[Objective] The aim of this study was to realize efficient expression of the porcine reproductive and respiratory syndrome virus （PRRSV） ORF7 gene in genetic engineering bacteria and analYze the immunological activi...[Objective] The aim of this study was to realize efficient expression of the porcine reproductive and respiratory syndrome virus （PRRSV） ORF7 gene in genetic engineering bacteria and analYze the immunological activity of the recombinant protein after purification. [ Method] The constructed recombinant expression vector pET-ORF7 was transformed into Escherichia co1BL21 （DE3） and induced by IPTG under the optimal condition. After analysis of SDS-PAGE and Western Blot, the expression products were purified by Ni-NTA His · Bind Resin chrom- atographic column under denaturing condition and renatured by gradient dialysis. Subsequently, the immunological activity of the renatured recombinant protein was detected by Westem Blot and indirect ELISA. [ Result] The recombinant plasmid pET-ORF7 expressed in E. coli successfully, and the fusion protein was in the form of inclusion body. By SDS-PAGE detection, the molecular weight of the expression protein was approximate 33 kD, according with the expectation. Analysis by Bandscan software showed that the expressed fusion protein was about 50% of total bacterial protein of BL21 （DE3）. Wastem Blot and indirect ELISA detection showed that the renatured protein could react with PRRSV positive serum specifically, indicating its good immunological activity. [ Conclusion] This study lays a foundation for the preparation of PRRSV monoclonal antibody and diagnostic kit.展开更多
Oyster,as a common aquatic food,play an important role in shellfish allergy.In this study,2 tropomyosin(TM)isoforms TM-αand TM-β(TM-α/-β)in Alectryonella plicatula were identified.The sequences of 852 bp encoding ...Oyster,as a common aquatic food,play an important role in shellfish allergy.In this study,2 tropomyosin(TM)isoforms TM-αand TM-β(TM-α/-β)in Alectryonella plicatula were identified.The sequences of 852 bp encoding 284 amino acids of TM-α/-βand 2 recombinant proteins were obtained,respectively.There were 12 amino acid differences between TM-α/-β.The results of immunological experiments indicated that TM-βhad stronger immunobinding activity and immunoreactivity than those of TM-α.Structural analysis showed that TM-βhad moreα-helix and higher surface hydrophobicity than TM-α.Sequences and epitopes alignment with shellfish TMs revealed that amino acids of TM-βwere more frequently recognized as IgE epitopes in other shellfish TMs than TM-α.Differences in structure and sequence account for the higher immunological activity of TM-βcompared to TM-α.These findings provide a theoretical basis for enriching the understanding of shellfish TM and accurate diagnosis of allergic components.展开更多
T-cell activation requires the formation of the immunological sy napse(IS)bet ween a T-cll and anantigen-presenting cell(AP C)to control the development of the adaptive immune response.How-ever,calcium release,an init...T-cell activation requires the formation of the immunological sy napse(IS)bet ween a T-cll and anantigen-presenting cell(AP C)to control the development of the adaptive immune response.How-ever,calcium release,an initial signal of T-cell activation,has been found to occur before IS for-mation.The mechanism for triggering the calcium signaling and relationship bet ween calciumrelease and IS format ion remains unclear.Herein,using live-cell imaging,we found that int ercellularadhesion molecule 1(ICAM-1),an essential mdlecule for IS formation,accumulated and then wasdepleted at the center of the synapse before complete IS formation.During the proces of ICAM1depletion,calcium was released.if ICAM-1 failed to be depleted from the center of the synapse,thesustained calcium signaling could not be induced.Moreover,depletion of ICAM-1 in ISs preferen-tially ccurred with the contact of antigen-specific T-cels and dendritic clls(DCs).Blocking thebinding ofICA M-1 and lymphocy te finction-associated antigen 1(LFA-1),ICAM-1 failed to depleteat the center of the synapse,and calcium release in T-clls decreased.In studying the mechanism ofhow the depletion ofiCA M1 could influence calcium release in T-clls,we found that the movementof ICAM-1 was associat ed with the localization of LFA-1 in the IS,which afected the localization ofcalcium microdomains,ORAIl and mitochondria in IS.Therefore,the depletion of ICAM-1 in the center of the synapse is an important factor for an initial sust ained calcium release in T-cells.展开更多
