RESUMEN
In recent decades, immunotoxins have attracted significant attention in treatment of a wide range of diseases including cancers due to their natural origins and their role in blocking crucial pathways within the cells. Ribosome inactivating proteins (RIPs) are efficient molecules in blocking protein synthesis through interactions with ribosomal rRNA molecules. cDNA molecule encoding HER2 scFv antibody fragment originated from trastuzumab attached to the mature alpha luffin gene fragment was subcloned into pET28a expression vector and expressed in different E. coli expression hosts. Identity of the expressed recombinant protein was investigated through western blotting and the fusion protein was purified using Ni-NTA affinity chromatography. The biological activity (toxicity) of the protein was investigated on DNA and RNA samples. A 58 kDa protein was expressed in E. coli. The best protein expression level was achieved in 0.2 mM IPTG at 30 °C in TB medium using E. coli BL21 (DE3) host strain. The fusion protein showed RNase and DNA glycosylase activity on tested RNA and DNA samples. DNA glycosylase activity of the recombinant fusion protein showed that alpha luffin part of this protein is active in conjugation to the scFv molecule and the expressed protein can be further studied in targeted biological in vitro assays.
Asunto(s)
Clonación Molecular/métodos , Escherichia coli/genética , Inmunotoxinas/genética , Proteínas Inactivadoras de Ribosomas Tipo 1/genética , Anticuerpos de Cadena Única/genética , Trastuzumab/genética , Línea Celular , Vectores Genéticos/genética , Humanos , Inmunotoxinas/farmacología , Receptor ErbB-2/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/farmacología , Proteínas Inactivadoras de Ribosomas Tipo 1/farmacología , Anticuerpos de Cadena Única/farmacología , Trastuzumab/farmacologíaRESUMEN
BACKGROUND AND PURPOSE: Aflatoxins are naturally produced by some species of Aspergillus, such as A. flavus and A. parasiticus. Aflatoxins reportedly have carcinogenic effects on human, poultry, and livestock, and therefore could be linked to severe human illnesses. Aflatoxin biosynthesis pathway involves different clustered genes, including structural, regular, and unassigned genes. The present study was conducted to detect aflR, aflP, and aflD as three important genes contributing to aflatoxin B1 production cycle in Aspergillus species isolated from the feedstuffs of animal husbandry. MATERIALS AND METHODS: This study was conducted on 25 isolates of A. flavus, A. parasiticus, A. nomius, and A. nidulans, isolated from animal feedstuff as a test group. The test group was compared with two standard strains (i.e., A. flavus and A. parasiticus) as aflatoxigenic reference organisms and negative controls (i.e., A. fumigatus, A. fusarium, and A. penicillium) in terms of the presence of aflR, aflP, and aflD genes using polymerase chain reaction (PCR). The determination of the toxigenicity and aflatoxin production of isolated Aspergillus species was accomplished using thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). RESULTS: The results obtained by the amplification of the selected genes by PCR method for the detection of aflatoxigenic Asprgillus species were significantly correlated with TLC and HPLC results. Accordingly, all samples, having positive results for aflatoxin B1 production in TLC and HPLC, were able to show the amplification of three target genes. However, 4 cases out of 6 (66%) non-aflatoxigenic isolates were positive for three or two genes. CONCLUSION: Based on the findings, the molecular detection of aflatoxin biosynthesis genes (i.e., aflP, aflD, and aflR) could be considered as a quick and reliable method for the detection of aflatoxigenic Aspergillus. Furthermore, this method could be useful in planning and implementing strategies targeted toward improving the safety of human or animal food.
RESUMEN
Tuberculosis (TB) remains as a major public health problem worldwide. Identification and selection of immunodominant antigens of Mycobacterium tuberculosis (MTB), capable of efficiently inducing a protective immune response is the ultimate goal of TB vaccine development studies. Accordingly, this study was designed to produce a novel M. tuberculosis fusion protein consisted of MTB ESAT-6 (early secreted antigenic target-6 kDa), as a potent immunogenic protein, fused to C-terminus of MTB HSP70 (HSP70(359-610)), as an appropriate carrier and adjuvant. The constructed gene was inserted into a prokaryotic expression vector (pQE30); consequently, the recombinant fusion protein with a 6xHis-tag was successfully over expressed in Escherichia coli M15. Inclusion bodies from bacterial cell lysates were solubilized and the recombinant fusion protein was easily purified by Ni-NTA affinity chromatography under denaturing conditions followed by urea gradient dialysis. The purified and refolded protein was then applied for immunization of mice that resulted in the detection of high titers of specific antibodies, high level of IFN-γ and cell proliferation. The results of our study could confirm the capability of E6H70C fusion protein, as a potential tuberculosis vaccine candidate, for the efficient induction of specific immune responses in a mouse model. However, further investigation need to evaluate the protectivity of this recombinant protein in host model.
