Maryam Sadat Nabavinia1, Mohammad Ramezani2, Aida Gholoobi3, Mahboubeh Naderinasab4, Zahra Meshkat4. 1. Department of Pharmaceutical Biotechnology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran. 2. Nanotechnology Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran. 3. Department of Modern Sciences and Technologies, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran. 4. Antimicrobial Resistance Research Center, Mashhad University of Medical Sciences, Mashhad, Iran.
Abstract
BACKGROUND: With one-third of the world's population infected, tuberculosis (TB) is one of the most common infectious diseases and a major public health problem, especially in developing countries. The efficacy of the BCG vaccine for controlling the disease in adults is poor. The development of an effective TB vaccine is a global objective. An effective tuberculosis vaccine should stimulate cellular immunity. DNA vaccines are a new generation of vaccines with the potential to achieve this goal. The aim of this study was to produce a DNA vaccine of Mtb72F. METHODS: mtb32C, mtb39, and mtb32N were cloned into pcDNA3.1 using restriction enzyme digestion and T4 DNA ligase. Colony-PCR and restriction enzyme digestion were performed to detect transformed bacteria. DNA sequencing confirmed the desired gene insertion into the vector. A Chinese hamster ovary (CHO) cell line was transfected with the recombinant plasmid and RT-PCR was performed to assess gene expression. RESULTS: Gel electrophoresis showed the expected amplified gene fragments of 429, 614, and 1200 base pairs (bps) for mtb32C, mtb32N, and mtb39, respectively. Enzyme digestion and gel electrophoresis showed the expected fragments, indicating the desired gene position and orientation in the recombinant plasmid. This finding was verified by DNA sequencing, and RT-PCR demonstrated gene expression in the CHO cell line. CONCLUSION: An Mtb72F DNA plasmid was successfully constructed. This plasmid may be a candidate for animal immunizations.
BACKGROUND: With one-third of the world's population infected, tuberculosis (TB) is one of the most common infectious diseases and a major public health problem, especially in developing countries. The efficacy of the BCG vaccine for controlling the disease in adults is poor. The development of an effective TB vaccine is a global objective. An effective tuberculosis vaccine should stimulate cellular immunity. DNA vaccines are a new generation of vaccines with the potential to achieve this goal. The aim of this study was to produce a DNA vaccine of Mtb72F. METHODS: mtb32C, mtb39, and mtb32N were cloned into pcDNA3.1 using restriction enzyme digestion and T4 DNA ligase. Colony-PCR and restriction enzyme digestion were performed to detect transformed bacteria. DNA sequencing confirmed the desired gene insertion into the vector. A Chinese hamster ovary (CHO) cell line was transfected with the recombinant plasmid and RT-PCR was performed to assess gene expression. RESULTS: Gel electrophoresis showed the expected amplified gene fragments of 429, 614, and 1200 base pairs (bps) for mtb32C, mtb32N, and mtb39, respectively. Enzyme digestion and gel electrophoresis showed the expected fragments, indicating the desired gene position and orientation in the recombinant plasmid. This finding was verified by DNA sequencing, and RT-PCR demonstrated gene expression in the CHO cell line. CONCLUSION: An Mtb72F DNA plasmid was successfully constructed. This plasmid may be a candidate for animal immunizations.
Entities:
Keywords:
Cloning; DNA vaccine; Mycobacterium tuberculosis; mtb32C; mtb32N; mtb39
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