Many eukaryotic genes are members of multi-gene families due to gene duplications, which generate new copies that allow functional divergence. However, the relationship between
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
Objectives： To clone and express Tp0453 outer membrane protein of Treponema pallidum, and to evaluate its significance in the serodiagnosis of syphilis. Methods： The immuno-dominant epitope of Tp0453 gene was amplif...Objectives： To clone and express Tp0453 outer membrane protein of Treponema pallidum, and to evaluate its significance in the serodiagnosis of syphilis. Methods： The immuno-dominant epitope of Tp0453 gene was amplified from the complete genome of T.pallidum by polymerase chain reactions （PCR）, subcloned into the expression vector Pqe32 to generate recombinant plasmid Pqe32/Tp0453, and was then expressed in E. coli M15. The fusion protein was purified with Ni-NTA affinity purification. Indirect ELISA was developed to detect human serum IgG antibody to T. pallidum. Results： The recombinant Tp0453 protein was successfully expressed and purified. The recombinant protein had a molecular weight of approximately 32KDa.Indirect ELISA to the recombinant protein was developed.Sixty control sera were tested by ELISA and yielded a sensitivity of 100% （30/30） and a specificity of 100% （30/30）. While testing for T. pallidum in human sera, the sensitivity and specificity of ELISA were 96.8% and 100%, respectively, when compared with TPPA test results. The concordance of results between the ELISA test and the TPPA test was 98.2%. Conclusion： The recombinant Tp0453 outer membrane protein elicited a strong immunoreaction to anti-T.pallidum IgG antibody and has great potential use in ELISA for the serodiagnosis of syphilis.展开更多
P65 protein, the major immunodominant protein of Mycoplasma hyopneu-moniae （Mhp） exhibiting no cross-reaction with other mycoplasmas, is general y used as a target protein for Mhp detection. In this study, BALB/c mi...P65 protein, the major immunodominant protein of Mycoplasma hyopneu-moniae （Mhp） exhibiting no cross-reaction with other mycoplasmas, is general y used as a target protein for Mhp detection. In this study, BALB/c mice were immunized with prokaryotical y expressed P65 recombinant protein to prepare monoclonal anti-body. After screening with Mhp whole-cel protein and P65 protein, a specific hy-bridoma cel line, 3G12, was obtained by ELISA. Identification results indicated that the antibody secreted by 3G12 hybridoma cel s could react with P65 protein and Mhp whole-cel protein. According to indirect ELISA assay, 3G12 cel culture super-natant possessed a titer of 1∶12 800 against P65 protein and 1∶3 200 against Mhp whole-cel protein; 3G12 ascites possessed a titer of above 1∶4 000 000 against P65 protein and above 1∶20 000 against Mhp 168 whole-cel protein. After long-term in vitro culture and continuous freezing-thawing, 3G12 cel line could stably secrete antibodies. A monoclonal antibody against P65 protein and Mhp whole-cel protein was successful y obtained in the present study, which provided basis for further in-vestigating the pathogenic mechanism of Mhp and establishing diagnostic methods of Mycoplasmal pneumonia of swine （MPS）.展开更多
Aim: To produce biologically active recombinant human (rh) ZP proteins in a human cell for use in sperm function tests. Methods: The human embryonic kidney cell line 293T was employed to produce rhZP1, rhZP2 and rhZP3...Aim: To produce biologically active recombinant human (rh) ZP proteins in a human cell for use in sperm function tests. Methods: The human embryonic kidney cell line 293T was employed to produce rhZP1, rhZP2 and rhZP3 proteins individually and together by co-expression. Presence of these proteins in the culture medium and cell lysate was assessed by Western blotting analysis. The effect of the recombinant proteins on the human AR was assessed. Results: RhZP2 and rhZP3 were secreted into the culture medium, whereas rhZPl was found only in the cell lysate. Interestingly, when all zona pellucida proteins were co-expressed in the same cells, rhZPl was also secreted into the culture medium. However, despite the presence of all three ZP proteins in sufficient concentration and evidence of heavy glycosylation on gel electrophoresis, biological activity to induce the AR was not observed. Conclusion: RhZP1, rhZP2 and rhZP3 were successfully expressed in the human embryonic kidney cell line 293T. It appears that an interaction amongst these proteins may be required for release of rhZPl from the cell. Although this approach is not satisfactory for producing active human ZP proteins, it makes a significant contribution to the understanding of the structural and functional characteristics of the ZP proteins.展开更多
