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Abstract Background and Objective: Increased antibiotic resistant strains and inadequacy of current vaccines against pneumococcal infections necessitate the study of novel protein antigens. It seems that minor autolysin of Streptococcus pneumoniae may have antigenicity. Thus, we aimed at cloning its gene for the first time. Material and Methods: After DNA extraction of Streptococcus pneumoniae (ATCC 49619), Specific primers were designed for amplifying minor autolysin gene fragment, using PCR. The purified gene fragment was inserted into pET21a vector and was transformed into bacterial competent cells by heat shock technique. The presence of gene and absence of mutation in the recombinant vector were checked out with sequencing and enzymatic digestion methods. The gene sequence was finally analyzed by bioinformatic tools. Results: The gene of minor autolysin was cloned successfully and the result of enzymatic digestion was the indication of complete isolation of this gen from plasmid. . Bioinformatics studies revealed that the mature protein was lacking signal peptide and the gene encoded 318 amino acids with a molecular weight of 36.4 kDa. Conclusion: The presentation and characterization of novel antigens such as minor autolysin could help us with finding new approaches for preventing and controlling pneumococcal infection. Keywords: Streptococcus Pneumoniae, Minor Autolysin, Cloning

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Background and objectives: HIV-1 Nef and Vpr antigens have been described as suitable candidates for therapeutic HIV vaccine development. The aim of this study was to generate Nef-Vpr fusion gene construct and to clone the construct into pET-23a (+), a prokaryotic expression vector.
Methods: HIV-1 Nef and Vpr genes were PCR-amplified from the pNL4-3 plasmid using specific primers and Pfu DNA polymerase. Results of PCR amplification were visualized by electrophoresis on 0.8% agarose gel. At first, the amplified Nef fragment was cloned into NheI and BamHI restriction sites of pET-23a expression vector. Next, cloning of Vpr gene was performed into BamHI and HindIII restriction sites of the pET-23a-Nef vector. Finally, purity of the recombinant pET-23-Nef-Vpr construct was determined by NanoDrop spectrophotometry.
Results: PCR amplification of Nef and Vpr genes was confirmed by detection of ~ 620 bp and ~ 291 bp bands, respectively. Cloning of the Nef-Vpr construct into the vector was confirmed by detection of a ~ 911 bp fragment following enzymatic digestion with NheI and HindIII and sequencing.
Conclusion: The successful construction of recombinant fusion plasmid encoding a chimeric Nef-Vpr gene was performed in a prokaryotic expression vector for development of HIV-1 recombinant protein vaccine in near future.

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Background: Recently, a rare heterozygous DYRK1B mutation (R102C) has been shown to be completely associated with a familial form of metabolic syndrome. The present study was done to create the R102C mutation in the mouse DYRK1B gene, produce recombinant lentiviruses and infect target cells with these viruses as a strategy for long-term gene expression.
Materials & methods: The DYRK1B mutation (R102C) gene created by Overlap Extension-PCR (OE-PCR) was cloned into the LeGO-iG2 transfer vector containing the GFP marker. To produce recombinant lentiviruses, the transfer vector carrying DYRK1B R102C and psPAX2 (packaging vector), and pMD2 (envelope vector) were simultaneously transferred into HEK-293T cells.
Results: OE-PCR method and sequencing showed that carried out the intended mutation was correct. Also, to observe the DYRK1B expression, which shows gene transfer success, a fluorescence microscope was used to check the GFP marker. Flow cytometry technique was used to determine the lentiviral titer. The infection efficiency of HEK-293T cells with lentivirus was (10⁸ TU/ml).
Conclusion: DYRK1B contributes to the development of metabolic syndrome, central obesity, early-onset coronary artery disease, hypertension, type 2 diabetes, and adipogenesis; thus, Therefore, curiosity about this gene can be effective in the development of therapeutic agents against these diseases. Lentiviruses containing DYRK1B R102C mutation have many benefits for in-vitro and in-vivo metabolic syndrome research. This study shows the successful use of recombinant lentiviral vectors for transferring genes into eukaryotic cells.