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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.