Recombinant tissue plasminogen activator （rPA） has been used as a thrombolytic agent. However, considerable improvements have been done to prolong its plasma half-life （tl/2） and reduce its side effects, such as i...Recombinant tissue plasminogen activator （rPA） has been used as a thrombolytic agent. However, considerable improvements have been done to prolong its plasma half-life （tl/2） and reduce its side effects, such as intracranial hemorrhage. Based on these improvements, a mutant ofrPA, mrPA, was designed by mutating its PAI-1 binding site to extend its tl/2. Furthermore, a fusion protein conjugating mrPA with NR3 was designed, which was a rAcAp5 mutant with a platelet GPIIb/IIIa-binding RGD motif, to enhance the ability of targeted-thrombus and thrombolysis. The synthesized DNA sequences coding the two proteins were amplified by PCR, cloned into pET30a to construct recombinant plasmids pET30a-mrPA and pET30a-mrPA-NR3, and transformed into E. coli BL21 （DE3）. The two proteins were expressed in inclusion bodies induced by isopropy113-D-1-thiogalactopyranoside. After purified to qualified purity using one-step Ni affinity chromatography, the denatured proteins were refolded by dialysis. Their thrombolytic effects in vitro and in vivo were evaluated. In vitro 3.5 and 7 pmol/L of mrPA significantly reduced thrombus weight; 1.75, 3.5 and 7 ~tmol/L of mrPA-NR3 also significantly reduced the thrombus weight, and mrPA-NR3 displayed stronger thrombolytic effects than mrPA at 7 lamol/L. In vivo both mrPA and mrPA-NR3 showed significantly thrombolytic effect at 60-240 ktmol/kg in thrombolytic model of inferior vena cava. Importantly, mrPA-NR3 exhibited more potent thrombolytic effect than both mrPA and rhM-tPA （positive control） at 240 p.mol/kg. In addition, these two novel proteins did not increase bleeding time while they exerted thrombolytic effect. In conclusion, we engineered two novel proteins and proved that fusion protein had better thrombolytic effect than non-fusion protein, and the results suggest that dual thrombolytic mechanism or thrombus-target potentiated the thrombolytic effect ofrPA and alleviated hemorrhage side reaction. This study may shed light on the development of novel thrombolytic agents with targeted thrombolysis and reduced side effects.展开更多
Induction of tumor vasculature occlusion by targeting a thrombogen to newly formed blood vessels in tumor tissues represents an intriguing approach to the eradication of primary solid tumors. In the current study, we ...Induction of tumor vasculature occlusion by targeting a thrombogen to newly formed blood vessels in tumor tissues represents an intriguing approach to the eradication of primary solid tumors. In the current study, we construct and express a fusion protein containing vascular endothelial growth factor (VEGF) and tissue factor (TF) to explore whether this fusion protein has the capability of inhibiting tumor growth in a colon carcinoma model. The murine cDNA of VEGF A and TF were amplified by reverse transcriptase polymerase chain reaction (RT-PCR), and then cloned into prokaryotic expression plasmid pQE30 with a linker. The expression product recombinant VEGF-TF (rVEGF-TF) was purified and proved to have comparable enzyme activity to a commercial TF and the capability of specific binding to tumor vessels. Significant decrease of tumor growth was found in the mice administered with rVEGF-TF on Day 6 after initiated rVEGF-TF treatment (P<0.05), and the tumor masses in 2 of 10 mice were almost disappeared on Day 14 after the first treatment. In addition, valid thrombogenesis and tumor necrosis were observed in the tumor tissues injected with rVEGF-TF. Our results demonstrate that occlusion of tumor vasculature with rVEGF-TF is potentially an effective approach for cancer therapy.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 unclear. 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 Trail-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 diagnostic reagents. METHODS: Fourteen clones encompassing HGV gene fragm...AIM: To study the epitope distribution of hepatitis G virus (HGV) and to seek for the potential recombinant antigens for the development of HGV diagnostic 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 E1 (G1), one from E2 (G31), three from NS3 (G6, G61, G7), one from NS5B (G821) and one chimeric fragment from NS3 and NS5B (G61-821) could be expressed well and showed obvious immunoreactivity by Western blotting. One clone with HGV 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 G1, G31, G61, G821 and G61-821 were detected in indirected ELISA as coating antigen respectively. Only recombinant G1 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 E1, E2, NS3 and NS5 fragment of HGV contain antigenic epitopes, which could be produced in prokaryotically expressed recombinant proteins. A high-yield recombinant protein (G1) 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
Approximately 40-50 β-defensins are predominantly expressed in the male reproductive system of mammals. This selective expression raises the question as to the roles of these molecules in innate immunity and fertilit...Approximately 40-50 β-defensins are predominantly expressed in the male reproductive system of mammals. This selective expression raises the question as to the roles of these molecules in innate immunity and fertility in the male reproductive tract. Rat β-defensin 22 is an epididymis-specific β-defensin expressed in segments 12-14 of the epididymis. This protein contains both β-defensin and lectin signature sequences, yet its antimicrobial activity and carbohydrate-binding ability have not been shown. We herein demonstrated the antimicrobial activity of recombinant rat β-defensin 22 against Escherichia coliand Candida albicans. Its lectinJike activity was also investigated by demonstrating its binding ability with heparin beads. This heparin-binding activity implies some potential roles for this defensin in determining the fertilisation capabilities of sperm.展开更多
