To obtain the recombination protein of renalase and prepare the monoclonal antibody, the human renalase gene was amplified from human kidney by specific primer and cloned the DNA fragments into the pET22b. After verif...To obtain the recombination protein of renalase and prepare the monoclonal antibody, the human renalase gene was amplified from human kidney by specific primer and cloned the DNA fragments into the pET22b. After verification of the positive clones, the gene was transformed to E. coli BL21 to express the protein with 6His on C terminal. The Balb/c mouse was immunized with the purified protein to prepare the monoclonal antibody by hybidoma technique. The renalase protein was reconstructed and 2 strains of the hybidoma which can stable secrete renalase were obtained. The monoclonal antibody can both react with the both recombinant and human serum renalase.展开更多
[ Objective] The aim of the study was to obtain monoclonal antibody against chicken CD8 molecule. [ Method] A fragment of chicken CD8 a/pha gene was amplified by PCR with a pair of designed primers. Then two recombina...[ Objective] The aim of the study was to obtain monoclonal antibody against chicken CD8 molecule. [ Method] A fragment of chicken CD8 a/pha gene was amplified by PCR with a pair of designed primers. Then two recombinant plasmids containing the amplified fragment were constructed. After prokaryotic expression and purification, the obtained recombinant protein was used to immunize Balb/c mice. Finally, the spleen cells were fused with myeloma cells （SP2/0）, and antibody titer of culture supematant was detected by ELISA. [ Result] A 510-bp gene fragment was amplified by PCR. The recombinant plasmid pET-32a-CD8 alpha was transformed into E. coli, and 39 kDa His-CD8 alpha fusion protein was induced to expression. After subcloning, the culture supernetant was detected by ELISA. A hybridoma cell strain, which could stably excrete antibody against CD8 alpha protein, was obtained and named Cll. The ELISA titer of cell supematant was higher than 1 ： 640. [ Conclusion] A hybridoma cell strain has been established using the CD8 alpha expressed in prokaryoUc system as immunogen.展开更多
HMBOX1 was a novel transcription factor possibly involving in function of pancreas and cytotoxicity of NK cells. For function determination, recombinant human HMBOX1 protein was obtained and purified, and the monoclon...HMBOX1 was a novel transcription factor possibly involving in function of pancreas and cytotoxicity of NK cells. For function determination, recombinant human HMBOX1 protein was obtained and purified, and the monoclonal antibodies against HMBOX1 were prepared. The full-length cDNA fragment encoding HMBOX1 was amplified from NK-92 cells and inserted into prokaryotic expression vector pET22b. The pET22b-HMBOX1-6his vector was then transformed into E. coli Rosetta （DE3） and induced by 1 mM IPTG for 4 h at 37℃. The fusion HMBOX1 protein was mainly expressed in inclusion bodies, which was purified and refolded using Ni^2＋-affinity chromatography. With the purified fusion HMBOX1 protein as antigen, monoclonal antibodies against HMBOX1 were generated, providing a potentially useful tool for further study in HMBOX1 functions. Using these anti-HMBOX1 mAbs, we identified that HMBOX1 is located in both cytoplasm and nucleus and could be detected in 10 human normal tissues, including cerebrum, pancreas, kidney and liver tissues. Moreover, the expression in hepatic carcinoma was significantly lower than that in adjacent tissues.展开更多
To prepare monoclonal antibody specific to epidermal growth factor receptor(EGFR)intracellular domain,its gene was amplified from total RNA of A431 cell by RT-PCR.Then the gene was cloned into prokaryotic vector pET30...To prepare monoclonal antibody specific to epidermal growth factor receptor(EGFR)intracellular domain,its gene was amplified from total RNA of A431 cell by RT-PCR.Then the gene was cloned into prokaryotic vector pET30a(+).The recombinant plasmid was transformed into E.coli BL21(DE3)strain for protein expression. Recombinant protein was induced with IPTG and purified using Ni^(2+)NTA agarose.Then the anti-EGFR monoclonal antibody(mAb)was prepared with classical hybridoma technique.Positive clones were selected using indirect enzyme-linked immunoabsorbent assay(ELISA).Totally 4 hybridoma clones were obtained and these mAbs were IgGl(3 clones)and IgG2a(I clone),respectively.Their light chains were all kappa chains. Western blotting analysis and confocal immunofluorescence assays demonstrated that mAbs could specifically recognize EGFR expressing on A431 carcinoma cell line.The mAbs will be useful in the study of EGFR-mediated signal transduction.Cellular & Molecular Immunology.2004;1(2):137-141.展开更多
