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Construction of the Chimeric HSP70 - E7 Vector and Evaluation of its Protein Production
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H Razavi-Nikoo   , H Soleimanjahi * , F Fotouhi , A Ghaemi , M Fazeli |
| Department of Virology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran |
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Abstract: (5726 Views) |
| Background and Aims: Since the produced recombinant proteins by molecular genetics techniques commonly have some limitations in the application, chimeric protein are introduced. Chimeric proteins have found widespread application for the study of protein folding, structure stability, function and immunogenisity. Methods: According to the known Immunomodulatory effect and structure of HSP70 molecule, full length human HSP70 was selected and a hybrid was made with HPV-E7gene.The recombinant plasmid pCDNA3.1/ E7-HSP70 was constructed using sequential PCR and cloning steps. The entire upstream and downstream sequences of the target molecules were synthesized separately. The sequential cloning was performed for cloning of the entire sequences of the target molecule of HPV16-E7 fragment in pcDNA3.1. Target DNA was visualized by staining with ethidium bromide. The Cos-7 cell lines were transfected with fusion proteins in 6 well microtiter plates. After 48 hours of transfection, the target cells were removed and to SDS-PAGE analysis for mRNA detection. Immunoreactivity of the protein product was assayed by Western blotting using monoclonal antibody. Results: The sequencing analysis showed that E7 gene was fused in frame to the HSP70 gene in pcDNA3.1/E7-HSP70. Western blot analysis of recombinant fusion protein using HSP 70 monoclonal antibody showed desired band as expected. The chimeric structure was expressed in cells, as expected. The resulting E7- HSP70 fusion gene would be a useful construct for future research. Conclusion: In order to change and enhance of the tropism and immunogenicity of recombinant protein, chimeric E7- HSP70 Vector was constructed. Since monovalent molecule and vaccines were clinically ineffective or poorly immunogenic, so applications of covalently linked product are introduced. The successful expression of the E7- Hsp70 fusion protein can be used as a molecular target for establishment of DNA and recombinant protein vaccine in future research. |
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| Keywords: pcDNA3.1, HSP70, chimeric vector |
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Full-Text [PDF 315 kb]
(1225 Downloads)
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Type of Study: Original article |
Subject:
General
Received: 2014/10/12 | Accepted: 2014/10/12 | Published: 2014/10/12
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