New strategies in vaccine development are urgently needed to combat emerging influenza viruses and to reduce the risk of pandemic disease surfacing. Being conserved, the M2 e protein, is a potential candidate for univ...New strategies in vaccine development are urgently needed to combat emerging influenza viruses and to reduce the risk of pandemic disease surfacing. Being conserved, the M2 e protein, is a potential candidate for universal vaccine development against influenza A viruses. Mycobacterium tuberculosis Hsp70(mHsp70) is known to cultivate the function of immunogenic antigen-presenting cells, stimulate a strong cytotoxic T lymphocyte(CTL) response, and stop the induction of tolerance. Thus, in this study, a recombinant protein from the extracellular domain of influenza A virus matrix protein 2(M2e), was fused to the C-terminus of Mycobacterium tuberculosis Hsp70(Hsp70c), to generate a vaccine candidate. Humoral immune responses, IFN-γ-producing lymphocyte, and strong CTL activity were all induced to confirm the immunogenicity of M2 e.Hsp70c(Hsp70359–610). And challenge tests showed protection against H1N1 and H9N2 strains in vaccinated groups. Finally these results demonstrates M2 e.Hsp70c fusion protein can be a candidate for a universal influenza A vaccine.展开更多
Objective: To produce fluorescent tagged recombinant erythroferrone protein(ERFE_eGFP) for laboratory investigations. Methods: Erythroferrone(ERFE) gene was fused to green fluorescent protein(eGFP) gene and cloned in ...Objective: To produce fluorescent tagged recombinant erythroferrone protein(ERFE_eGFP) for laboratory investigations. Methods: Erythroferrone(ERFE) gene was fused to green fluorescent protein(eGFP) gene and cloned in a pSecTag2Hygro plasmid. The constructed plasmid was amplified in Escherichia coli DH5α and the eGFP-fused ERFE(ERFE_eGFP) protein was expressed in human embryonic kidney(HEK293T) cell line. Results: The plasmid constructed from colony C6 contained ERFE_eGFP with the correct restriction sizes of 4.2 kb and expressed secretory ERFE_eGFP fusion protein(approximately size of 75 kDa) in HEK293T cell line. Conclusions: ERFE_eGFP recombinant protein is successfully expressed as a secretory functional protein and could be sensitively detected using fluorometry. This fusion protein might benefit future applications for localization of cellular ERFE receptors and competitive immunoassay of ERFE concentration.展开更多
It is well known that Tn5B1-4 (commercially known as the High Five) cell line is highly susceptible to baculovirus and provides superior production of recombinant proteins when compared to other insect cell lines.But ...It is well known that Tn5B1-4 (commercially known as the High Five) cell line is highly susceptible to baculovirus and provides superior production of recombinant proteins when compared to other insect cell lines.But the characteristics of the cell line do not always remain stable and may change upon continuous passage.Recently an alphanodavirus,named Tn5 Cell Line Virus (or TNCL Virus),was identified in High Five cells in particular.Therefore,we established a new cell line,QB-Tn9-4s,from Trichoplusia ni,which was determined to be free of TNCL virus by RT-PCR analysis.In this paper,we describe the development of a novel cell clone,QB-CL-B,from a low passage QB-Tn9-4s cell line and report its susceptibility to AcMNPV,and the level of recombinant protein production.This cell clone was similar to its parental cells QB-Tn9-4s and Tn5B1-4 cells in morphology and growth rate;although it also showed approximately the same responses to AcMNPV infection and production of occlusion bodies,there were higher levels of recombinant protein production in comparison to QB-Tn9-4s (parental cells) and High5 cells.展开更多
INTRODUCTIONAlthough reliable assays for the detection ofhepatitis C virus and E virus became available, still10% 20% hepatitis are not caused byhepatitis A-E virus[1-3]. In 1996, two research groups isolatedthis agen...INTRODUCTIONAlthough reliable assays for the detection ofhepatitis C virus and E virus became available, still10% 20% hepatitis are not caused byhepatitis A-E virus[1-3]. In 1996, two research groups isolatedthis agent independently and almost simultaneouslyand named hepatitis G virus and GB virus C,respectively[4-7].展开更多
Summary： A new type of TGF-β3 fusion protein with targeted therapy function was constructed, and its feasibility and target specificity of inducing chondrogenesis were investigated by transfecting LAP-MMP-mTGF-β3 g...Summary： A new type of TGF-β3 fusion protein with targeted therapy function was constructed, and its feasibility and target specificity of inducing chondrogenesis were investigated by transfecting LAP-MMP-mTGF-β3 gene into adipose-derived stem cells （ADSCs）. The recombinant pIRES- EGFP-MMP was constructed by inserting the sense and antisense DNA of encoding the amino acid of the synthetic MMP enzyme cutting site into the eukaryotic expression vector pIRES-EGFE LAP and mTGF-β3 fragments were obtained by using RT-PCR and inserted into the upstream and downstream of MMP from pIRES-EGFP-MMP respectively, and the recombinant plasmid of pIRES-EGFP- LAP-MMP-mTGF-β3 was constructed, which was transferred to ADSCs. The ADSCs were cultured and divided in three groups： experimental group （MMP group）, negative control group （no MMP） and non-transfection group. The morphological changes were observed microscopically, and the expression of proteoglycan and type II collagen （Col II） was detected by using Alcian blue staining and immuno- histochemistry staining at 7th, 14th and 21st day after culture. The recombinant plasmid of pIRES-EGFP-LAP-MMP-mTGF-β3 was correctly constructed by methods of enzyme cutting and se- quencing analysis. The mTGF-β3 fusion protein was successfully expressed after transfection, and in the presence of the MMP, active protein mTGF-β3 was generated, which significantly promoted differ- entiation of ADSCs into chondrocytes and the expression of cartilage matrix. The novel fusion protein LAP-MMP-mTGF-β3 can targetedly induce differentiation of ADSCs into chondrocytes, which would open up prospects for target therapy of cartilage damage repair in future.展开更多