Immunoglobulin heavy chain Fd gene and K chain gene were amplified by Polymerase Chain Reaction (PCR) from total RNA of a hybridom acell line secreting anti-stomach cancer monoclonal antibody 3G9. Sequences analysis s...Immunoglobulin heavy chain Fd gene and K chain gene were amplified by Polymerase Chain Reaction (PCR) from total RNA of a hybridom acell line secreting anti-stomach cancer monoclonal antibody 3G9. Sequences analysis showed that VH, D, JH genes were derived from VH7183, DFL16. 1, and JH3, and that V. and J, from VkⅡ and Jk2.3G9 Fd and K DNA segments were cloned Into expression vector pComb3. Soluble Fab was expressed by IPTG Induction and proved by western blotting. The specific antigen binding activity of the bacterially produced 3G9 Fab was demonstrated by enzyme- linked Immunofiltration展开更多
[ Objective] To compare the characteristics of monoclonal antibodies against infectious bursal disease virus （IBDV） prepared with two different immunogens, VP2 protein expressed by prokaryotic system and purified IB...[ Objective] To compare the characteristics of monoclonal antibodies against infectious bursal disease virus （IBDV） prepared with two different immunogens, VP2 protein expressed by prokaryotic system and purified IBDV. [Methed] IBDV VP2 gene was amplified by RT-PCR and expressed in a prokaryotic system. The recombinant protein was purified by affinity chromatography. IBDV was pudfied by ultracentrifugation. Balb/c mice were immunized with the purified recombinant protein and IBDV, respectively. The monoclonal antibodies were screened by ELISA. [ Result] Two cell lines secreting antibodies against IBDV VP2 protein were obtained, and their ELISA titers were 1：2 × 10^4. Four cell lines secreting antibodies against I BDV were produced, and their ELISA titers were 1：2× 10^6, 1：6 × 10^4, 1：1× 10^5 and 1：4 × 10^3, respectively. All monoclonal antibodies specifically bound to their own immunogen but did not react with other viruses or proteins. After 10 -20 passages, these cell lines still secreted antibodies stably. [Condusion] The monoclonal antibodies prepared with the recombinant IBDV VP2 protein or purified IBDV can induce immune resoonse in mice. and VP2 soecific monoclonal antibodies can be obtained with VP2 orotein expressed in the Drokarvotic system as immunoQen.展开更多
INTRODUCTIONCalmodulin（CaM）,widely distributed in almost alleukaryotic cells,is a major intracellular calcium receptorresponsible for mediating the Ca<sup>2+</sup> signal to a multitude ofdifferent enzym...INTRODUCTIONCalmodulin（CaM）,widely distributed in almost alleukaryotic cells,is a major intracellular calcium receptorresponsible for mediating the Ca<sup>2+</sup> signal to a multitude ofdifferent enzyme systems and is thought to play a vital rolein the regulation of cell proliferative cycle.Recently,展开更多
Objective: To study the expression level of TRF1 (telomeric repeat binding factor 1) protein in human acute leukemia and relationship between expression level of TRF1 protein and telomerase. Methods: A quantitative We...Objective: To study the expression level of TRF1 (telomeric repeat binding factor 1) protein in human acute leukemia and relationship between expression level of TRF1 protein and telomerase. Methods: A quantitative Western-Blot technique was developed using anti-TRF133-277 monoclonal antibody and GST-TRF1 purity protein as a standard to further determine the ex-pression level of TRF1 protein in total proteins extracted from clinical specimens. Results: Bone marrow tissues of 20 acute leukemia patients were studied, 11 healthy donors’ bone marrows