ABSTRACT
Chlamydia trachomatis is the most reported bacterial sexually transmitted disease, especially among young women worldwide. The aim of this study was comparison to evaluate the prevalence of Chlamydia trachomatis infection in woman with tubal infertility by means of PCR and cell culture techniques. Fifty-one women with confirmed TFI were enrolled in this study in [avicenna infertility Clinic] between January 2010 and January 2011. Cervical swab and cytobrush specimens were collected from each patient by gynecologists and sent to laboratory in transport media. Detection of Chlamydia trachomatis in samples was performed using PCR and bacteria culture in MacCoy cell line. The data were analyzed by Fisher's exact test and independent t-test. Statistical significance was established at a p-value <0.05. A significant relation was observed between increased the age of first intercourse and chlamydial infection. Six [11.7%] samples had positive PCR result, whereas cell culture results were positive in only 2 [3.9%] samples. A significant relation was also identified between the duration of infertility and infection [p<0.05] by PCR versus cell culture method. The results showed that PCR is a rapid method, compared to cell culture for detecting Chlamydial organism. It also became clear that the age at first intercourse is important to predict the likelihood of Chlamydia trachomatis
Subject(s)
Humans , Female , Chlamydia trachomatis , Infertility, Female/etiology , Infertility, Female/diagnosis , Cell Culture Techniques , Polymerase Chain ReactionABSTRACT
Helicobacter pylori is a widely distributed Gram negative bacterium that infects the human stomach and duodenum. Some antibiotic regimens are subjected to cure the infection but the cost of drugs, poor patient compliance and emerging of antibiotic-resistant strains are limiting the usefulness of these antibiotic therapies. Therefore, interest in developing a H. pylori vaccine is growing up rapidly. The aim of this study was to construct a recombinant vector containing fusion genes encoding a fragment of B subunit from Helicobacter pylori [H. pylori] urease [UreB332] and Helicobacter pylori adhesion A [HpaA] and expressed it in E. coli BL21, as well as determining its antigenicity as a vaccine candidate of H. pylori. The target genes encoding UreB332 and HpaA amplified from standard H. pylori chromosome by PCR, digested by restricted endonuclease enzyme and inserted into the prokaryotic expression vector pET28a [+] which was digested by corresponding restricted endonuclease enzyme. The target fusion protein was expressed in the BL21 [DE3] E. coli. Furthermore, UreB332-HpaA antigenicity was studied by western blotting after Ni-NTA agarose resin purification. Enzyme digestion analysis, PCR and sequencing showed that the target genes were inserted correctly into the recombinant vector. The fusion protein UreB332-HpaA was recognized by the rabbit anti H. pylori polyclonal antibody and the human sera infected with H. pylori. Our results in addition to favorable properties of HpaA and UreB antigens, support the application of rUreB332-HpaA fusion protein, as a good candidate for the development of H. pylori vaccine
ABSTRACT
Helicobacter pylori is a widely distributed gram negative bacterium that infects the human stomach and duodenum. Some antibiotic regimens are subjected to cure the infection but the cost of drugs, poor patient compliance and emerging of antibiotic-resistant strains are limiting the usefulness of these antibiotic therapies. Therefore, interest in a H. pylori vaccine is growing up rapidly. We selected a fragment of B subunit of H. pylori urease enzyme consist of four important epitopes, involving in elevating host immune responses. This 1070bp fragment was amplified by PCR from genomic DNA isolated from H. pylori 22596 and then cloned into the pET28a expression vector. UreB229-561 was expressed and then affinity-purified by Ni2+-Sepharose resin. The recombinant UreB229-561 was reacted with the serum of/. pylori-infected human and rabbit anti-H. pylori polyclonal antibody in western-blot analysis. Having transformed competent E.coli DH5 alpha with ligation product of digested ureB fragment and pET28a, plasmid extraction from single colonies appeared in LB-agar plate after 18-24 h incubation at 37°C, using plasmid extraction kit [Bioneer, Korea]. Applying both infected human serum and rabbit anti-H. pylori polyclonal antibody, brown strip corresponding to the location of the recombinant protein appeared on PVDF membrane after adding DAB solution, hence confirming the antigenicity of the protein. This recombinant fragment showed urease activity. Our findings confirmed that a prokaryotic expression system of rUreB[229-561] was successfully constructed. The results of SDS-PAGE showed that our constructed prokaryotic expression system pET28 alpha- ureB[229-561]-BL21DE3 efficiently produces target recombinant protein in the form of dissoluble inclusion body. Therefore we can suggest that these epitopes can effectively be a vaccine candidate