In this study, a panel of monoclonal antibodies （mAbs） against Porcine reproductive and respiratory syndrome virus（PRRSV）, named as 8C9 and4B4, were produced by fusing SP2/0 myeloma cells and spleen cells of BALB/...In this study, a panel of monoclonal antibodies （mAbs） against Porcine reproductive and respiratory syndrome virus（PRRSV）, named as 8C9 and4B4, were produced by fusing SP2/0 myeloma cells and spleen cells of BALB/c mice immunized with the PRRSV （TCID50=5.5）, screened by the indirect ELISA and subjected to several limiting dilutions, mAbs were then identified by biological characterization. Among the two fusion cell strains, 8C9 belonged to the IgG1 subclass and 4B4 belonged to the IgG2a subclass. The titers in cell culture supematant and abdomen liquor reached to 1：104and 1：105, respectively. The specificity test indicated that the two cells had specific reactions for the PRRSV and GP5 protein respectively, and no reaction with Classical swine fever virus （CSFV） or Swine vesicular disease virus （SVDV）. The molecular weights of the heavy chain and light chain were about 45.0 kDa and 25.0 kDa, respectively. In neutralization activity tests, the results showed that the prepared mAb 4B4 can protect 50% of cells with no CPE in dilution up to 1：512, but mAB 8C9 has no neutralization activities to PRRSV.展开更多
Objective To express the recombinant human bone morphogenetic protein-7 (rhBMP-7) in Chinese hamster ovary (CHO) cells and to establish the in vitro biological activity assay of rhBMP-7. Methods Human BMP-7 cDNA was s...Objective To express the recombinant human bone morphogenetic protein-7 (rhBMP-7) in Chinese hamster ovary (CHO) cells and to establish the in vitro biological activity assay of rhBMP-7. Methods Human BMP-7 cDNA was subcloned into pcDNA3.1 mammalian expression vector and transfected to CHO cells by using the lipofectin transfection method. BMP-7 expression cell culture supernatants were harvested and purified for target protein. To analyze the bioactivity of the secreted rhBMP-7, a novel in vitro assay was established by measuring its alkaline phosphatase (ALP) stimulating of osteoblast cell line, W-20-17. Results BMP-7 stably expressing cell clone was selected, which secreted mature disulfide-linked homodimer form of hBMP-7 and had an apparent molecular weight of 36kDa. rhBMP-7 with >95% purity was obtained using 3 step chromatography method. Bioactivity assay showed that the purified protein specifically stimulated W-20-17 cell producing ALP, with a 4-fold increase of ALP activity at 100ng/ml or more, and the EC50 of 15.6ng/ml. Conclusion Purified rhBMP-7 from this CHO expression system has significant biological activity in induction of osteoblast phenotype, which demonstrates potential bone regeneration activity.展开更多
To clone and express the recombinant outer membrane protein Tp0453 of Treponema pallidum and to analyze the immuno-reactivity and immunogenicity of the expressed protein, the immuno-dominant epitope of the Tp0453 was ...To clone and express the recombinant outer membrane protein Tp0453 of Treponema pallidum and to analyze the immuno-reactivity and immunogenicity of the expressed protein, the immuno-dominant epitope of the Tp0453 was amplified from the complete genome of T.pallidum by PCR, subcloned into expression vector pQE32 to generate the recombinant plasmid pQE32/Tp0453, then expressed in E.coli M15 and analyzed by SDS/PAGE and Western blotting. The fusion protein expressed was purified with Ni-NTA affinity chromatography. Its immuno-reactivity was assayed by indirect ELISA, and the immunogenicity was determined by immunization with this fusion protein in New Zealand rabbits. In the present study, a fusion protein of molecular weight about 32 kDa was obtained. As demonstrated by Western blotting, the recombinant protein could react specifically with positive IgG sera of patients with syphilis, and the antibodies against T.pallidum in human sera were successfully detected by indirect ELISA. Both the sensitivity and specificity of ELISA based on the Tp0453 fusion protein as were 100% (30/30) when detected with control sera. In comparison with the results of IgG ELISA with those of TPPA. It was found that the sensitivity of ELISA was 96.8% and the specificity was 100%. The difference of ELISA and TPPA was not significant, and the concordance of results between ELISA and TPPA was 98.2%. In addition, specific humoral responses could be elicited by immunization with the recombinant fusion protein in New Zealand rabbits with a specific antibody titer of 1∶1280 after 3 successive doses of immunization. These results demonstrate that the expressed recombinant fusion protein shows excellent immuno-competence and provide foundation to develop a quick diagnostic kid applied to detect the presence of T.pallidum infections.展开更多
文摘Many eukaryotic genes are members of multi-gene families due to gene duplications, which generate new copies that allow functional divergence. However, the relationship between
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 .
