Producción y evaluación del antígeno recombinante TES-30 de Toxocara canis para el inmunodiagnóstico de toxocariasis / Production and evaluation of the recombinant antigen TES-30 of Toxocara canis for the immunodiagnosis of toxocariasis

Introducción. Toxocara canis es un nematodo patógeno de cánidos que accidentalmente puede ser transmitido a los humanos. A pesar de la importancia de la serología para el diagnóstico de esta zoonosis, los kits diagnósticos usan antígenos crudos de excreción-secreción, en su mayoría glucoproteínas que no son específicas de especie, por lo cual pueden presentarse reacciones cruzadas con anticuerpos generados contra otros parásitos. Objetivos. Producir el antígeno recombinante TES-30 de T. canis y evaluarlo para el inmunodiagnóstico de la toxocariasis. Materiales y métodos. Se clonó el gen que codifica TES-30 en el vector de expresión pET28a (+), usando oligonucleótidos de cadena sencilla unidos mediante reacción en cadena de la polimerasa (PCR). La proteína rTES-30 se purificó por cromotografia de afinidad (Ni 2+ ). La reacción serológica de rTES-30 se evaluó mediante immunoblot . Teniendo en cuenta que no existe una prueba de referencia , se observó el comportamiento del antigeno en comparación con la prueba de rutina para el inmunodiagnóstico de la toxocariasis, es decir, la técnica ELISA convencional con antígenos de excreción-secreción. Resultados. El rTES-30 se produjo a partir de un cultivo de Escherichia coli LB, con un rendimiento de 2,25 mg/l y 95 % de pureza. La concordancia de la reacción entre el immunoblot rTES-30 y la ELISA convencional, fue de 73 % (46/63) y de 100 % con los 21 sueros no reactivos. De los 21 sueros con diagnóstico de otras parasitosis, 19 fueron reactivos con ELISA, mientras que tan solo siete fueron positivos con el immunoblot rTES-30. La concordancia entre la ELISA y el immunoblot fue moderada (índice kappa de 0,575; IC 95% 0,41-0,74). Conclusiones. Los datos presentados respaldan la utilidad del immunoblot r TES-3 0 para la confirmación de los posibles positivos por ELISA, no solo en los estudios epidemiológicos, sino también, como candidato para el desarrollo de pruebas diagnósticas de la toxocariasis ocular en Colombia.

Introduction: Toxocara canis is a pathogenic nematode of canines which can be accidentally transmitted to humans. Although serology is the most important diagnostic tool for this zoonosis, diagnostic kits use crude excretion/secretion antigens, most of them being glycoproteins which are not species-specific and may cross-react with antibodies generated against other parasites. Objectives: To produce the rTES-30 recombinant antigen of Toxocara canis and evaluate it in the immunodiagnosis of toxocariasis. Materials and methods: The gene that codes for TES-30 was cloned in the expression vector pET28a (+) using single-stranded oligonucleotides united by PCR. The protein rTES-30 was purified by Ni 2+ affinity chromotography. Seroreactivity of rTES-30 was evaluated by immunoblot. Given that there is no gold standard test, the behaviour of the antigen was compared with the method that is routinely used to immunodiagnose toxocariasis, i.e., the conventional ELISA technique using excretion/secretion antigens. Results: The rTES-30 was produced from an Escherichia coli LB culture which yielded 2.25 mg/L of the antigen with a purity of 95%. The results obtained showed 73% (46/63) concordance of reactivity between the rTES-30 immunoblot and the conventional ELISA, and 100% concordance with the non-reactive sera (21). Nineteen of the 21 sera positive for other parasitoses reacted with ELISA, while only seven of these were positive with the rTES-30 immunoblot. Concordance between the ELISA and the immunoblot was moderate (kappa coefficient: 0.575; 95% CI: 0.41- 0.74). Conclusions: The data presented show the potential of the rTES-30 inmunoblot for confirmation of possible ELISA positives, not only in epidemiological studies, but also as a candidate for the development of diagnostic tests for ocular toxocariasis in Colombia.

