Saghar Zonnar1, Said Abdul Ghafour Saeedy2, Fahimeh Nemati3, Mohammad Javad Motamedi4, Hamidreza Raeespour5, Jafar Amani6. 1. Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran. 2. Department of Paraclinic, School of Medicine, Herat University, Herat, Afghanistan. 3. Department of Biotechnology, Faculty of Advanced Science and Technology, Tehran Medical Sciences, Islamic Azad University (IAUPS), Tehran, Iran. 4. Department of Plant Bioproducts, Institute of Agricultural Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran. 5. Department of Genetics, Islamic Azad University, Varamin Pishva Branch, Tehran, Iran. 6. Applied Microbiology Research Center, Systems Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran.
Abstract
Objectives: Atherosclerosis is the main cause of cardiovascular disease (CVD) which has a key role in the development of coronary artery disease (CAD). Based on clinical studies, HSP60 is the only HSP that can cause atherosclerosis. In this paper, the expression level of HSP60 and the pathogenic degree of its cloned part was investigated in atherosclerosis condition. Materials and Methods: After the designation of the specific primers for HSP60, PCR was done by the Pfu enzyme. Subsequently, the PCR products were cloned into a prokaryotic expression vector pET-28a. The resultant recombinant vector was transferred in BL21 and purified. Purification of protein was done by the Nickel affinity column. After confirmation of Western blotting and HSP60 protein purification, purified protein concentration was measured by the Bradford method, and purity was analyzed by SDS PAGE 12%. New Zealand rabbits were tested as an animal model. At the next step, the recombinant protein was injected into the animal model that was on a fatty diet. Results: The prokaryotic expression plasmid pET28a-hps60 was successfully constructed, the HSP60 protein was expressed and purified in Escherichia coli BL21 (DE3). We found that the rabbit that was receiving the recombinant vaccine with the fatty diet showed a lower amount of fat deposition at the media endothelial level than the rabbit which received only the fatty diet. Conclusion: Taking recombinant protein concomitant with a fatty diet, causes betterment of atherosclerosis via decreasing aggregation of cholesterol and thickness of the endothelial media.
Objectives: Atherosclerosis is the main cause of cardiovascular disease (CVD) which has a key role in the development of coronary artery disease (CAD). Based on clinical studies, HSP60 is the only HSP that can cause atherosclerosis. In this paper, the expression level of HSP60 and the pathogenic degree of its cloned part was investigated in atherosclerosis condition. Materials and Methods: After the designation of the specific primers for HSP60, PCR was done by the Pfu enzyme. Subsequently, the PCR products were cloned into a prokaryotic expression vector pET-28a. The resultant recombinant vector was transferred in BL21 and purified. Purification of protein was done by the Nickel affinity column. After confirmation of Western blotting and HSP60 protein purification, purified protein concentration was measured by the Bradford method, and purity was analyzed by SDS PAGE 12%. New Zealand rabbits were tested as an animal model. At the next step, the recombinant protein was injected into the animal model that was on a fatty diet. Results: The prokaryotic expression plasmid pET28a-hps60 was successfully constructed, the HSP60 protein was expressed and purified in Escherichia coli BL21 (DE3). We found that the rabbit that was receiving the recombinant vaccine with the fatty diet showed a lower amount of fat deposition at the media endothelial level than the rabbit which received only the fatty diet. Conclusion: Taking recombinant protein concomitant with a fatty diet, causes betterment of atherosclerosis via decreasing aggregation of cholesterol and thickness of the endothelial media.
Authors: G H M van Puijvelde; T van Es; E J A van Wanrooij; K L L Habets; P de Vos; R van der Zee; W van Eden; Th J C van Berkel; J Kuiper Journal: Arterioscler Thromb Vasc Biol Date: 2007-09-27 Impact factor: 8.311