- Email: [email protected]
Cloning, expression and purification of the recombinant NS3, NT-gp96 and CT-gp96 proteins
Abstracts
Conclusion: Art v1 has a tendency towards binding to HLA-DRB1*01, HLA-DRB1*04 or HLA-DRB1*13. Individuals with HLA-DRB1*01 or HLADRB1*13 might be more susceptible to allergy to A. vulgaris, genetically. Keywords: Artemisia vulgaris, Allergic rhinitis, HLA-DRB1 doi:10.1016/j.clinbiochem.2011.08.670
E.Poster – [A-10-178-1] Cloning, expression and purification of the recombinant NS3, NT-gp96 and CT-gp96 proteins Mohammadreza Hajizadeha, Pooneh Mokaramb, Zohreh Mostafavi-Pourb, Sima Rafatic, Azam Boahassanic a Recombinant Protein Laboratory in Biochemistry Department, Medical School, Shiraz University of Medical Science, Iran b Medical Science c Molecular Immunology and Vaccine Research Laboratory, Pasteur Institute of Iran, Tehran, Iran E-mail addresses: [email protected] (M. Hajizadeh), [email protected] (P. Mokaram), [email protected] (Z. Mostafavi-Pour), [email protected] (S. Rafati), [email protected] (A. Boahassani) Introduction: Hepatitis C virus (HCV) belongs to the Flaviviridae family that is a major risk factor in the development of hepatocellular carcinoma (HCC). HCV encodes several structural and non-structural proteins that interfere with the host cell antiviral response. Non-structural protein 3 (NS3) has two distinct activities, protease and helicase. NS3 gene is highly conserved and is an important target for antiviral therapy within different HCV genotypes. GP96 acts as a chaperone and immunization with gp96 induced potent CTL responses to peptides of viral antigens. Methods and results: The coding sequence of NS3 (195 amino acids attached to the N-terminal region of NS3) was designed. Then this construct was cloned into pCR2.1 vector by MWG Company. For generation of pQE30 NS3 we sub-cloned the mentioned sequence in expression vector pQE30. The DNA encoding the N-terminal (64–1077) and C-terminal fragments (1645–2552) of Gp96, were cloned into expression vector pQE30 in Pasteur Institute of Iran. The ligation mixture of each gene was used to transform E. coli M15 strain. Protein expressions were induced individually using 1 mM IPTG. Recombinant proteins were purified by affinity chromatography on Ni-NTA resin using 6XHis-tag. Protein samples were analyzed by SDS-PAGE in a gel containing 12% (w/v) polyacrylamide and western blot analysis was done. Conclusion: The capacity of gp96 to bind a broad array of peptides provides the conceptual framework for the vaccination strategy of combining NS3 with gp96. Keywords: HCV, NS3, GP96 doi:10.1016/j.clinbiochem.2011.08.671
Poster – [A-10-187-1] Assessment of Th17 and Treg cytokines and molecules in glomerolunephritise class IV in SLE patients Samaneh Soltania, Mahmoud Mahmoudib, Maryam Rastinb, Maryam Sahebaric, Zahra Mirfeizid, Fatemeh Nazemiand, Nafiseh Tabasib, Shahrzad Zamanib, Mitra Masoudianb, Darush Haghmoradb, Abbasali Aminib a Buali Research Institute, Buali Square, Mashhad, Iran b Buali Research Institute, Buali Square, Iran c Ghaem Hospital, Iran d Emam Rexa Hospital, Iran
S273
E-mail addresses: [email protected] (S. Soltani), [email protected] (M. Mahmoudi), [email protected] (M. Rastin), [email protected] (M. Sahebari), [email protected] (Z. Mirfeizi), [email protected] (F. Nazemian), [email protected] (N. Tabasi), [email protected] (S. Zamani), [email protected] (M. Masoudian), [email protected] (D. Haghmorad), [email protected] (A. Amini) Introduction: Systemic lupus erythematosus is an auto immune disease with kidney involvement and unknown etiology. Alteration in immune responses have an important role in the pathogenesis of disease. Inflammatory conditions and cytokines affect lymphocytes sub groups. In the presence of IL6‚ IL17 and TGF-B ‚naive T lymphocytes may convert to TH17 cells‚ which play an important role in the pathogenesis of glomerulonephritise. TGF-B in the absence of IL6 converts T cells to T regulatory cells which have immunomodulatory effects. The study group was comprised of 20 glomerulonephritise class IV patients and 20 sex and age matched non nephritises SLE patients. 