Objective：With the regular mixed lymphocytes culture （MLC） to detect the allograft rejection, the reactivity of the activated lymphocytes （primed lymphocytes） of a recipient shows sometimes increase and sometimes...Objective：With the regular mixed lymphocytes culture （MLC） to detect the allograft rejection, the reactivity of the activated lymphocytes （primed lymphocytes） of a recipient shows sometimes increase and sometimes decrease against the antigens from the donor, which is inconsistent with the clinical results. In order to establish a convenient method for testing the specificity of the activated lymphocytes in vitro, so as to know the rejection occurred or not by testing the existence of the specific activated lymphocytes against donor＇s HLA antigens in the recipient＇s peripheral blood. Methods： Anti-IL-2 neutralizing monoclonal antibody （anti-IL-2 N-mAb） and immunosuppressors were introduced in this test system in the presence of specific stimulators and activated lymphocytes. Results ： When the activated lymphocytes were chosen from the one-way MLC 4 d to undergo re-stimulation by specific stimulators, the activity of activated lymphocytes in the treatment group was suppressed significantly compared with that in the control group. The result of this test method is consistent with the biopsy in the clinical diagnosis of rejection. Conclusion：h suggests that the activated lymphocytes can be inactivated by specific antigens in certain conditions. This can be a useful tool to define the specificity of the activated lymphocytes.展开更多
In this study,the effects of three different concentrations（0.5,0.25 and 0.125μl/L）of dichlorvos solution on phenoloxidase（PO）activity,hemolysin activity,peroxidase（POD）activity and antibacterial activity in th...In this study,the effects of three different concentrations（0.5,0.25 and 0.125μl/L）of dichlorvos solution on phenoloxidase（PO）activity,hemolysin activity,peroxidase（POD）activity and antibacterial activity in the serum of Macrobrachium nipponense during four days were investigated.The results indicated that phenoloxidase activity,hemolysin activity,peroxidase activity and antibacterial activity in the serum of Macrobrachium nipponense were improved under the stress of dichlorvos during a short time.With the extension of stress duration and increase of dichlorvos concentration,the activities of various immunological indices were inhibited due to the cumulative effect of dichlorvos in vivo;overall,the reduction increased gradually with the extension of stress duration.展开更多
Objectives To investigate the anti-inflammatory components from the leaves of Liriodendron chinensis. Methods The 95% alcohol extract from the leaves of L. chinensis was subjected to column chromatography, and the str...Objectives To investigate the anti-inflammatory components from the leaves of Liriodendron chinensis. Methods The 95% alcohol extract from the leaves of L. chinensis was subjected to column chromatography, and the structures of purified compounds were determined by spectral methods. The bioassay was performed through the inhibitory effects on the inflammatory cells activated by lipopolysaccharide （LPS）. Results Nine compounds were isolated, including octacosanoic acid （1）, stearic acid （2）, （2R-2-hydroxy-N-[（2S,3S,4R,8E- l ,3,4-tri-hydroxyicos-8-en- 2-yl]tetracosanamide （3）, （2 R）- 2- hyd roxy- N- [（2 S, 3 S,4 R,8 E）- 1 - O-β- D-glucopyranosyloxy-3,4-dihydroxy- octadec-8-en-2-yl]eicosanamide （4）, （2R）-2-hydroxy-N-[（2S,3S,4R,8E）-l-O-β-D- glucopyranosyloxy-3,4-dihydroxyoctadec-8-en-2-yl]hexadecanamide （5）, dicentrinone （6）, liriodenine （7）, daucosterol （8）, and liriodendritol （9） and among which five compounds could significantly lower the content of nitric oxide （NO） from peritoneal macrophages of rats induced by LPS and reduce the splenic lymphocyte proliferation in mice. This is the first report on the occurrence of ceramides and dicentrinone in the plants of Liriodendron Linn. Conclusion The five compounds are likely to be anti-inflammatory compounds concerning to their pronounced inhibitory action on the activated inflammatory cells. This assessment might provide a basis for searching the potent active compounds used for the treatment of inflammation.展开更多
To prepare an effective immune preparation to treat hepatitis B, hens were immunized with hepatitis B vaccines, and then anti-HBV egg yolk extraction （anti-HBV EYE） was refined from egg yolk by a dialyzable method. ...To prepare an effective immune preparation to treat hepatitis B, hens were immunized with hepatitis B vaccines, and then anti-HBV egg yolk extraction （anti-HBV EYE） was refined from egg yolk by a dialyzable method. Its chemical characteristics were identified by ultraviolet spectrum, HPLC, Lowry analysis and pharmacopocia-raleted methods. The specific immunological activity was examined by leukocyte adherence inhibition （LAI） in vitro and delayed type hypersensitivity （DTH） in vivo. Anti-HBV EYE was a small dialyzable substance with molecular weight less than 12 kD containing 18 kinds of amino acids. The preparation could obviously inhibit LAI and DTH which was similar to hepatitis B virus-specific transfer factor of pig spleen. However, there were no similar effects observed in the nonspecific transfer factor （NTF） group, control egg yolk extraction （CEYE） group and hepatitis A virus （HAV） group. The results suggested that anti-HBV EYE contained hepatitis B virus-specific transfer factor （STF） and had the antigen-specific cell immune activity similar to PSHBV-TF. The STF obtained from egg yolk of the hens immunized with specific antigen, might be a potential candidate for immunoregulation in hepatitis B prevention and treatment.展开更多
