Mature porcine interleukin-2 （pIL-2） gene was amplified by PCR from the plasmid pGEM-T-pIL2 and cloned into the baculovirus pFastBacTM Dual vector of the Bac-to-Bac baculovirus expression system under the control of...Mature porcine interleukin-2 （pIL-2） gene was amplified by PCR from the plasmid pGEM-T-pIL2 and cloned into the baculovirus pFastBacTM Dual vector of the Bac-to-Bac baculovirus expression system under the control of the PH promoter. Recombinant plL-2 （rpIL-2） expressed in Sf9 insect cells was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunofluorescence assay. Western blot analysis confirmed that the rpIL-2 protein had a molecular mass of 20 kDa, which was larger than the molecular mass of the mature protein predicted based on its peptide sequence. The rpIL-2 protein induced in vitro proliferation of ConA-stimulated porcine splenocytes and enhanced in vivo protective immune responses induced by vaccinating the pigs with inactivated oil emulsion vaccine against swine influenza virus. The results showed that the rpIL-2 expressed in Sf9 insect cells has immunoenhancement effects; the finding lays the foundation for the preparation of a specific recombinant IL-2 protein and the development of a novel immune adjuvant of vaccines against various infectious porcine pathogens to increase the immunoprotective efficacy of vaccines.展开更多
Porcine interleukin-2 and porcine interleukin-6 cDNA sequences were cloned into the expressing vectors pET-28a and pGEX-KG respectively. They were expressed in E. coli BL21(DE3)with high-level production. The gene del...Porcine interleukin-2 and porcine interleukin-6 cDNA sequences were cloned into the expressing vectors pET-28a and pGEX-KG respectively. They were expressed in E. coli BL21(DE3)with high-level production. The gene deleted vaccine of pseudorabies virus Ea strain(TK-/gG-/LacZ+)was mixed with the two different purified recombinant proteins each, or both, with the doses of 2, 5 or 10 μg ml-1. Ten groups of pseudorabies negative antibody swines were immuned twice with tested vaccines with different doses, or control vaccine, respectively. The antibody liters of the test groups were detected by neutralization test, and the daily weight gains of swines were calculated and analyzed statistically. In the study, all the neutralizing antibody ti-ters in test groups were higher than the control group, and the recombinant proteins appeared a dose dependent adjuvant effect. The tested vaccines with 2 μg ml-1 pIL-2 and with 10 μg ml-1 pIL-2/pIL-6 got significant and extremely significant differences, compared with the vaccines without pILs. The difference of the daily weight gain indicated the potential positive influence of pIL-2 and pIL-6 on immune protection.展开更多
Background Oxidative stress,caused by an imbalance in the production and elimination of intracellular reactive oxygen species(ROS),has been recognized for its detrimental effects on mammalian embryonic development.Lut...Background Oxidative stress,caused by an imbalance in the production and elimination of intracellular reactive oxygen species(ROS),has been recognized for its detrimental effects on mammalian embryonic development.Luteolin(Lut)has been documented for its protective effects against oxidative stress in various studies.However,its specific role in embryonic development remains unexplored.This study aims to investigate the influence of Lut on porcine embryonic development and to elucidate the underlying mechanism.Results After undergoing parthenogenetic activation(PA)or in vitro fertilization,embryos supplemented with 0.5μmol/L Lut displayed a significant enhancement in cleavage and blastocyst formation rates,with an increase in total cell numbers and a decrease in the apoptosis rate compared to the control.Measurements on D2 and D6 revealed that embryos with Lut supplementation had lower ROS levels and higher glutathione levels compared to the control.Moreover,Lut supplementation significantly augmented mitochondrial content and membrane potential.Intriguingly,activation of the Nrf2/Keap1 signaling pathway was observed in embryos supplemented with Lut,leading to the upregulation of antioxidant-related gene transcription levels.To further validate the relationship between the Nrf2/Keap1 signaling pathway and effects of Lut in porcine embryonic development,we cultured PA embryos in a medium supplemented with brusatol,with or without the inclusion of Lut.The positive effects of Lut on developmental competence were negated by brusatol treatment.Conclusions Our findings indicate that Lut-mediated activation of the Nrf2/Keap1 signaling pathway contributes to the enhanced production of porcine embryos with high developmental competence,and offers insight into the mechanisms regulating early embryonic development.展开更多
Diabetic cardiomyopathy is a disorder of the cardiac muscle that affects patients with diabetes.The exact mechanisms underlying diabetic cardiomyopathy are mostly unknown,but several factors have been implicated in th...Diabetic cardiomyopathy is a disorder of the cardiac muscle that affects patients with diabetes.The exact mechanisms underlying diabetic cardiomyopathy are mostly unknown,but several factors have been implicated in the pathogenesis of the disease and its progression towards heart failure,including endothelial dysfunction,autonomic neuropathy,metabolic alterations,oxidative stress,and alterations in ion homeostasis,especially calcium transients[1].In Military Medical Research,Jiang et al.[2]sought to determine the functional role of complement factor D(Adipsin)in the pathophysiology of diabetic cardiomyopathy.展开更多