Translational Read-Through of a Nonsense Mutation Causing Bartter Syndrome

Bartter syndrome (BS) is classified into 5 genotypes according to underlying mutant genes and BS III is caused by loss-of-function mutations in the CLCNKB gene encoding for basolateral ClC-Kb. BS III is the most common genotype in Korean patients with BS and W610X is the most common CLCNKB mutation in Korean BS III. In this study, we tested the hypothesis that the CLCNKB W610X mutation can be rescued in vitro using aminoglycoside antibiotics, which are known to induce translational read-through of a nonsense mutation. The CLCNKB cDNA was cloned into a eukaryotic expression vector and the W610X nonsense mutation was generated by site-directed mutagenesis. Cultured polarized MDCK cells were transfected with the vectors, and the read-through was induced using an aminoglycoside derivative, G418. Cellular expression of the target protein was monitored via immunohistochemistry. While cells transfected with the mutant CLCNKB failed to express ClC-Kb, G418 treatment of the cells induced the full-length protein expression, which was localized to the basolateral plasma membranes. It is demonstrated that the W610X mutation in CLCNKB can be a good candidate for trial of translational read-through induction as a therapeutic modality.

Human SNF2L Gene Is Regulated Constitutively and Inducibly in Neural Cells via a cAMP-Response Element

PURPOSE: SNF2L belongs to Imitation Switch family and plays an essential role in neural tissues and gonads. In our previous studies, we have demonstrated that the basal transcription of human SNF2L gene is regulated by two cis-elements, cAMP response element (CRE)- and Sp1-binding sites. Recent studies suggested that cyclic adenosine monophosphate (cAMP) stimulation significantly up-regulated SNF2L expression in ovarian granulose cells. These data suggested that protein kinase-mediated signal pathways might also regulate SNF2L expression in neural cells. We therefore investigated the effects of agents that activate protein kinases A on SNF2L gene expression in neural cells. MATERIALS AND METHODS: To increase intracellular cAMP levels, all neural cells were treated with forskolin and dbcAMP, two cAMP response activators. We exmined the effects of cAMP on the promoter activity of human SNF2L gene by luciferase reporter gene assays, and further examined the effects of cAMP on endogenous SNF2L mRNA levels by qPCR. RESULTS: Transient expression of a luciferase fusion gene under the control of the SNF2L promoter was significantly increased by treatment of rat primary neurons with forskolin or dbcAMP, but not PC12, C6 and SH-SY5Y cells. Consistently, treatment with forskolin or dbcAMP could enhance endogenous SNF2L mRNA levels also only in rat primary neurons. CONCLUSION: These results suggest that the CRE consensus sequence in the SNF2L proximal promoter most likely confers constitutive activation and regulation by cAMP in neural cells.

Expression of mouse beta defensin 2 in Escherichia coli and its broad-spectrum antimicrobial activity

Mature mouse beta defensin 2 (mBD2) is a small cationic peptide with antimicrobial activity. Here we established a prokaryotic expression vector containing the cDNA of mature mBD2 fused with thioredoxin (TrxA), pET32a-mBD2. The vector was transformed into Escherichia Coli (E. coli) Rosseta-gami (2) for expression fusion protein. Under the optimization of fermentation parameters: induce with 0.6 mM isopropylthiogalactoside (IPTG) at 34ºC in 2×YT medium and harvest at 6 h postinduction, fusion protein TrxA-mBD2 was high expressed in the soluble fraction (>95 percent). After cleaved fusion protein by enterokinase, soluble mature mBD2 was achieved 6 mg/L with a volumetric productivity. Purified recombinant mBD2 demonstrated clear broad-spectrum antimicrobial activity for fungi, bacteria and virus. The MIC of antibacterial activity of against Staphylococcus aureus was 50 µg/ml. The MIC of against Candida albicans (C. albicans) and Cryptococcus neoformans (C. neoformans) was 12.5µg/ml and 25µg/ml, respectively. Also, the antimicrobial activity of mBD2 was effected by NaCl concentration. Additionally, mBD2 showed antiviral activity against influenza A virus (IAV), the protective rate for Madin-Darby canine kidney cells (MDCK) was 93.86 percent at the mBD2 concentration of 100 µg/ml. These works might provide a foundation for the following research on the mBD2 as therapeutic agent for medical microbes.

Cloning and expression of antiviral/ribosome-inactivating protein from Bougainvillea xbuttiana.