基金Financially supported by Key grant from the Education Committee of Hunan Province (No. 02A046)
文摘Objectives： To clone and express Tp0453 outer membrane protein of Treponema pallidum, and to evaluate its significance in the serodiagnosis of syphilis. Methods： The immuno-dominant epitope of Tp0453 gene was amplified from the complete genome of T.pallidum by polymerase chain reactions （PCR）, subcloned into the expression vector Pqe32 to generate recombinant plasmid Pqe32/Tp0453, and was then expressed in E. coli M15. The fusion protein was purified with Ni-NTA affinity purification. Indirect ELISA was developed to detect human serum IgG antibody to T. pallidum. Results： The recombinant Tp0453 protein was successfully expressed and purified. The recombinant protein had a molecular weight of approximately 32KDa.Indirect ELISA to the recombinant protein was developed.Sixty control sera were tested by ELISA and yielded a sensitivity of 100% （30/30） and a specificity of 100% （30/30）. While testing for T. pallidum in human sera, the sensitivity and specificity of ELISA were 96.8% and 100%, respectively, when compared with TPPA test results. The concordance of results between the ELISA test and the TPPA test was 98.2%. Conclusion： The recombinant Tp0453 outer membrane protein elicited a strong immunoreaction to anti-T.pallidum IgG antibody and has great potential use in ELISA for the serodiagnosis of syphilis.
基金Supported by National Natural Science Foundation of China(31100136,3111339)Independent Innovation Fund of Agricultural Science and Technology of Jiangsu Province[CX(13)3066]~~
文摘P65 protein, the major immunodominant protein of Mycoplasma hyopneu-moniae （Mhp） exhibiting no cross-reaction with other mycoplasmas, is general y used as a target protein for Mhp detection. In this study, BALB/c mice were immunized with prokaryotical y expressed P65 recombinant protein to prepare monoclonal anti-body. After screening with Mhp whole-cel protein and P65 protein, a specific hy-bridoma cel line, 3G12, was obtained by ELISA. Identification results indicated that the antibody secreted by 3G12 hybridoma cel s could react with P65 protein and Mhp whole-cel protein. According to indirect ELISA assay, 3G12 cel culture super-natant possessed a titer of 1∶12 800 against P65 protein and 1∶3 200 against Mhp whole-cel protein; 3G12 ascites possessed a titer of above 1∶4 000 000 against P65 protein and above 1∶20 000 against Mhp 168 whole-cel protein. After long-term in vitro culture and continuous freezing-thawing, 3G12 cel line could stably secrete antibodies. A monoclonal antibody against P65 protein and Mhp whole-cel protein was successful y obtained in the present study, which provided basis for further in-vestigating the pathogenic mechanism of Mhp and establishing diagnostic methods of Mycoplasmal pneumonia of swine （MPS）.
文摘Aim: To produce biologically active recombinant human (rh) ZP proteins in a human cell for use in sperm function tests. Methods: The human embryonic kidney cell line 293T was employed to produce rhZP1, rhZP2 and rhZP3 proteins individually and together by co-expression. Presence of these proteins in the culture medium and cell lysate was assessed by Western blotting analysis. The effect of the recombinant proteins on the human AR was assessed. Results: RhZP2 and rhZP3 were secreted into the culture medium, whereas rhZPl was found only in the cell lysate. Interestingly, when all zona pellucida proteins were co-expressed in the same cells, rhZPl was also secreted into the culture medium. However, despite the presence of all three ZP proteins in sufficient concentration and evidence of heavy glycosylation on gel electrophoresis, biological activity to induce the AR was not observed. Conclusion: RhZP1, rhZP2 and rhZP3 were successfully expressed in the human embryonic kidney cell line 293T. It appears that an interaction amongst these proteins may be required for release of rhZPl from the cell. Although this approach is not satisfactory for producing active human ZP proteins, it makes a significant contribution to the understanding of the structural and functional characteristics of the ZP proteins.
