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Isolation and Cloning of Streptococcus Pneumoniae Surface Protein (PSP) gene in Escherichia Coli DH5 Alpha to Obtain Vaccine
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Hajar Bagheri    , Hamid Azadegan Qomi , Kiomars Amini |
| Associate Professor, Department of Microbiology, Faculty of Basic Sciences, Saveh Branch, Islamic Azad University, Saveh, Iran , Dr_kumarss_amini@yahoo.com |
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Abstract: (1424 Views) |
Background and Aim: Streptococcus pneumoniae or pneumococcus, gram-positive diplococcus with a diameter of 0.5 to 1.2 μm, alpha hemolytic and oxygen tolerant of Streptococcus and causes diseases such as meningitis and pneumonia. The bacterium carries the Pneumococcal A surface protein gene (PspA) from S, and several studies show that PspA is a very promising target for new vaccine formulations. Therefore, the aim of this study was to isolate and clone the Streptococcus pneumoniae surface protein (psp) gene in Escherichia coli DH5α in order to provide a vaccine for children.
Methods: Sampling of a total of 60 pneumococcal isolates was isolated from nasopharyngeal samples of healthy children under 6 years of age. In order to isolate and initially identify Streptococcus pneumoniae, culture medium and biochemical tests were used and DNA extraction was performed. Then, strains with pspC gene were identified by PCR. The pspC gene of the positive strains was transfected into Escherichia coli host bacteria by vector and cloned by TA cloning technique. Finally, the expression of genes in Escherichia coli DH5α was measured by Real time PCR technique. ClustalX and Mega5 software were used to draw the phylogenetic tree.
Results: A total of 12 pneumococcal isolates were isolated from the screening of clinical specimens sent to the laboratory, which were identified based on morphological, microscopic and biochemical characteristics. Of these, 1 isolate had pspC gene. After cloning, pspC genes were isolated from cloned selection colonies (blue / white). In order to confirm the results of DNA cloning, it was extracted from suspected colonies and PCR by real time test. Finally, sequencing images, m13 and proliferation curves, and confirmed gene expression in Escherichia coli DH5α.
Conclusion: In this study, by genomic evaluation of pneumococci isolated from children under 6 years of age, pspC gene with immunization potential was extracted from this bacterium in order to make a vaccine. And cloning was successful. Finally, by examining the phylogenetic tree drawn in this study, the degree of similarity and kinship of Streptococcus pneumoniae with other species was shown. |
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| Keywords: Streptococcus pneumoniae, PspC, Clonin |
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Full-Text [PDF 1638 kb]
(752 Downloads)
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Type of Study: Research |
Subject:
Special
Received: 2021/12/14 | Accepted: 2021/12/01 | Published: 2021/12/01
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