were taken as a control. The expression level of TRF1 protein was significantly higher (P<0.01) in normal bone marrow ((2.217±0.462) μg/μl) than that of acute leukemia patients ((0.754±0.343) μg/μl). But there was no remarkable difference between ALL and ANLL patients ((0.618±0.285) μg/μl vs (0.845±0.359) μg/μl, P>0.05). After chemotherapy, TRF1 expression level of patients with complete remission elevated ((0.772±0.307) μg/μl vs (1.683±0.344) μg/μl, P<0.01), but lower than that of normal ((2.217±0.462) μg/μl, P<0.01). There was no significantly difference after chemotherapy ((0.726±0.411) μg/μl vs (0.895±0.339) μg/μl, P>0.05). TRF1 expression level of patients with complete remission is higher than that of patients without complete remission ((1.683±0.344) μg/μl vs (0.895±0.339) μg/μl, P<0.01). All samples were determined for telomerase activity. It was confirmed that the activity of telomerase in normal bone marrow was lower than that of acute leukemia patients ((0.125±0.078) μg/μl vs (0.765±0.284) μg/μl, P<0.01). There was no significant difference of expression level of TRF1 protein between ALL and ANLL patients ((0.897±0.290) μg/μl vs (0.677±0.268) μg/μl, P>0.05). After chemotherapy, telomerase activity of patients with complete remission decreased ((0.393±0.125) μg/μl), but was still higher than that of normal ((0.125±0.078) μg/μl, P<0.01). Conclusion: The expression level of TRF1 protein has corre-lativity to the activity of telomerase (P<0.001).展开更多
The genome of rice dwarf phytoreovirus (RDV) is composed of 12 double-stranded RNA segments, of which segment S6 encodes a non-structural protein Pns6 identified as the movement protein. In this report, Pns6 with a 6-...The genome of rice dwarf phytoreovirus (RDV) is composed of 12 double-stranded RNA segments, of which segment S6 encodes a non-structural protein Pns6 identified as the movement protein. In this report, Pns6 with a 6-histidine tag at the N-terminal was expressed in E. coli after induction under low temperature (18℃) and low concentration (0.4 mmol/L and 0.2 mmol/L) of IPTG, and then purified by Ni-chelated affinity chromatography. Stability analysis indicated that the expressed HisPns6 protein was stable at 37℃ after 24 h treatment. This recombinant protein was then used to make monoclonal antibody. Total 18 hybridoma clones were obtained. The specificity of antibodies was tested by Western blot using native Pns6 extracted from RDV-infected rice leaves, and 15 positive clones were confirmed. Mapping of the antigenic sites of Pns6 using antibodies showed that the most sensitive antigen determinant is located in the C-terminal region (the 296th-509th amino acids) of Pns6, which is confirms bioinformatics analysis.展开更多
目的研究和分析Doublecortin样激酶-1(Doublecortin Like Disease 1,DCLK1)单克隆抗体在结肠癌中的表达情况与相关影响因素。方法选取2018年7月—2022年6月哈尔滨工业大学附属黑龙江省医院收治的73例结直肠癌患者作为研究对象,根据患者...目的研究和分析Doublecortin样激酶-1(Doublecortin Like Disease 1,DCLK1)单克隆抗体在结肠癌中的表达情况与相关影响因素。方法选取2018年7月—2022年6月哈尔滨工业大学附属黑龙江省医院收治的73例结直肠癌患者作为研究对象,根据患者肿瘤组织中DCLK1表达水平,将其分为DCLK1低表达组(n=31)与DCLK1高表达组(n=42),分析DCLK1单克隆抗体在结直肠癌中表达的影响因素。结果DCLK1高表达组与DCLK1低表达组肿瘤直径、肿瘤分期(Tumor Node Metastasis,TNM)、浸润深度、淋巴结是否转移、有无脉管、肿瘤病灶是否远处转移及肿瘤分化程度对比,差异有统计学意义(P均<0.05)。多因素Logistic回归分析研究结果表明,肿瘤组织中DCLK1高表达水平影响因素包括淋巴结转移、浸润深度、TNM分期、肿瘤直径、肿瘤病灶远处转移、肿瘤分化程度(OR=1.079、1.235、1.548、1.784、1.745、2.026,P均<0.05)。结论肿瘤组织中DCLK1高表达水平影响因素包括淋巴结转移、浸润深度、TNM分期、肿瘤直径、肿瘤病灶远处转移、肿瘤分化程度,临床可通过检测上述指标的方式判断肿瘤组织中DCLK1表达水平。展开更多
Background:Monoclonal antibody therapy has an important role to play as a post-exposure prophylactic and therapeutic for the treatment of viral infections,including emerging infections.For example,several patients of ...Background:Monoclonal antibody therapy has an important role to play as a post-exposure prophylactic and therapeutic for the treatment of viral infections,including emerging infections.For example,several patients of the present Ebola virus outbreak in West Africa were treated with ZMapp,a cocktail of three monoclonal antibodies which are expressed in Nicotiana benthamiana.Discussion:The majority of monoclonal antibodies in clinical use are expressed in mammalian cell lines which offer native folding and glycosylation of the expressed