文摘[Objective] The aim of this study was to realize efficient expression of the porcine reproductive and respiratory syndrome virus （PRRSV） ORF7 gene in genetic engineering bacteria and analYze the immunological activity of the recombinant protein after purification. [ Method] The constructed recombinant expression vector pET-ORF7 was transformed into Escherichia co1BL21 （DE3） and induced by IPTG under the optimal condition. After analysis of SDS-PAGE and Western Blot, the expression products were purified by Ni-NTA His · Bind Resin chrom- atographic column under denaturing condition and renatured by gradient dialysis. Subsequently, the immunological activity of the renatured recombinant protein was detected by Westem Blot and indirect ELISA. [ Result] The recombinant plasmid pET-ORF7 expressed in E. coli successfully, and the fusion protein was in the form of inclusion body. By SDS-PAGE detection, the molecular weight of the expression protein was approximate 33 kD, according with the expectation. Analysis by Bandscan software showed that the expressed fusion protein was about 50% of total bacterial protein of BL21 （DE3）. Wastem Blot and indirect ELISA detection showed that the renatured protein could react with PRRSV positive serum specifically, indicating its good immunological activity. [ Conclusion] This study lays a foundation for the preparation of PRRSV monoclonal antibody and diagnostic kit.
基金supported by the National Natural Scientific Foundation of China(32072336,32472449).
文摘Oyster,as a common aquatic food,play an important role in shellfish allergy.In this study,2 tropomyosin(TM)isoforms TM-αand TM-β(TM-α/-β)in Alectryonella plicatula were identified.The sequences of 852 bp encoding 284 amino acids of TM-α/-βand 2 recombinant proteins were obtained,respectively.There were 12 amino acid differences between TM-α/-β.The results of immunological experiments indicated that TM-βhad stronger immunobinding activity and immunoreactivity than those of TM-α.Structural analysis showed that TM-βhad moreα-helix and higher surface hydrophobicity than TM-α.Sequences and epitopes alignment with shellfish TMs revealed that amino acids of TM-βwere more frequently recognized as IgE epitopes in other shellfish TMs than TM-α.Differences in structure and sequence account for the higher immunological activity of TM-βcompared to TM-α.These findings provide a theoretical basis for enriching the understanding of shellfish TM and accurate diagnosis of allergic components.
基金supported by the National Major Scientic Research Program of China(Grant No.2011CB910404)the National Nature Science Foundation of China(Grant Nos.61227017,31400772 and 81273215)+3 种基金the National Science Fund for Distinguished Young Scholars(Grant No.61425006)the grants of the Project for Laureate of Taishan Scholar(Grant No.ts201511075)the Innovation Project of Shandong Academy of Medical Sciences,the Projects of medical and health technology development program in Shandong province(No.2015WS0194)the science and technology program from Shandong Academy of Medical Sciences(No.2015-25).
文摘T-cell activation requires the formation of the immunological sy napse(IS)bet ween a T-cll and anantigen-presenting cell(AP C)to control the development of the adaptive immune response.How-ever,calcium release,an initial signal of T-cell activation,has been found to occur before IS for-mation.The mechanism for triggering the calcium signaling and relationship bet ween calciumrelease and IS format ion remains unclear.Herein,using live-cell imaging,we found that int ercellularadhesion molecule 1(ICAM-1),an essential mdlecule for IS formation,accumulated and then wasdepleted at the center of the synapse before complete IS formation.During the proces of ICAM1depletion,calcium was released.if ICAM-1 failed to be depleted from the center of the synapse,thesustained calcium signaling could not be induced.Moreover,depletion of ICAM-1 in ISs preferen-tially ccurred with the contact of antigen-specific T-cels and dendritic clls(DCs).Blocking thebinding ofICA M-1 and lymphocy te finction-associated antigen 1(LFA-1),ICAM-1 failed to depleteat the center of the synapse,and calcium release in T-clls decreased.In studying the mechanism ofhow the depletion ofiCA M1 could influence calcium release in T-clls,we found that the movementof ICAM-1 was associat ed with the localization of LFA-1 in the IS,which afected the localization ofcalcium microdomains,ORAIl and mitochondria in IS.Therefore,the depletion of ICAM-1 in the center of the synapse is an important factor for an initial sust ained calcium release in T-cells.
