Interleukin 21 （IL-21） is a novel type I cytokine that is significantly homologous to IL-2, IL-4 and IL-15. Its receptor complex contains γc chain which is also a component of receptors for IL-2, IL-4, IL-7, IL-9 a...Interleukin 21 （IL-21） is a novel type I cytokine that is significantly homologous to IL-2, IL-4 and IL-15. Its receptor complex contains γc chain which is also a component of receptors for IL-2, IL-4, IL-7, IL-9 and IL-15, so there may be overlapping or relevancies in their biological functions. IL-21 is capable of co-stimulating mature T cells, B cells, NK cells, and of stimulating CD16 expression on the surface of NK cells to induce ADCC in innate immune response. It can also strengthen the anti-tumor effect of the cellular immunity, especially v/a enhancing the activities of NK and antigen specific CTL cells. Thus, IL-21 is a potential useful therapeutic molecule for immunotherapy of malignancies, by eliciting innate and adaptive anti-tumor immune responses in tumor-bearing hosts. In order to study the biological functions of IL-21, we constructed a mIL-21 prokaryotic expression plasmid and expressed the recombinant mIL-21 protein in E. coli in present study. The recombinant plasmid pET28a/mIL-21 with a carboxyl terminal His-tag was subcloned from the pcDNA3.1/mIL-21 and expressed in E. coli. The induced protein was detected by SDS-PAGE, and identified by Western-blot assay with anti-mIL-21 antibody. The recombinant protein was purified v/a Ni^＋ affinity chromatography, and renatured with GSH/GSSG system. Our mouse T cell proliferation experiment showed that the recombinant mIL-21 protein could enhance the mouse T cell proliferation either by itself alone or in the presence of Con A.展开更多
INTRODUCTIONHelicobacter pylori (H . pylori) is associated with the development of chronic gastritis ,peptic ulcer and gastric cancer and gastric MALT lymphoma[1-9],H .pylori has many antigens ,including urease ,heat ...INTRODUCTIONHelicobacter pylori (H . pylori) is associated with the development of chronic gastritis ,peptic ulcer and gastric cancer and gastric MALT lymphoma[1-9],H .pylori has many antigens ,including urease ,heat shock protein and vacuolating cytotoxin and so on ,and urease is an important factor in the colinization of the gastric mucosa and suspected to cause damage to the gastric mucosa[10-14].At the same time ,urdase is also one of the important protective antigens .展开更多
To construct a recombinant plasmid Pet23a-M, the gene encoding severe acute respiratory syndrome (SARS) coronavirus membrane protein was amplified by RT-PCR and cloned into the expression plasmid Pet23a. Results of re...To construct a recombinant plasmid Pet23a-M, the gene encoding severe acute respiratory syndrome (SARS) coronavirus membrane protein was amplified by RT-PCR and cloned into the expression plasmid Pet23a. Results of restriction endonuclease analysis, PCR detection and DNA sequencing analysis revealed that the cloned DNA sequence was the same as that reported. The recombinants were transformed into Escherichia coli (E.Coli) BL21 (DE3) and induced by Isopropyl-β-D-thiogalactopyranoside (IPTG). The expression of 27 kD (1 kD=0.9921 ku) protein was detected by SDS-PAGE and pured by metal chelated chromatography. Results of Western-blot showed that this expressed protein could react with antibodies in sera of SARS patients during convalescence. This provided the basis for the further study on SARS virus vaccine and diagnostic agents.展开更多
Objective: To express nitroreductase gene NOR1 in Escherichia coli and to purify the expressed protein in order to get the polyclonal antibody of NOR1. Methods: The full length of NOR1 gene was amplified by reverse tr...Objective: To express nitroreductase gene NOR1 in Escherichia coli and to purify the expressed protein in order to get the polyclonal antibody of NOR1. Methods: The full length of NOR1 gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and digested with BamHI and XhoI restriction endonucleases. The plasmid pGEX-4T-2 was also digested with BamHI and XhoI, then the NOR1 gene was inserted into vector pGEX-4T-2. The recombinant expression vector pGEX-4T-2/NOR1 was identified by sequencing and restriction enzymes digestion. E.coli Jm105 transformed with the recombinant plasmid was induced by IPTG to express the GST fusion protein. The purified targeted protein obtained by affinity chromatography was used to immunize New Zealand rabbits to acquire antiserum. Antiserum was analyzed with immunoblot. Results: The 1.25 kb NOR1 gene was successfully isolated. After induction, a new anticipated protein of 74 kDa appeared on sodium dodecylsulfate polyacrylamide (SDS-PAGE). The result was confirmed by Western blot analysis, and the purified targeted protein was obtained by affinity chromatography. The titer of antiserum was 1:8. Conclusion: A high level of expression of GST-NOR1 is obtained in JM 105, and its antiserum can be prepared successfully.展开更多