antibody.Monoclonal antibody expression in vegetal systems offers advantages over expression in mammalian cell lines,including improved potential for scale up and reduced costs.In this paper,I highlight the advantages of an upcoming protozoal system for the expression of recombinant antibody formats.Leishmania tarentolae offers a robust,economical expression of proteins with mammalian glycosylation patterns expressed in stable cell lines and grown in suspension culture.Several advantages of this system make it particularly suited for use in developing contexts.Summary:Given the potential importance of monoclonal antibody therapy in the containment of emerging viral infections,novel and alternative strategies to improve production must be explored.展开更多
基金the Scientific Research Project of Shanghai Municipal Health Bureau (2008Y034)the Natural Scientific Research Project of Shanghai(05ZR14086)
文摘To obtain the recombination protein of renalase and prepare the monoclonal antibody, the human renalase gene was amplified from human kidney by specific primer and cloned the DNA fragments into the pET22b. After verification of the positive clones, the gene was transformed to E. coli BL21 to express the protein with 6His on C terminal. The Balb/c mouse was immunized with the purified protein to prepare the monoclonal antibody by hybidoma technique. The renalase protein was reconstructed and 2 strains of the hybidoma which can stable secrete renalase were obtained. The monoclonal antibody can both react with the both recombinant and human serum renalase.
基金supported by the grants of the National Natural Science Foundation of China (30671537)
文摘[ Objective] The aim of the study was to obtain monoclonal antibody against chicken CD8 molecule. [ Method] A fragment of chicken CD8 a/pha gene was amplified by PCR with a pair of designed primers. Then two recombinant plasmids containing the amplified fragment were constructed. After prokaryotic expression and purification, the obtained recombinant protein was used to immunize Balb/c mice. Finally, the spleen cells were fused with myeloma cells （SP2/0）, and antibody titer of culture supematant was detected by ELISA. [ Result] A 510-bp gene fragment was amplified by PCR. The recombinant plasmid pET-32a-CD8 alpha was transformed into E. coli, and 39 kDa His-CD8 alpha fusion protein was induced to expression. After subcloning, the culture supernetant was detected by ELISA. A hybridoma cell strain, which could stably excrete antibody against CD8 alpha protein, was obtained and named Cll. The ELISA titer of cell supematant was higher than 1 ： 640. [ Conclusion] A hybridoma cell strain has been established using the CD8 alpha expressed in prokaryoUc system as immunogen.
基金Acknowledgements This work was supported by grants from National Natural Science Foundation of China （#30671901 #30628014）, National 863 Science Program by Ministry of Science and Technology of China （#2007AA021010, 2007AA021109）, National 973 Basic Science Project by Ministry of Science ＆ Technology of China （#2010CB911901）.
文摘HMBOX1 was a novel transcription factor possibly involving in function of pancreas and cytotoxicity of NK cells. For function determination, recombinant human HMBOX1 protein was obtained and purified, and the monoclonal antibodies against HMBOX1 were prepared. The full-length cDNA fragment encoding HMBOX1 was amplified from NK-92 cells and inserted into prokaryotic expression vector pET22b. The pET22b-HMBOX1-6his vector was then transformed into E. coli Rosetta （DE3） and induced by 1 mM IPTG for 4 h at 37℃. The fusion HMBOX1 protein was mainly expressed in inclusion bodies, which was purified and refolded using Ni^2＋-affinity chromatography. With the purified fusion HMBOX1 protein as antigen, monoclonal antibodies against HMBOX1 were generated, providing a potentially useful tool for further study in HMBOX1 functions. Using these anti-HMBOX1 mAbs, we identified that HMBOX1 is located in both cytoplasm and nucleus and could be detected in 10 human normal tissues, including cerebrum, pancreas, kidney and liver tissues. Moreover, the expression in hepatic carcinoma was significantly lower than that in adjacent tissues.