10 cc bloods were collected from each patients .Lymphocytes isolated using ficoll and RNA extracted by Trizol‚ then c DNA was synthesized using MMULV‚ then with specific primer and probes the expression levels of FOXP3‚ TGF-B‚ IFNγ‚ IL6‚ IL17 genes by Real time PCR ‚Taqman method were analyzed. Our results showed that the expression levels of IL6 ‚IL17 IFNγ‚ TGF-B genes in SLE patients with glomerulonephritise class IV were higher than non nephritis patients, but FOXP3 was decreased in the nephritic SLE patients more than nonnephritic patients. In conclusion in this study we found that cytokines related to TH17 cells increase, and Treg related genes decrease in SLE patients with kidney injuries. Keywords: Glomerolunephritise, Cytokines, TH17, Treg, Lupus doi:10.1016/j.clinbiochem.2011.08.672
Oral – [A-10-199-1] The response of macrophage cells to sense and antisense double-stranded RNA Mohajeri Nasrina,b, Bandehpour Mojganb, Mosaffa Narimanc, Kochaki Amenehb, Yarian Fatemehb a Department of Cellular & Molecular Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran b Department of Cellular and Molecular Biology Research Center, Iran c Department of Immunology Research Center, Shahid Beheshti University of Medical Science, Tehran, Iran E-mail address: [email protected] (M. Nasrin) Introduction: Silencing of gene expression by siRNA (small interfering RNA) is a powerful approach to study the genetic analysis and functional roles of mammalian genes. The stimulation of innate immunity by these sequences depends on sequence, structure of siRNA and method of cell type delivery. Methods: After cloning of sense and antisense stranded as mammalian two hybrid system plasmids, vectors were transfected into human blood macrophage on the basis of 6 experimental and control. Groups: LPS/antisense, LPS/sense, LPS/dsRNA and antisense, sense, dsRNA. After 24 h of transfection, cytokine response was assessed by ELISA. Results: Among the proinflammatory cytokine :IL-1β, IL-8, IL-12, TNF-α and IFN-γ, only IL-8 was elevated in LPS/sense with significant difference comparable by sense and dsRNA.
Conclusion: Art v1 has a tendency towards binding to HLA-DRB1*01, HLA-DRB1*04 or HLA-DRB1*13. Individuals with HLA-DRB1*01 or HLADRB1*13 might be more susceptible to allergy to A. vulgaris, genetically. Keywords: Artemisia vulgaris, Allergic rhinitis, HLA-DRB1 doi:10.1016/j.clinbiochem.2011.08.670
E.Poster – [A-10-178-1] Cloning, expression and purification of the recombinant NS3, NT-gp96 and CT-gp96 proteins Mohammadreza Hajizadeha, Pooneh Mokaramb, Zohreh Mostafavi-Pourb, Sima Rafatic, Azam Boahassanic a Recombinant Protein Laboratory in Biochemistry Department, Medical School, Shiraz University of Medical Science, Iran b Medical Science c Molecular Immunology and Vaccine Research Laboratory, Pasteur Institute of Iran, Tehran, Iran E-mail addresses: [email protected] (M. Hajizadeh), [email protected] (P. Mokaram), [email protected] (Z. Mostafavi-Pour), [email protected] (S. Rafati), [email protected] (A. Boahassani) Introduction: Hepatitis C virus (HCV) belongs to the Flaviviridae family that is a major risk factor in the development of hepatocellular carcinoma (HCC). HCV encodes several structural and non-structural proteins that interfere with the host cell antiviral response. Non-structural protein 3 (NS3) has two distinct activities, protease and helicase. NS3 gene is highly conserved and is an important target for antiviral therapy within different HCV genotypes. GP96 acts as a chaperone and immunization with gp96 induced potent CTL responses to peptides of viral antigens. Methods and results: The coding sequence of NS3 (195 amino acids attached to the N-terminal region of NS3) was designed. Then this construct was cloned into pCR2.1 vector by MWG Company. For generation of pQE30 NS3 we sub-cloned the mentioned sequence in expression vector pQE30. The DNA encoding the N-terminal (64–1077) and C-terminal fragments (1645–2552) of Gp96, were cloned into expression vector pQE30 in Pasteur Institute of Iran. The ligation mixture of each gene was used to transform E. coli M15 strain. Protein expressions were induced individually using 1 mM IPTG. Recombinant proteins were purified by affinity chromatography on Ni-NTA resin using 6XHis-tag. Protein samples were analyzed by SDS-PAGE in a gel containing 12% (w/v) polyacrylamide and western blot analysis was done. Conclusion: The capacity of gp96 to bind a broad array of peptides provides the conceptual framework for the vaccination strategy of combining NS3 with gp96. Keywords: HCV, NS3, GP96 doi:10.1016/j.clinbiochem.2011.08.671