Trail, a tumor necrosis factor-related apoptosis-inducing ligand, is a novel potent endogenous activator of the cell death pathway through the activation of cell surface death receptors Trail-R1 and Trail-R2. Its role...Trail, a tumor necrosis factor-related apoptosis-inducing ligand, is a novel potent endogenous activator of the cell death pathway through the activation of cell surface death receptors Trail-R1 and Trail-R2. Its role, like FasL in activation-induced cell death (AICD), has been demonstrated in immune system. However the mechanism of Trail induced apoptosis remains nuclear. In this report, the recombinant Trail protein was expressed and purified. The apoptosis-inducing activity and the regulation mechanism of recombinant Trail on Jurkat T cells were explored in vitro. Trypan blue exclusion assay demonstrated that the recombinant Trail protein actively killed Jurkat T cells in a dose-dependent manner. Trail-induced apoptosis in Jurkat T cells were remarkably reduced by Bcl-2 over expression in BcL-2 gene transfected cells. Treatment with PMA (phorbol 12-myristate 13-acetate), a PKC activator, suppressed nail-induced apoptosis in Jurkat T cells. The inhibition of apoptosis by PMA was abolished by pretreatment with Bis, a PKC inhibitor. Taken together, it was suggested that Bcl-2 over-expression and PMA activated PKC actively down-regulated the Trail-mediated apoptosis in Jurkat T cell.展开更多
AIM To study the epitope distribution of hepatitis G virus (HGV) and to seek for the potential recombinant antigens for the development of HGV diagnositic reagents.METHODS Fourteen clones encompassing HGV gene fragmen...AIM To study the epitope distribution of hepatitis G virus (HGV) and to seek for the potential recombinant antigens for the development of HGV diagnositic reagents.METHODS Fourteen clones encompassing HGV gene fragments from core to NS3 and NS5 were constructed using prokaryotic expression vector pRSET and (or)pGEX. and expressed in E. coli. Western blotting and ELISA were used to detect the immunoreactivity of these recombinant proteins.``RESULTS One clone with HGV fragment from core to El(Gl). one from E2 (G31), three from NS3 (G6, G61, G7),one from NS5B (G821) and one chimeric fragment from NS3and NS5B (G61 821) could be expressed well and showed obvious immunoreactivity by Western blotting.One clone with I-KGV framment from NS5B (G82) was also well expressed, but could not show immunoreactivity by Western blotting. No obvious expression was found in the other six clones. All the expressed recombinant proteins were in inclusion body form, except the protein G61 which could be expressed in soluble form. Further purified recombinant proteins Gl, G,31, G61, G821 and G61 821were detected in indirected ELISA as coating antigen respectively. Only recombinant Gl could still show immunoreactivity, and the other four recombinant proteins failed to react to the HGV antibody positive sera.Western blotting results indicated that the immunoactivity of these four recombinant proteins were lost during purification.``CONCLUSION Core to El, E2. NS3 and NS5 fragment of HGV contain antigenic epitopes, which could be produced in prokaryotically expressed recombinant proteins. A high. yield recombinant protein (Gl) located in HGV core to E1 could remain its epitope after purification, which showed the potential that G1 could be used as a coating antigen to develop an ELISA kit for HGV specific antibody diagnosis.展开更多
目的构建结核分枝杆菌培养滤液蛋白21(Culture filtrate protein 21,CFP21)原核表达载体,获得CFP21蛋白。方法以结核分枝杆菌H37Rv株基因组DNA为模板,PCR扩增cfp21基因,质粒PET28a(+)为表达载体,构建PET28a(+)/cfp21质粒,转化入大肠埃希...目的构建结核分枝杆菌培养滤液蛋白21(Culture filtrate protein 21,CFP21)原核表达载体,获得CFP21蛋白。方法以结核分枝杆菌H37Rv株基因组DNA为模板,PCR扩增cfp21基因,质粒PET28a(+)为表达载体,构建PET28a(+)/cfp21质粒,转化入大肠埃希菌DH5α中,抽提质粒,PCR扩增,测序,转化到大肠埃希菌BL21中,用IPTG诱导表达,SDS-PAGE分析,并用His Bind蛋白纯化试剂盒纯化CFP21。结果构建、表达和纯化了CFP21,分子量约为24kD,主要以包涵体形式存在。结论目的基因克隆入宿主菌中并表达成功,纯化得到CFP21蛋白,为CFP21的进一步研究奠定基础。展开更多