基金National Natural Science Foundation of China(Grant No.81573333 and 81503060)
文摘Recombinant tissue plasminogen activator （rPA） has been used as a thrombolytic agent. However, considerable improvements have been done to prolong its plasma half-life （tl/2） and reduce its side effects, such as intracranial hemorrhage. Based on these improvements, a mutant ofrPA, mrPA, was designed by mutating its PAI-1 binding site to extend its tl/2. Furthermore, a fusion protein conjugating mrPA with NR3 was designed, which was a rAcAp5 mutant with a platelet GPIIb/IIIa-binding RGD motif, to enhance the ability of targeted-thrombus and thrombolysis. The synthesized DNA sequences coding the two proteins were amplified by PCR, cloned into pET30a to construct recombinant plasmids pET30a-mrPA and pET30a-mrPA-NR3, and transformed into E. coli BL21 （DE3）. The two proteins were expressed in inclusion bodies induced by isopropy113-D-1-thiogalactopyranoside. After purified to qualified purity using one-step Ni affinity chromatography, the denatured proteins were refolded by dialysis. Their thrombolytic effects in vitro and in vivo were evaluated. In vitro 3.5 and 7 pmol/L of mrPA significantly reduced thrombus weight; 1.75, 3.5 and 7 ~tmol/L of mrPA-NR3 also significantly reduced the thrombus weight, and mrPA-NR3 displayed stronger thrombolytic effects than mrPA at 7 lamol/L. In vivo both mrPA and mrPA-NR3 showed significantly thrombolytic effect at 60-240 ktmol/kg in thrombolytic model of inferior vena cava. Importantly, mrPA-NR3 exhibited more potent thrombolytic effect than both mrPA and rhM-tPA （positive control） at 240 p.mol/kg. In addition, these two novel proteins did not increase bleeding time while they exerted thrombolytic effect. In conclusion, we engineered two novel proteins and proved that fusion protein had better thrombolytic effect than non-fusion protein, and the results suggest that dual thrombolytic mechanism or thrombus-target potentiated the thrombolytic effect ofrPA and alleviated hemorrhage side reaction. This study may shed light on the development of novel thrombolytic agents with targeted thrombolysis and reduced side effects.
基金the National Natural Science Foundation of China (Nos. 30560160 and 30560048)the New Century Excellent Talents in University of China (No. NCET-05-0757)the Education Department of Hainan Province, China (No. Hjkj200422)
文摘Induction of tumor vasculature occlusion by targeting a thrombogen to newly formed blood vessels in tumor tissues represents an intriguing approach to the eradication of primary solid tumors. In the current study, we construct and express a fusion protein containing vascular endothelial growth factor (VEGF) and tissue factor (TF) to explore whether this fusion protein has the capability of inhibiting tumor growth in a colon carcinoma model. The murine cDNA of VEGF A and TF were amplified by reverse transcriptase polymerase chain reaction (RT-PCR), and then cloned into prokaryotic expression plasmid pQE30 with a linker. The expression product recombinant VEGF-TF (rVEGF-TF) was purified and proved to have comparable enzyme activity to a commercial TF and the capability of specific binding to tumor vessels. Significant decrease of tumor growth was found in the mice administered with rVEGF-TF on Day 6 after initiated rVEGF-TF treatment (P<0.05), and the tumor masses in 2 of 10 mice were almost disappeared on Day 14 after the first treatment. In addition, valid thrombogenesis and tumor necrosis were observed in the tumor tissues injected with rVEGF-TF. Our results demonstrate that occlusion of tumor vasculature with rVEGF-TF is potentially an effective approach for cancer therapy.
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 unclear. 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 Trail-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 diagnostic 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 E1 (G1), one from E2 (G31), three from NS3 (G6, G61, G7), one from NS5B (G821) and one chimeric fragment from NS3 and NS5B (G61-821) could be expressed well and showed obvious immunoreactivity by Western blotting. One clone with HGV 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 G1, G31, G61, G821 and G61-821 were detected in indirected ELISA as coating antigen respectively. Only recombinant G1 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 E1, E2, NS3 and NS5 fragment of HGV contain antigenic epitopes, which could be produced in prokaryotically expressed recombinant proteins. A high-yield recombinant protein (G1) 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Approximately 40-50 β-defensins are predominantly expressed in the male reproductive system of mammals. This selective expression raises the question as to the roles of these molecules in innate immunity and fertility in the male reproductive tract. Rat β-defensin 22 is an epididymis-specific β-defensin expressed in segments 12-14 of the epididymis. This protein contains both β-defensin and lectin signature sequences, yet its antimicrobial activity and carbohydrate-binding ability have not been shown. We herein demonstrated the antimicrobial activity of recombinant rat β-defensin 22 against Escherichia coliand Candida albicans. Its lectinJike activity was also investigated by demonstrating its binding ability with heparin beads. This heparin-binding activity implies some potential roles for this defensin in determining the fertilisation capabilities of sperm.