文摘Objective：With the regular mixed lymphocytes culture （MLC） to detect the allograft rejection, the reactivity of the activated lymphocytes （primed lymphocytes） of a recipient shows sometimes increase and sometimes decrease against the antigens from the donor, which is inconsistent with the clinical results. In order to establish a convenient method for testing the specificity of the activated lymphocytes in vitro, so as to know the rejection occurred or not by testing the existence of the specific activated lymphocytes against donor＇s HLA antigens in the recipient＇s peripheral blood. Methods： Anti-IL-2 neutralizing monoclonal antibody （anti-IL-2 N-mAb） and immunosuppressors were introduced in this test system in the presence of specific stimulators and activated lymphocytes. Results ： When the activated lymphocytes were chosen from the one-way MLC 4 d to undergo re-stimulation by specific stimulators, the activity of activated lymphocytes in the treatment group was suppressed significantly compared with that in the control group. The result of this test method is consistent with the biopsy in the clinical diagnosis of rejection. Conclusion：h suggests that the activated lymphocytes can be inactivated by specific antigens in certain conditions. This can be a useful tool to define the specificity of the activated lymphocytes.
文摘In this study,the effects of three different concentrations（0.5,0.25 and 0.125μl/L）of dichlorvos solution on phenoloxidase（PO）activity,hemolysin activity,peroxidase（POD）activity and antibacterial activity in the serum of Macrobrachium nipponense during four days were investigated.The results indicated that phenoloxidase activity,hemolysin activity,peroxidase activity and antibacterial activity in the serum of Macrobrachium nipponense were improved under the stress of dichlorvos during a short time.With the extension of stress duration and increase of dichlorvos concentration,the activities of various immunological indices were inhibited due to the cumulative effect of dichlorvos in vivo;overall,the reduction increased gradually with the extension of stress duration.
基金Priority academic program development of Jiangsu Higher Education Institutions(ysxk-2014)
文摘Objectives To investigate the anti-inflammatory components from the leaves of Liriodendron chinensis. Methods The 95% alcohol extract from the leaves of L. chinensis was subjected to column chromatography, and the structures of purified compounds were determined by spectral methods. The bioassay was performed through the inhibitory effects on the inflammatory cells activated by lipopolysaccharide （LPS）. Results Nine compounds were isolated, including octacosanoic acid （1）, stearic acid （2）, （2R-2-hydroxy-N-[（2S,3S,4R,8E- l ,3,4-tri-hydroxyicos-8-en- 2-yl]tetracosanamide （3）, （2 R）- 2- hyd roxy- N- [（2 S, 3 S,4 R,8 E）- 1 - O-β- D-glucopyranosyloxy-3,4-dihydroxy- octadec-8-en-2-yl]eicosanamide （4）, （2R）-2-hydroxy-N-[（2S,3S,4R,8E）-l-O-β-D- glucopyranosyloxy-3,4-dihydroxyoctadec-8-en-2-yl]hexadecanamide （5）, dicentrinone （6）, liriodenine （7）, daucosterol （8）, and liriodendritol （9） and among which five compounds could significantly lower the content of nitric oxide （NO） from peritoneal macrophages of rats induced by LPS and reduce the splenic lymphocyte proliferation in mice. This is the first report on the occurrence of ceramides and dicentrinone in the plants of Liriodendron Linn. Conclusion The five compounds are likely to be anti-inflammatory compounds concerning to their pronounced inhibitory action on the activated inflammatory cells. This assessment might provide a basis for searching the potent active compounds used for the treatment of inflammation.
文摘To prepare an effective immune preparation to treat hepatitis B, hens were immunized with hepatitis B vaccines, and then anti-HBV egg yolk extraction （anti-HBV EYE） was refined from egg yolk by a dialyzable method. Its chemical characteristics were identified by ultraviolet spectrum, HPLC, Lowry analysis and pharmacopocia-raleted methods. The specific immunological activity was examined by leukocyte adherence inhibition （LAI） in vitro and delayed type hypersensitivity （DTH） in vivo. Anti-HBV EYE was a small dialyzable substance with molecular weight less than 12 kD containing 18 kinds of amino acids. The preparation could obviously inhibit LAI and DTH which was similar to hepatitis B virus-specific transfer factor of pig spleen. However, there were no similar effects observed in the nonspecific transfer factor （NTF） group, control egg yolk extraction （CEYE） group and hepatitis A virus （HAV） group. The results suggested that anti-HBV EYE contained hepatitis B virus-specific transfer factor （STF） and had the antigen-specific cell immune activity similar to PSHBV-TF. The STF obtained from egg yolk of the hens immunized with specific antigen, might be a potential candidate for immunoregulation in hepatitis B prevention and treatment.