文摘Interleukin 21 （IL-21） is a novel type I cytokine that is significantly homologous to IL-2, IL-4 and IL-15. Its receptor complex contains γc chain which is also a component of receptors for IL-2, IL-4, IL-7, IL-9 and IL-15, so there may be overlapping or relevancies in their biological functions. IL-21 is capable of co-stimulating mature T cells, B cells, NK cells, and of stimulating CD16 expression on the surface of NK cells to induce ADCC in innate immune response. It can also strengthen the anti-tumor effect of the cellular immunity, especially v/a enhancing the activities of NK and antigen specific CTL cells. Thus, IL-21 is a potential useful therapeutic molecule for immunotherapy of malignancies, by eliciting innate and adaptive anti-tumor immune responses in tumor-bearing hosts. In order to study the biological functions of IL-21, we constructed a mIL-21 prokaryotic expression plasmid and expressed the recombinant mIL-21 protein in E. coli in present study. The recombinant plasmid pET28a/mIL-21 with a carboxyl terminal His-tag was subcloned from the pcDNA3.1/mIL-21 and expressed in E. coli. The induced protein was detected by SDS-PAGE, and identified by Western-blot assay with anti-mIL-21 antibody. The recombinant protein was purified v/a Ni^＋ affinity chromatography, and renatured with GSH/GSSG system. Our mouse T cell proliferation experiment showed that the recombinant mIL-21 protein could enhance the mouse T cell proliferation either by itself alone or in the presence of Con A.
基金Supported by the National Major Science and Technology Projects,No.96-901-01-54.
文摘INTRODUCTIONHelicobacter pylori (H . pylori) is associated with the development of chronic gastritis ,peptic ulcer and gastric cancer and gastric MALT lymphoma[1-9],H .pylori has many antigens ,including urease ,heat shock protein and vacuolating cytotoxin and so on ,and urease is an important factor in the colinization of the gastric mucosa and suspected to cause damage to the gastric mucosa[10-14].At the same time ,urdase is also one of the important protective antigens .
文摘To construct a recombinant plasmid Pet23a-M, the gene encoding severe acute respiratory syndrome (SARS) coronavirus membrane protein was amplified by RT-PCR and cloned into the expression plasmid Pet23a. Results of restriction endonuclease analysis, PCR detection and DNA sequencing analysis revealed that the cloned DNA sequence was the same as that reported. The recombinants were transformed into Escherichia coli (E.Coli) BL21 (DE3) and induced by Isopropyl-β-D-thiogalactopyranoside (IPTG). The expression of 27 kD (1 kD=0.9921 ku) protein was detected by SDS-PAGE and pured by metal chelated chromatography. Results of Western-blot showed that this expressed protein could react with antibodies in sera of SARS patients during convalescence. This provided the basis for the further study on SARS virus vaccine and diagnostic agents.
文摘Objective: To express nitroreductase gene NOR1 in Escherichia coli and to purify the expressed protein in order to get the polyclonal antibody of NOR1. Methods: The full length of NOR1 gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and digested with BamHI and XhoI restriction endonucleases. The plasmid pGEX-4T-2 was also digested with BamHI and XhoI, then the NOR1 gene was inserted into vector pGEX-4T-2. The recombinant expression vector pGEX-4T-2/NOR1 was identified by sequencing and restriction enzymes digestion. E.coli Jm105 transformed with the recombinant plasmid was induced by IPTG to express the GST fusion protein. The purified targeted protein obtained by affinity chromatography was used to immunize New Zealand rabbits to acquire antiserum. Antiserum was analyzed with immunoblot. Results: The 1.25 kb NOR1 gene was successfully isolated. After induction, a new anticipated protein of 74 kDa appeared on sodium dodecylsulfate polyacrylamide (SDS-PAGE). The result was confirmed by Western blot analysis, and the purified targeted protein was obtained by affinity chromatography. The titer of antiserum was 1:8. Conclusion: A high level of expression of GST-NOR1 is obtained in JM 105, and its antiserum can be prepared successfully.