文摘New strategies in vaccine development are urgently needed to combat emerging influenza viruses and to reduce the risk of pandemic disease surfacing. Being conserved, the M2 e protein, is a potential candidate for universal vaccine development against influenza A viruses. Mycobacterium tuberculosis Hsp70(mHsp70) is known to cultivate the function of immunogenic antigen-presenting cells, stimulate a strong cytotoxic T lymphocyte(CTL) response, and stop the induction of tolerance. Thus, in this study, a recombinant protein from the extracellular domain of influenza A virus matrix protein 2(M2e), was fused to the C-terminus of Mycobacterium tuberculosis Hsp70(Hsp70c), to generate a vaccine candidate. Humoral immune responses, IFN-γ-producing lymphocyte, and strong CTL activity were all induced to confirm the immunogenicity of M2 e.Hsp70c(Hsp70359–610). And challenge tests showed protection against H1N1 and H9N2 strains in vaccinated groups. Finally these results demonstrates M2 e.Hsp70c fusion protein can be a candidate for a universal influenza A vaccine.
文摘Objective: To produce fluorescent tagged recombinant erythroferrone protein(ERFE_eGFP) for laboratory investigations. Methods: Erythroferrone(ERFE) gene was fused to green fluorescent protein(eGFP) gene and cloned in a pSecTag2Hygro plasmid. The constructed plasmid was amplified in Escherichia coli DH5α and the eGFP-fused ERFE(ERFE_eGFP) protein was expressed in human embryonic kidney(HEK293T) cell line. Results: The plasmid constructed from colony C6 contained ERFE_eGFP with the correct restriction sizes of 4.2 kb and expressed secretory ERFE_eGFP fusion protein(approximately size of 75 kDa) in HEK293T cell line. Conclusions: ERFE_eGFP recombinant protein is successfully expressed as a secretory functional protein and could be sensitively detected using fluorometry. This fusion protein might benefit future applications for localization of cellular ERFE receptors and competitive immunoassay of ERFE concentration.
基金supported in part by the Chinese National Basic Research Program(973)2009CB118900Chinese National Science Foundation Project30771451Boyce Thompson Institute Project BTI-QAU1-23-2007
文摘It is well known that Tn5B1-4 (commercially known as the High Five) cell line is highly susceptible to baculovirus and provides superior production of recombinant proteins when compared to other insect cell lines.But the characteristics of the cell line do not always remain stable and may change upon continuous passage.Recently an alphanodavirus,named Tn5 Cell Line Virus (or TNCL Virus),was identified in High Five cells in particular.Therefore,we established a new cell line,QB-Tn9-4s,from Trichoplusia ni,which was determined to be free of TNCL virus by RT-PCR analysis.In this paper,we describe the development of a novel cell clone,QB-CL-B,from a low passage QB-Tn9-4s cell line and report its susceptibility to AcMNPV,and the level of recombinant protein production.This cell clone was similar to its parental cells QB-Tn9-4s and Tn5B1-4 cells in morphology and growth rate;although it also showed approximately the same responses to AcMNPV infection and production of occlusion bodies,there were higher levels of recombinant protein production in comparison to QB-Tn9-4s (parental cells) and High5 cells.
基金supported by the National Key Technology Research and Development Program of the Ministry of Science and Technology of China (No. 2015BAD16B01)TianjinKey Technology Research and Development Support Program (No. 13ZCDNC01900)
基金Supported by the Neational Natural Science Foundation of China, No. 39825116, 39970394.
文摘INTRODUCTIONAlthough reliable assays for the detection ofhepatitis C virus and E virus became available, still10% 20% hepatitis are not caused byhepatitis A-E virus[1-3]. In 1996, two research groups isolatedthis agent independently and almost simultaneouslyand named hepatitis G virus and GB virus C,respectively[4-7].
