into further understandings of its biological functions.

Molecular phylogenetic analysis of Cysticercus ovis from Egypt based on MT-CO1 gene sequences / Análise filogenética molecular de Cysticercus ovis do Egito com base nas sequências do gene MT-CO1

Abstract Cysticercus ovis or sheep measles is the larval stage of Taenia ovis, which is the intestinal tapeworm of dogs. It is found in the cardiac and skeletal muscles of sheep and can be the cause of partial or total condemnation of carcasses at abattoirs. The aim of the current work was to determine the prevalence of C. ovis among sheep in Upper Egypt and to present the molecular and phylogenetic analysis of this using the amplified Mitochondrial Cytochrome Oxidase subunit 1 (MT-CO1) gene. A total of 1885 sheep slaughtered at local abattoirs of 4 different governorates of Upper Egypt (Asuit, Sohag, Qena and Aswan) were carefully examined for C. ovis. The overall prevalence of infection was 2.02%. The highest rate of infection was observed in adult animals over 4 years of age (44.73%). There was no significant effect of animal sex on infection rates. The phylogenic analysis of C. ovis Egyptian isolates showed very close similarity to the New Zealand isolate (AB731675). This is the first report showing the genetic analysis of C. ovis in Egypt, which provides a very powerful tool for taxonomy and definitive diagnosis of C. ovis, which could be helpful for preventive and control programs.

Resumo Cysticercus ovis "sheep measles" é o estágio larval da Taenia ovis, encontrada nos músculos de carneiros, causado pela ingestão de ovos de Taenia ovis, parasita de cães. O objetivo do presente trabalho foi determinar a prevalência de C. ovis entre ovinos no Alto Egito e apresentar as análises moleculares e filogenéticas, utilizando o gene da subunidade mitocondrial citocromo-oxidase amplificada 1 (MT-CO1). Um total de 1885 ovinos abatidos em matadouros locais de 4 províncias diferentes do Alto Egito (Asuit, Sohag, Qena e Aswan) foram cuidadosamente examinados para C. ovis. A prevalência geral de infecção foi de 2,02%. A maior taxa de infecção foi observada em animais adultos com mais de 4 anos de idade (44,73%). Não houve efeito significativo do sexo nas taxas de infecção. A análise filogenética de isolados egípcios de C. ovis mostrou uma similaridade muito próxima ao isolado da Nova Zelândia (AB731675). Este é o primeiro relato mostrando a análise genética de C. ovis no Egito, fornecendo uma ferramenta para taxonomia e diagnóstico definitivo de C. ovis, podendo ser útil para programas preventivo e de controle.

Identificación morfológica y molecular de Cysticercus fasciolaris aislado de un roedor (Rattus norvegicus) de la provincia de Buenos Aires (Argentina) / [Morphological and molecular identification of Cysticercus fasciolaris isolated from rodent hosts (Rattus norvegicus) in Buenos Aires province (Argentina)].

In a rodent (Rattus norvegicus) survey in Buenos Aires province, metacestodes of tapeworms were found encysted in the liver of the host. The aim of this work was the morphological and molecular identification of this parasite. To achieve the molecular characterization of the parasite, ribosomal (28S) and mitochondrial (COI) DNA were amplified and sequenced. Based on both morphological and molecular data using bioinformatic tools, the metacestode was identified as Cysticercus fasciolaris. The adult form of this tapeworm (Taenia taeniaeformis) commonly infects felid and canid mammalian hosts. This is the first report on the molecular identification of Cysticercus fasciolaris in Buenos Aires province (Argentina).

Taeniasis and cysticercosis in Asia and the Pacific: present state of knowledge and perspectives.

Several topics on taeniasis and cysticercosis in Asia and the Pacific are overviewed. In Asia and the Pacific, three human taeniid species have been recognized: Taenia solium, Taenia saginata and Taenia asiatica. The first topic is on evolution of T. solium. Mitochondrial DNA polymorphisms of T. solium worldwide are discussed with emphasis of two specific genotypes: American-African and Asian. The second topic is recent major advances in sero- and molecular-diagnosis of T. solium cysticercosis in humans, pigs and dogs. The third is the present situation of T. solium taeniasis/cysticercosis in Papua (Irian Jaya), Indonesia. The forth is the present situation of T. solium cysticercosis and T. saginata taeniasis in Bali, Indonesia. The fifth is the present situation of T. asiatica taeniasis in Asia and the Pacific and in North Sumatra, Indonesia. The sixth is on the debate of the exact definition of T. asiatica. Because T. asiatica can not be differentiated from T. saginata morphologically, it is time to re-evaluate T. saginata in Asia and the Pacific. New and broad-based surveys across this region are necessary from epidemiological and public health perspectives, based on evidence.

Studies on the development of DNA vaccine against Cysticercus cellulosae infection and its efficacy.

DNA vaccine against Cysticercus cellulosae infection was developed and its efficacy was tested. A pair of primers specific to antigen B gene of C. cellulosae was designed which amplified the gene successfully with RT-PCR. The gene was ligated to PV93 vector, and the recombinant of antigen B gene and PV93 was transformed to JM83 cells. The transformed JM83 cells were cultured in a large scale and the plasmid purified. Based on the recombinant plasmid. a DNA vaccine was developed and used to vaccinate two groups of experimental pigs. In each group, there was a routine vaccine, an enhanced vaccine and a control group. Groups 1 and 2 were challenged at 4 months and at 14 days post vaccination respectively with eggs of Taenia solium. The antibody response was also tested with ELISA. The results suggested that all animals vaccinated AgB gene DNA vaccine, no matter by routine or enhanced vaccine, their antibodies reached maximum peak 23 days post vaccination and decreased gradually. When the animals were challenged 4 months after vaccination, they had strong immunity and the parasites decrease rates were 91.2% and 93.1% respectively. When pigs vaccinated with AgB gene DNA vaccine were challenged 14 days post vaccination with 18,000 eggs/pig. The animals showed strong immunity and the parasite decrease rates were 99.5% and 84.9% respectively. However at that time, the antibodies did not reach the peak. While in the control group, the number of C. cellulosae was as many as 2,500. It was concluded that the pigs vaccinated with DNA vaccine had strong immunity against infection of eggs of T. solium.