Analysis of immumoreactivity of heterologously expressed non-structural protein 4B [NS4B] from hepatitis C virus [HCV] genotype 1a

Background: Detection of hepatitis C virus specific antibodies is the initial step in chronic HCV diagnosis. HCV NS4B is among the most immunogenic HCV antigens and has been widely used in commercial Enzyme Immunoassays [EIA]. Additionally, NS4B, a key protein in the virus replication, can be an alternative target for antiviral therapy

Objectives: Development of a new method for high-level expression and purification of NS4B coding region was the aim of the report

Materials and Methods: Viral RNA was purified from the serum of an HCV positive patient and NS4B coding region was amplified using nested RT-PCR. PCR products were cloned into pET102/D-TOPO expression vector and transformed into E. coli BL21. Induction was performed by adding 1 mM isopropyl-beta-D-thiogalactopyranoside [IPTG] to the culture medium. Immunoreactivity of the purified recombinant proteins was evaluated by immunoblotting and indirect enzymelinked immunosorbent assay [ELISA]

Results: The recombinant NS4B protein was expressed and its immunoreactivity was confirmed by ELISA and western blotting

Conclusions: The directional TOPO cloning provides an efficient and easy platform for heterologous expression of immunoreactive HCV NS4B

Development of a new indirect ELISA method for detection of anti-tuberculosis antibodies in human serum

Tuberculosis is a crucial health problem. Establishing a rapid, reliable and still inexpensive diagnostic method for tuberculosis seems to be substantial in developing countries where TB has very high incidence rate. An Indirect Enzyme-linked immunosorbent Assay [ELISA] was established to detect serum antibodies against Mycobacterium tuberculosis. Three kinds of antigens were used to prepare the solid phase for antibody assay including: purified protein derivative [PPD], M. tuberculosis Bacilli, and Mycobacterium bovis Bacillus Calmette Guerin [BCG]. Sera of two main following groups were investigated in this study: sera samples from smear-positive, culture-positive and Tuberculin Skin Test-positive TB patients and sera samples from smear-negative, culture negative and TST-negative healthy individuals. Among the antigens used, BCG produced higher sensitivity and specificity in the assay. With PPD as the solid phase, higher sensitivity, but lower specificity was observed in comparison with BCG. Both, low response and noise [non-specific binding] were observed with TB bacilli as the solid phase in the assay. Using BCG solid phase system in this method resulted in higher sensitivity in comparison to single antigen solid phase systems. In addition, we were able to circumvent the problem of non-specific bindings in more popular multi-antigenic solid systems such as PPD. By using this new indirect ELISA, a rapid, reliable and still inexpensive diagnosis of tuberculosis might be possible. Although, further investigations are required to our result

Comparison of cell wall proteins in putative Candida albicans and Candida dubliniensis by using modified staining method and SDS PAGE

Candida species are among the most common causes of opportunistic fungal diseases. Among Candida species, Candida albicans is responsible for most infections. Having many strains, C. albicans is very polymorph. C. dubliniensis is very similar to albicans species both morphologically and physiologically. For an infection to occur, cell wall proteins play an important role as they enable yeast to adhere to host cells and begin pathogenesis. Therefore, we decided to extract these proteins and examine them through common molecular methods of protein analysis including SDS-PAGE. Initially cell wall proteins of two C. albicans strains [CBS 562 and PTCC6027] and one C. dubliniensis strain [CBS7987] were extracted by using a solution of beta-mercaptoethanol and ammonium carbonate. After dialysis against Tris-HCL buffer, SDS gel electrophoresis was performed on the proteins extract. Bands were then visualized by using three different staining methods among which one method provided improved detection. By using Coomassie Brilliant Blue staining method, proteins with molecular weight of 42, 66.2 and 200 kDa were detected. By using Silver staining method, proteins with molecular weight of 21.5, 28.5 and 37 kDa were detected. However, using combined Coomassie Brilliant Blue and Sliver staining method visualized more bands resulting in improved detection. To answer many existing questions about fungal diseases, fungi cell wall proteins are necessary to be examined. To commence such examinations, a simple step may be an SDS-PAGE performance on as many strains as possible. A combined staining method can enhance bands detection