基金supported by the National Natural Science Foundation of China(No.81101376)
文摘Summary： A new type of TGF-β3 fusion protein with targeted therapy function was constructed, and its feasibility and target specificity of inducing chondrogenesis were investigated by transfecting LAP-MMP-mTGF-β3 gene into adipose-derived stem cells （ADSCs）. The recombinant pIRES- EGFP-MMP was constructed by inserting the sense and antisense DNA of encoding the amino acid of the synthetic MMP enzyme cutting site into the eukaryotic expression vector pIRES-EGFE LAP and mTGF-β3 fragments were obtained by using RT-PCR and inserted into the upstream and downstream of MMP from pIRES-EGFP-MMP respectively, and the recombinant plasmid of pIRES-EGFP- LAP-MMP-mTGF-β3 was constructed, which was transferred to ADSCs. The ADSCs were cultured and divided in three groups： experimental group （MMP group）, negative control group （no MMP） and non-transfection group. The morphological changes were observed microscopically, and the expression of proteoglycan and type II collagen （Col II） was detected by using Alcian blue staining and immuno- histochemistry staining at 7th, 14th and 21st day after culture. The recombinant plasmid of pIRES-EGFP-LAP-MMP-mTGF-β3 was correctly constructed by methods of enzyme cutting and se- quencing analysis. The mTGF-β3 fusion protein was successfully expressed after transfection, and in the presence of the MMP, active protein mTGF-β3 was generated, which significantly promoted differ- entiation of ADSCs into chondrocytes and the expression of cartilage matrix. The novel fusion protein LAP-MMP-mTGF-β3 can targetedly induce differentiation of ADSCs into chondrocytes, which would open up prospects for target therapy of cartilage damage repair in future.
基金Chinese National Technology Researchand Development Program (863 Program,2006AA10A204)
文摘In this study, a panel of monoclonal antibodies （mAbs） against Porcine reproductive and respiratory syndrome virus（PRRSV）, named as 8C9 and4B4, were produced by fusing SP2/0 myeloma cells and spleen cells of BALB/c mice immunized with the PRRSV （TCID50=5.5）, screened by the indirect ELISA and subjected to several limiting dilutions, mAbs were then identified by biological characterization. Among the two fusion cell strains, 8C9 belonged to the IgG1 subclass and 4B4 belonged to the IgG2a subclass. The titers in cell culture supematant and abdomen liquor reached to 1：104and 1：105, respectively. The specificity test indicated that the two cells had specific reactions for the PRRSV and GP5 protein respectively, and no reaction with Classical swine fever virus （CSFV） or Swine vesicular disease virus （SVDV）. The molecular weights of the heavy chain and light chain were about 45.0 kDa and 25.0 kDa, respectively. In neutralization activity tests, the results showed that the prepared mAb 4B4 can protect 50% of cells with no CPE in dilution up to 1：512, but mAB 8C9 has no neutralization activities to PRRSV.
文摘Objective To express the recombinant human bone morphogenetic protein-7 (rhBMP-7) in Chinese hamster ovary (CHO) cells and to establish the in vitro biological activity assay of rhBMP-7. Methods Human BMP-7 cDNA was subcloned into pcDNA3.1 mammalian expression vector and transfected to CHO cells by using the lipofectin transfection method. BMP-7 expression cell culture supernatants were harvested and purified for target protein. To analyze the bioactivity of the secreted rhBMP-7, a novel in vitro assay was established by measuring its alkaline phosphatase (ALP) stimulating of osteoblast cell line, W-20-17. Results BMP-7 stably expressing cell clone was selected, which secreted mature disulfide-linked homodimer form of hBMP-7 and had an apparent molecular weight of 36kDa. rhBMP-7 with >95% purity was obtained using 3 step chromatography method. Bioactivity assay showed that the purified protein specifically stimulated W-20-17 cell producing ALP, with a 4-fold increase of ALP activity at 100ng/ml or more, and the EC50 of 15.6ng/ml. Conclusion Purified rhBMP-7 from this CHO expression system has significant biological activity in induction of osteoblast phenotype, which demonstrates potential bone regeneration activity.
文摘To clone and express the recombinant outer membrane protein Tp0453 of Treponema pallidum and to analyze the immuno-reactivity and immunogenicity of the expressed protein, the immuno-dominant epitope of the Tp0453 was amplified from the complete genome of T.pallidum by PCR, subcloned into expression vector pQE32 to generate the recombinant plasmid pQE32/Tp0453, then expressed in E.coli M15 and analyzed by SDS/PAGE and Western blotting. The fusion protein expressed was purified with Ni-NTA affinity chromatography. Its immuno-reactivity was assayed by indirect ELISA, and the immunogenicity was determined by immunization with this fusion protein in New Zealand rabbits. In the present study, a fusion protein of molecular weight about 32 kDa was obtained. As demonstrated by Western blotting, the recombinant protein could react specifically with positive IgG sera of patients with syphilis, and the antibodies against T.pallidum in human sera were successfully detected by indirect ELISA. Both the sensitivity and specificity of ELISA based on the Tp0453 fusion protein as were 100% (30/30) when detected with control sera. In comparison with the results of IgG ELISA with those of TPPA. It was found that the sensitivity of ELISA was 96.8% and the specificity was 100%. The difference of ELISA and TPPA was not significant, and the concordance of results between ELISA and TPPA was 98.2%. In addition, specific humoral responses could be elicited by immunization with the recombinant fusion protein in New Zealand rabbits with a specific antibody titer of 1∶1280 after 3 successive doses of immunization. These results demonstrate that the expressed recombinant fusion protein shows excellent immuno-competence and provide foundation to develop a quick diagnostic kid applied to detect the presence of T.pallidum infections.