基金supported by Science and Technology Commission of Shanghai Municipality(03JC14085)grant of National Natural Science Foundation#30170888the grant from E-institutes of Shanghai Universities Immunology Division.
文摘To prepare monoclonal antibody specific to epidermal growth factor receptor(EGFR)intracellular domain,its gene was amplified from total RNA of A431 cell by RT-PCR.Then the gene was cloned into prokaryotic vector pET30a(+).The recombinant plasmid was transformed into E.coli BL21(DE3)strain for protein expression. Recombinant protein was induced with IPTG and purified using Ni^(2+)NTA agarose.Then the anti-EGFR monoclonal antibody(mAb)was prepared with classical hybridoma technique.Positive clones were selected using indirect enzyme-linked immunoabsorbent assay(ELISA).Totally 4 hybridoma clones were obtained and these mAbs were IgGl(3 clones)and IgG2a(I clone),respectively.Their light chains were all kappa chains. Western blotting analysis and confocal immunofluorescence assays demonstrated that mAbs could specifically recognize EGFR expressing on A431 carcinoma cell line.The mAbs will be useful in the study of EGFR-mediated signal transduction.Cellular & Molecular Immunology.2004;1(2):137-141.
文摘Immunoglobulin heavy chain Fd gene and K chain gene were amplified by Polymerase Chain Reaction (PCR) from total RNA of a hybridom acell line secreting anti-stomach cancer monoclonal antibody 3G9. Sequences analysis showed that VH, D, JH genes were derived from VH7183, DFL16. 1, and JH3, and that V. and J, from VkⅡ and Jk2.3G9 Fd and K DNA segments were cloned Into expression vector pComb3. Soluble Fab was expressed by IPTG Induction and proved by western blotting. The specific antigen binding activity of the bacterially produced 3G9 Fab was demonstrated by enzyme- linked Immunofiltration
文摘[ Objective] To compare the characteristics of monoclonal antibodies against infectious bursal disease virus （IBDV） prepared with two different immunogens, VP2 protein expressed by prokaryotic system and purified IBDV. [Methed] IBDV VP2 gene was amplified by RT-PCR and expressed in a prokaryotic system. The recombinant protein was purified by affinity chromatography. IBDV was pudfied by ultracentrifugation. Balb/c mice were immunized with the purified recombinant protein and IBDV, respectively. The monoclonal antibodies were screened by ELISA. [ Result] Two cell lines secreting antibodies against IBDV VP2 protein were obtained, and their ELISA titers were 1：2 × 10^4. Four cell lines secreting antibodies against I BDV were produced, and their ELISA titers were 1：2× 10^6, 1：6 × 10^4, 1：1× 10^5 and 1：4 × 10^3, respectively. All monoclonal antibodies specifically bound to their own immunogen but did not react with other viruses or proteins. After 10 -20 passages, these cell lines still secreted antibodies stably. [Condusion] The monoclonal antibodies prepared with the recombinant IBDV VP2 protein or purified IBDV can induce immune resoonse in mice. and VP2 soecific monoclonal antibodies can be obtained with VP2 orotein expressed in the Drokarvotic system as immunoQen.
