جستجوی مقالات مرتبط با کلیدواژه « sds-page » در نشریات گروه « پزشکی »

Tuberculosis is one of the major global health problems with high mortality. Establishing a rapid and low-cost identification method is significant, especially in developing countries. In this study, we aimed to design an indirect ELISA system with low molecular weight antigens secreted by Mycobacterium tuberculosis strain C, for identification of Mycobacterium tuberculosis Complex infection in human.

The secretory proteins from culture filtrate of Mtb strain C extracted by Sephadex-G50 were used as target antigen in designing an indirect ELISA system. Then, human sera with tuberculin skin test, culture, acid-fast bacilli smear, and PCR test results were selected to detect human serum antibodies against these low molecular weight proteins.

During gel chromatography and SDS-PAGE (12.5%) analysis, the purified protein F2 fractions with approximately 0.7 mg/ml and molecular weights in the range of 14-40 kDa were used as the target antigen in designing the indirect ELISA system. The ELISA system designed by F2 showed 78% sensitivity and 92% specificity in identifying tuberculosis.

The ELISA results based on low molecular weight proteins, with the sensitivity and specificity achieved in this study, might support the hypothesis that the Mtb culture filtrate antigens could be used as a rapid and sensitive assay in ELISA test for detection of pulmonary TB.

Streptococci are Gram-positive and anaerobic bacteria that are isolated from different sources. These bacteria are capable of producing superantigens, toxins, and biologically-active substances and are therefore very important in the field of infectious disease. Streptococcal pyrogenic exotoxins A (speA) is produced by various bacteria, including Streptococcus dysgalactiae. This study aimed at the isolation, cloning, and production of speA from streptococci from the skin of psoriasis patients.

In this descriptive-laboratory study, samples were taken from 60 psoriasis patients. S. dysgalactiae isolated were identified by different tests. The speA gene was cloned by the TA cloning method using PTG-19 vector into the Escherichia coli X11 blue as host. Expression of the cloned gene in recombinant colonies was evaluated by SDS-PAGE.

Screening of white recombinant colonies confirmed the presence of speA genes. Expression of the speA gene in E. coli X11 blue was confirmed by SDS-PAGE.

Streptococcus superantigens can be considered as a rich source of vaccine production for different infections caused by these bacteria. By utilizing different cloning hosts and investigating optimal production conditions, S. dysgalactiae could be a candidate for future studies on vaccine production.

Pertussis is still one of the major public health problems. The increase of the disease emerged in recent decades due to the replacement of the reactogenic whole cell vaccine with the safer acellular vaccine and the genetic diversity of the bacterium. As outer membrane vesicles (OMVs) obtained from Bordetella pertussis contains surface immunogenic antigen in its structure, it has an acceptable characteristic that could be considered as a good candidate for pertussis vaccine.

Vaccinal strain BP134 strain of B. pertussis was cultured under standard conditions and OMVs were isolated by modifying the method without the use of ultracentrifuge. The isolated vesicles were examined by transmission electron microscopy and protein content was measured by the Bradford method. The expression of virulence factors was confirmed by SDS-PAGE and protein expression was confirmed by Western immunoblot. Pyrogenicity test and abnormal toxicity test were performed on extracted vesicles.

The morphology of the vesicles was confirmed in the range of 40 to 200 nm. The protein concentration of the extracted vesicles was determined as 600 μg. Expression analysis by SDS-PAGE and western blot confirmed the presence of virulence factors, pertussis toxin, filamentous hemagglutinin, and pertactin using monoclonal antibodies in OMVs of the vaccinal strain. Pyrogenicity test and abnormal toxicity test were negative.

The proposed method is a simple and efficient method for isolation of the B. pertussis OMVs. The OMVs extracted from B. pertussis could be a candidate for a new generation of pertussis vaccine alone or in combination with adjuvants to design future acellular vaccines.

Strongyloides stercoralis is prevalent in tropical and subtropical regions of the world. S. stercoralis is the only nematodes with the ability to multiply in its host's body via autoinfection transmission. Larvae detection in faeces is difficult partly because of low egg production and irregular larvae excretion in faeces. Serologic tests (ELISA, IFA) are also diagnostic, however, S. stercoralis antigens are not available as a diagnostic tool. In the present study, we analyzed filariform larva (L3) proteins of S. stercoralis by the immuno blot technique.

Stool samples were examined by direct smear, formalin-ether and agar plate method to identify the patients. Sera were stored at 20°С. Filariform larvae were obtained by agar plate culture, which was incubated for 6-7days at 25ºC, then frozen at – 70°С. Finally, larvae were suspended at a concentration level of 12000 in 250l PBS, containing protease inhibitors and then sonicated. Protein level was measured by Bradford method. Proteins of S. stercoralis filariform larvae were separated by SDS-PAGE, blotted onto nitrocellulose paper. Western blot analysis of these antigens was achieved using infected human sera (0.1, 0.01, 0.001 dilution) with strongyloidiasis, toxocariasis, hydatidosis, amoebiasis and normal human serum as control.

Four immunodominant proteins (23, 28, 30, 41 kDa) were recognized with strongyloidiasis sera in 0.1diluted serum. None of the proteins reacted to normal human and amebiasis serum, but some showed reaction with serum of hydatidosis and toxocariosis. Having increased the level of serum dilution, only 41 kDa protein was recognized by strongyloidiasis sera. Other sera did not show any reaction to the parasite’s proteins. Therefore, the 41 kDa protein presents as most important immunodaminant protein in this study.

The identification of immunodominant proteins, which have adapted themselves to the physiological and genetically conditions of the host is an appropriate diagnostic approach. Indeed, recombinant protein, such as 41 kDa, could enhance the sensitivity and specificity of serologic tests.