基金Major State BasicResearch (973) Program of China, (G1999053905).
文摘Trail, a tumor necrosis factor-related apoptosis-inducing ligand, is a novel potent endogenous activator of the cell death pathway through the activation of cell surface death receptors Trail-R1 and Trail-R2. Its role, like FasL in activation-induced cell death (AICD), has been demonstrated in immune system. However the mechanism of Trail induced apoptosis remains nuclear. In this report, the recombinant Trail protein was expressed and purified. The apoptosis-inducing activity and the regulation mechanism of recombinant Trail on Jurkat T cells were explored in vitro. Trypan blue exclusion assay demonstrated that the recombinant Trail protein actively killed Jurkat T cells in a dose-dependent manner. Trail-induced apoptosis in Jurkat T cells were remarkably reduced by Bcl-2 over expression in BcL-2 gene transfected cells. Treatment with PMA (phorbol 12-myristate 13-acetate), a PKC activator, suppressed nail-induced apoptosis in Jurkat T cells. The inhibition of apoptosis by PMA was abolished by pretreatment with Bis, a PKC inhibitor. Taken together, it was suggested that Bcl-2 over-expression and PMA activated PKC actively down-regulated the Trail-mediated apoptosis in Jurkat T cell.
基金Supported by National 863 Project,No.102-07-02-079th Five-Year Sci-Tech Plan,No.96-906A-03-08
文摘AIM To study the epitope distribution of hepatitis G virus (HGV) and to seek for the potential recombinant antigens for the development of HGV diagnositic reagents.METHODS Fourteen clones encompassing HGV gene fragments from core to NS3 and NS5 were constructed using prokaryotic expression vector pRSET and (or)pGEX. and expressed in E. coli. Western blotting and ELISA were used to detect the immunoreactivity of these recombinant proteins.``RESULTS One clone with HGV fragment from core to El(Gl). one from E2 (G31), three from NS3 (G6, G61, G7),one from NS5B (G821) and one chimeric fragment from NS3and NS5B (G61 821) could be expressed well and showed obvious immunoreactivity by Western blotting.One clone with I-KGV framment from NS5B (G82) was also well expressed, but could not show immunoreactivity by Western blotting. No obvious expression was found in the other six clones. All the expressed recombinant proteins were in inclusion body form, except the protein G61 which could be expressed in soluble form. Further purified recombinant proteins Gl, G,31, G61, G821 and G61 821were detected in indirected ELISA as coating antigen respectively. Only recombinant Gl could still show immunoreactivity, and the other four recombinant proteins failed to react to the HGV antibody positive sera.Western blotting results indicated that the immunoactivity of these four recombinant proteins were lost during purification.``CONCLUSION Core to El, E2. NS3 and NS5 fragment of HGV contain antigenic epitopes, which could be produced in prokaryotically expressed recombinant proteins. A high. yield recombinant protein (Gl) located in HGV core to E1 could remain its epitope after purification, which showed the potential that G1 could be used as a coating antigen to develop an ELISA kit for HGV specific antibody diagnosis.
文摘目的构建结核分枝杆菌培养滤液蛋白21(Culture filtrate protein 21,CFP21)原核表达载体,获得CFP21蛋白。方法以结核分枝杆菌H37Rv株基因组DNA为模板,PCR扩增cfp21基因,质粒PET28a(+)为表达载体,构建PET28a(+)/cfp21质粒,转化入大肠埃希菌DH5α中,抽提质粒,PCR扩增,测序,转化到大肠埃希菌BL21中,用IPTG诱导表达,SDS-PAGE分析,并用His Bind蛋白纯化试剂盒纯化CFP21。结果构建、表达和纯化了CFP21,分子量约为24kD,主要以包涵体形式存在。结论目的基因克隆入宿主菌中并表达成功,纯化得到CFP21蛋白,为CFP21的进一步研究奠定基础。