A full-length cDNA encoding ribosome-inactivating/antiviral protein (RIP/AVP)from the leaves of Bougainvillea x buttiana was isolated.The cDNA consisted of 1364 nucleotides with an open reading frame (ORF)of 960 nucleotides encoding a 35.49 kDa protein of 319 amino acids.The deduced amino acid sequence has a putative active domain conserved in RIPs/AVPs and shows a varying phylogenetic relationship to the RIPs from other plant species.The deduced protein has been designated BBAP1 (Bougainvillea x buttiana antiviral protein1).The ORF was cloned into an expression vector and expressed in E.coli as a fusion protein of approximately 78 kDa.The cleaved and purified recombinant BBAP1 exhibited ribosome-inhibiting rRNA N-glycosidase activity,and imparted a high level of resistance against the tobacco mosaic virus (TMV).

Proteins in a DNA world: expression systems for their study / Proteínas en el mundo del DNA: Sistemas de expresión para su estudio

Every day, new proteins are discovered and the need to understand its function arises. Human proteins have a special interest, because to know its role in the cell may lead to the design of a cure for a disease. In order to obtain such information, we need enough protein with a high degree of purity, and in the case of the human proteins, it is almost impossible to achieve this by working on human tissues. For that reason, the use of expression systems is needed. Bacteria, yeast, animals and plants have been genetically modified to produce proteins from different species. Even "cell-free" systems have been developed for that purpose. Here, we briefly review the options with their advantages and drawback, and the purification systems and analysis that can be done to gain understanding on the function and structure of the protein of interest.

Cada día, nuevas proteínas son descubiertas y surge la necesidad de caracterizarlas, siendo las de origen humano las que presentan un mayor interés. Conocer su función nos ayudará a entender padecimientos y diseñar una posible cura. Sin embargo, obtener suficiente cantidad de proteínas humanas en cantidad para llevar a cabo los análisis pertinentes, presenta una gran dificultad. Por tal razón, es necesario el uso de sistemas de expresión de proteínas heterólogas. Bacterias, levaduras, animales y plantas han sido modificados genéticamente para expresar proteínas de otras especies, e incluso sistemas in vitro han sido desarrollados para producir proteínas. En este artículo se revisan brevemente las opciones con sus ventajas y desventajas, así como las estrategias de purificación y los análisis que se pueden llevar a cabo para avanzar en el conocimiento de la función y estructura de la proteína de interés.

In vivo determination of the gap2 gene promoter activity in Giardia lamblia

A shuttle vector for Escherichia coli and Giardia lamblia was modified to produce a reporter plasmid, which monitors the expression of prescribed gene in G. lamblia by measuring its luciferase activity. Promoter regions of the gap2 gene, one of the genes induced during encystation, were cloned into this plasmid, and the resultant constructs were then transfected into trophozoites of G. lamblia. Transgenic trophozoites containing one of the 3 gap2-luc reporters were induced to encystation, and characterized with respect to gap2 gene expression by measuring their luciferase activities. Giardia containing a gap2-luc fusion of 112-bp upstream region showed full induction of luciferase activity during encystation.

Distribution of Adenoviral Vector in Brain after Intravenous Administration

The delivery of transgenes to the central nervous system (CNS) can be a valuable tool to treat CNS diseases. Various systems for the delivery to the CNS have been developed; vascular delivery of viral vectors being most recent. Here, we investigated gene transfer to the CNS by intravenous injection of recombinant adenoviral vectors, containing green fluorescence protein (GFP) as a reporter gene. Expression of GFP was first observed 6 days after the gene transfer, peaked at 14 days, and almost diminished after 28 days. The observed expression of GFP in the CNS was highly localized to hippocampal CA regions of cerebral neocortex, inferior colliculus of midbrain, and granular cell and Purkinje cell layers of cerebellum. It is concluded that intravenous delivery of adenoviral vectors can be used for gene delivery to the CNS, and hence the technique could be beneficial to gene therapy.

Molecular cloning of Plasmodium falciparum blood stage antigens and application of the recombinant proteins in serodiagnosis.

A Plasmodium falciparum genomic DNA library was established in the expression vector lambda gt11, cloned in Escherichia coli. The library was screened with human hyperimmune sera by in situ hybridization. Twenty clones expressing P. falciparum sequences as polypeptides fused to beta-galactosidase were identified. One, CD3A/9025/60, reacted with all immune sera and expressed polypeptides that were larger than beta-galactosidase as well as reacting with antibodies to beta-galactosidase and to P. falciparum. When the fusion proteins were used as target antigens to diagnose malaria antibodies, a result was obtained which correlated well with indirect fluorescence assay.