Poster – [A-10-187-1] Assessment of Th17 and Treg cytokines and molecules in glomerolunephritise class IV in SLE patients Samaneh Soltania, Mahmoud Mahmoudib, Maryam Rastinb, Maryam Sahebaric, Zahra Mirfeizid, Fatemeh Nazemiand, Nafiseh Tabasib, Shahrzad Zamanib, Mitra Masoudianb, Darush Haghmoradb, Abbasali Aminib a Buali Research Institute, Buali Square, Mashhad, Iran b Buali Research Institute, Buali Square, Iran c Ghaem Hospital, Iran d Emam Rexa Hospital, Iran
S273
E-mail addresses: [email protected] (S. Soltani), [email protected] (M. Mahmoudi), [email protected] (M. Rastin), [email protected] (M. Sahebari), [email protected] (Z. Mirfeizi), [email protected] (F. Nazemian), [email protected] (N. Tabasi), [email protected] (S. Zamani), [email protected] (M. Masoudian), [email protected] (D. Haghmorad), [email protected] (A. Amini) Introduction: Systemic lupus erythematosus is an auto immune disease with kidney involvement and unknown etiology. Alteration in immune responses have an important role in the pathogenesis of disease. Inflammatory conditions and cytokines affect lymphocytes sub groups. In the presence of IL6‚ IL17 and TGF-B ‚naive T lymphocytes may convert to TH17 cells‚ which play an important role in the pathogenesis of glomerulonephritise. TGF-B in the absence of IL6 converts T cells to T regulatory cells which have immunomodulatory effects. The study group was comprised of 20 glomerulonephritise class IV patients and 20 sex and age matched non nephritises SLE patients. 10 cc bloods were collected from each patients .Lymphocytes isolated using ficoll and RNA extracted by Trizol‚ then c DNA was synthesized using MMULV‚ then with specific primer and probes the expression levels of FOXP3‚ TGF-B‚ IFNγ‚ IL6‚ IL17 genes by Real time PCR ‚Taqman method were analyzed. Our results showed that the expression levels of IL6 ‚IL17 IFNγ‚ TGF-B genes in SLE patients with glomerulonephritise class IV were higher than non nephritis patients, but FOXP3 was decreased in the nephritic SLE patients more than nonnephritic patients. In conclusion in this study we found that cytokines related to TH17 cells increase, and Treg related genes decrease in SLE patients with kidney injuries. Keywords: Glomerolunephritise, Cytokines, TH17, Treg, Lupus doi:10.1016/j.clinbiochem.2011.08.672
Oral – [A-10-199-1] The response of macrophage cells to sense and antisense double-stranded RNA Mohajeri Nasrina,b, Bandehpour Mojganb, Mosaffa Narimanc, Kochaki Amenehb, Yarian Fatemehb a Department of Cellular & Molecular Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran b Department of Cellular and Molecular Biology Research Center, Iran c Department of Immunology Research Center, Shahid Beheshti University of Medical Science, Tehran, Iran E-mail address: [email protected] (M. Nasrin) Introduction: Silencing of gene expression by siRNA (small interfering RNA) is a powerful approach to study the genetic analysis and functional roles of mammalian genes. The stimulation of innate immunity by these sequences depends on sequence, structure of siRNA and method of cell type delivery. Methods: After cloning of sense and antisense stranded as mammalian two hybrid system plasmids, vectors were transfected into human blood macrophage on the basis of 6 experimental and control. Groups: LPS/antisense, LPS/sense, LPS/dsRNA and antisense, sense, dsRNA. After 24 h of transfection, cytokine response was assessed by ELISA. Results: Among the proinflammatory cytokine :IL-1β, IL-8, IL-12, TNF-α and IFN-γ, only IL-8 was elevated in LPS/sense with significant difference comparable by sense and dsRNA.