Asunto(s)
Antígenos Bacterianos/genética , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/inmunología , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/inmunología , Vacunas contra la Tuberculosis/genética , Vacunas contra la Tuberculosis/inmunología , Animales , Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/aislamiento & purificación , Proteínas Bacterianas/aislamiento & purificación , Secuencia de Bases , Clonación Molecular , Citocinas/biosíntesis , ADN Bacteriano/genética , Femenino , Expresión Génica , Genes Bacterianos , Proteínas HSP70 de Choque Térmico/aislamiento & purificación , Inmunización , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/aislamiento & purificación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/aislamiento & purificación , Vacunas contra la Tuberculosis/aislamiento & purificación , Vacunas de Subunidad/genética , Vacunas de Subunidad/inmunología , Vacunas de Subunidad/aislamiento & purificaciónRESUMEN
Invasive aspergillosis increases in chronic immunosuppressive diseases such as cancer. There is little information about the mechanisms by which Aspergillus infection affects the immune regulation and microenvironment of cancer cells. Hence, this study was aimed at investigating the effect of invasive aspergillosis on immunosurveillance, metastasis, and prognosis of cancer in tumor-bearing mice. After implantation of mouse mammary tumor in BALB/c mice, they were infected with Aspergillus conidia intravenously. For comparison, groups of mice were experimentally infected with Aspergillus conidia or implanted with tumor cells separately. Seven days after Aspergillus infection, the serum levels of tissue inhibitor of metalloproteinase-1 (TIMP-1) were measured by ELISA, and subsequently regulatory T lymphocytes were analyzed by flow cytometry. The survival of animals and mean tumor size were then determined. Our results indicated that tumor sizes in mice increased significantly after infection with Aspergillus conidia. Moreover, invasive aspergillosis enhanced the population of regulatory lymphocytes and level of TIMP-1. This study supports the idea that massive Aspergillus infection could stimulate tumor growth and increases the possibility of a bad prognosis. As a result, treatment of Aspergillus infection could be considered an important issue for efficient cancer therapy.
Asunto(s)
Aspergilosis/complicaciones , Aspergilosis/inmunología , Neoplasias Mamarias Animales/inmunología , Neoplasias Mamarias Animales/microbiología , Microambiente Tumoral/inmunología , Animales , Aspergillus/metabolismo , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Interacciones Huésped-Patógeno , Tolerancia Inmunológica , Huésped Inmunocomprometido , Terapia de Inmunosupresión , Neoplasias Mamarias Animales/patología , Ratones , Ratones Endogámicos BALB C , Monitorización Inmunológica , Metástasis de la Neoplasia , Trasplante de Neoplasias , Linfocitos T Reguladores , Inhibidor Tisular de Metaloproteinasa-1/sangreRESUMEN
A regulatory or suppressor T cell is functionally defined as a T cell that inhibits an immune response by influencing the activity of another cell type. On the other hand, Th1 cells express IFN-gamma and mediate cellular immunity. Sclareol exhibits growth inhibition and cytotoxic activity against a variety of human cancer cell lines. In the first set of experiments, Sclareol was isolated from the plant Salvia sclarea and our study assessed the immuno-therapeutic effectiveness of Sclareol by direct intra-tumoral injection. Secondly, several immunological parameters such as splenocytes proliferation, intra-tumor CD4+CD25+Foxp3+ Treg cells, IFN-gamma and IL-4 secretion and tumor size were assessed to evaluate the anti-tumoral immune response. By all means, the findings confirmed that the activity of Sclareol could reduce the tumor growth in vivo against breast cancer.