基金the Natural Science Fundation of Jiangsu Province,№BK95141307
文摘INTRODUCTIONCalmodulin（CaM）,widely distributed in almost alleukaryotic cells,is a major intracellular calcium receptorresponsible for mediating the Ca<sup>2+</sup> signal to a multitude ofdifferent enzyme systems and is thought to play a vital rolein the regulation of cell proliferative cycle.Recently,
文摘Objective: To study the expression level of TRF1 (telomeric repeat binding factor 1) protein in human acute leukemia and relationship between expression level of TRF1 protein and telomerase. Methods: A quantitative Western-Blot technique was developed using anti-TRF133-277 monoclonal antibody and GST-TRF1 purity protein as a standard to further determine the ex-pression level of TRF1 protein in total proteins extracted from clinical specimens. Results: Bone marrow tissues of 20 acute leukemia patients were studied, 11 healthy donors’ bone marrows were taken as a control. The expression level of TRF1 protein was significantly higher (P<0.01) in normal bone marrow ((2.217±0.462) μg/μl) than that of acute leukemia patients ((0.754±0.343) μg/μl). But there was no remarkable difference between ALL and ANLL patients ((0.618±0.285) μg/μl vs (0.845±0.359) μg/μl, P>0.05). After chemotherapy, TRF1 expression level of patients with complete remission elevated ((0.772±0.307) μg/μl vs (1.683±0.344) μg/μl, P<0.01), but lower than that of normal ((2.217±0.462) μg/μl, P<0.01). There was no significantly difference after chemotherapy ((0.726±0.411) μg/μl vs (0.895±0.339) μg/μl, P>0.05). TRF1 expression level of patients with complete remission is higher than that of patients without complete remission ((1.683±0.344) μg/μl vs (0.895±0.339) μg/μl, P<0.01). All samples were determined for telomerase activity. It was confirmed that the activity of telomerase in normal bone marrow was lower than that of acute leukemia patients ((0.125±0.078) μg/μl vs (0.765±0.284) μg/μl, P<0.01). There was no significant difference of expression level of TRF1 protein between ALL and ANLL patients ((0.897±0.290) μg/μl vs (0.677±0.268) μg/μl, P>0.05). After chemotherapy, telomerase activity of patients with complete remission decreased ((0.393±0.125) μg/μl), but was still higher than that of normal ((0.125±0.078) μg/μl, P<0.01). Conclusion: The expression level of TRF1 protein has corre-lativity to the activity of telomerase (P<0.001).
基金Supported by the Special Commonweal Industry Research Program of Agriculture (Grant no. nyhyzx07-051)the National High Technology Research and Development Program of China (Grant No. 2007AA021505) to Prof. LI Yi
文摘The genome of rice dwarf phytoreovirus (RDV) is composed of 12 double-stranded RNA segments, of which segment S6 encodes a non-structural protein Pns6 identified as the movement protein. In this report, Pns6 with a 6-histidine tag at the N-terminal was expressed in E. coli after induction under low temperature (18℃) and low concentration (0.4 mmol/L and 0.2 mmol/L) of IPTG, and then purified by Ni-chelated affinity chromatography. Stability analysis indicated that the expressed HisPns6 protein was stable at 37℃ after 24 h treatment. This recombinant protein was then used to make monoclonal antibody. Total 18 hybridoma clones were obtained. The specificity of antibodies was tested by Western blot using native Pns6 extracted from RDV-infected rice leaves, and 15 positive clones were confirmed. Mapping of the antigenic sites of Pns6 using antibodies showed that the most sensitive antigen determinant is located in the C-terminal region (the 296th-509th amino acids) of Pns6, which is confirms bioinformatics analysis.
基金I wish to thank Dr Ian Brierley(Division of Virology,University of Cambridge)and Dr Nyarie Sithole(Department of Medicine,University of Cambridge)for useful discussions and advice.JDJ is funded by a Wellcome Trust Infection and Immunity PhD Studentship.
文摘Background:Monoclonal antibody therapy has an important role to play as a post-exposure prophylactic and therapeutic for the treatment of viral infections,including emerging infections.For example,several patients of the present Ebola virus outbreak in West Africa were treated with ZMapp,a cocktail of three monoclonal antibodies which are expressed in Nicotiana benthamiana.Discussion:The majority of monoclonal antibodies in clinical use are expressed in mammalian cell lines which offer native folding and glycosylation of the expressed antibody.Monoclonal antibody expression in vegetal systems offers advantages over expression in mammalian cell lines,including improved potential for scale up and reduced costs.In this paper,I highlight the advantages of an upcoming protozoal system for the expression of recombinant antibody formats.Leishmania tarentolae offers a robust,economical expression of proteins with mammalian glycosylation patterns expressed in stable cell lines and grown in suspension culture.Several advantages of this system make it particularly suited for use in developing contexts.Summary:Given the potential importance of monoclonal antibody therapy in the containment of emerging viral infections,novel and alternative strategies to improve production must be explored.