BACKGROUND Mycoplasma pneumoniae(MP)frequently causes respiratory infections in children,whereas Epstein-Barr virus(EBV)typically presents subclinical manifestations in immunocompetent pediatric populations.The incide...BACKGROUND Mycoplasma pneumoniae(MP)frequently causes respiratory infections in children,whereas Epstein-Barr virus(EBV)typically presents subclinical manifestations in immunocompetent pediatric populations.The incidence of MP and EBV coinfections is often overlooked clinically,with the contributory role of EBV in pulmonary infections alongside MP remaining unclear.AIM To evaluate the serum concentrations of interleukin-2(IL-2)and interleukin-12(IL-12)in pediatric patients with MP pneumonia co-infected with EBV and assess their prognostic implications.METHODS We retrospectively analyzed clinical data from patients diagnosed with MP and EBV co-infection,isolated MP infection,and a control group of healthy children,spanning from January 1,2018 to December 31,2021.Serum IL-2 and IL-12 levels were quantified using enzyme-linked immunosorbent assay.Logistic regression was employed to identify factors influencing poor prognosis,while receiver operating characteristic(ROC)curves evaluated the prognostic utility of serum IL-2 and IL-12 levels in co-infected patients.RESULTS The co-infection group exhibited elevated serum IL-2 and C-reactive protein(CRP)levels compared to both the MP-only and control groups,with a reverse trend observed for IL-12(P<0.05).In the poor prognosis cohort,elevated CRP and IL-2 levels,alongside prolonged fever duration,contrasted with reduced IL-12 levels(P<0.05).Logistic regression identified elevated IL-2 as an independent risk factor and high IL-12 as a protective factor for adverse outcomes(P<0.05).ROC analysis indicated that the area under the curves for IL-2,IL-12,and their combination in predicting poor prognosis were 0.815,0.895,and 0.915,respectively.CONCLUSION Elevated serum IL-2 and diminished IL-12 levels in pediatric patients with MP and EBV co-infection correlate with poorer prognosis,with combined IL-2 and IL-12 levels offering enhanced predictive accuracy.展开更多
[Objective] This study aimed to investigate the changes of the transcriptional levels of molecules associated with endogenous antigen processing and presenta- tion in porcine skin-derived dendritic cells infected with...[Objective] This study aimed to investigate the changes of the transcriptional levels of molecules associated with endogenous antigen processing and presenta- tion in porcine skin-derived dendritic cells infected with PCV2 in vivo. [Method] Healthy 40-day-old Landrace piglets were infected with porcine circovirus type 2 （PCV2） and euthanized on the 34, 7rd, 14th, 21st and 35th d post inoculation （DPI）. The porcine skin-derived dendritic cells （DCs） were collected to analyze the transcrip- tional levels of molecules （LMP7, UBP, MHC-I, calreticulin） associated with endogenous antigen processing and presentation by using real-time fluorescent quantitative PCR （real-time FQ-PCR）. [Result] The results showed that the level of LMP7 mR- NAs was reduced significantly on the 3DPI （P〈0.05）; the level of UBP mRNAs was consistently up-regulated, which increased significantly on the 21DPI and 35DPI （P〈 0.05）; the level of MHC-I mRNAs was significantly down-regulated on the 7DPI （P〈 0.05）; the level of calreticulin mRNAs was up-regulated slightly without significant dif- ference. [Conclusion] PCV2 can inhibit the endogenous antigen processing and presentation ability of porcine skin-derived DCs at early stages of infection.展开更多
BALB/c mice were immunized using synthetic tandem polypeptide of Cap protein epitope of porcine circovirus type 2 （PCV2） as the antigen. By using lym-phocyte hybridoma technique, a hybridoma cellline stably secretin...BALB/c mice were immunized using synthetic tandem polypeptide of Cap protein epitope of porcine circovirus type 2 （PCV2） as the antigen. By using lym-phocyte hybridoma technique, a hybridoma cellline stably secreting monoclonal an-tibody against PCV2-rCap protein was successful y obtained and named as 670#. The ascites titer of the obtained monoclonal antibody was 1∶100 000. Western blot results showed that the monoclonal antibody could react with prokaryotical y ex-pressed PET32a-ORF2 recombinant protein, eukaryotical y expressed ORF1-ORF2 tandem protein and PCV2 whole virus celllysate. Indirect EILSA demonstrated that the monoclonal antibody could bind with ORF1-ORF2 tandem protein. Indirect im-munofluorescence assay （IFA） indicated that the monoclonal antibody could identify native PCV2 virus. The preparation of this monoclonal antibody provided technical tools for epitope analysis and molecular diagnosis of PCV2 virus.展开更多
[Objective] This study aimed to investigate the genetic variation of porcine circovirus type 2 （PCV2） in China. [Method] The strain was isolated from infected samples by cel passage and preliminarily identified by P...[Objective] This study aimed to investigate the genetic variation of porcine circovirus type 2 （PCV2） in China. [Method] The strain was isolated from infected samples by cel passage and preliminarily identified by PCR and IFA. Ful-length genome of the isolated strain was obtained by specific amplification for homology and phylogenetic analysis. [Result] A PCV2 strain was successful y isolated and named 201105ZJ, which could proliferate in PK15 cel lines. Specific fragments could be amplified by specific PCR assay. According to results of IFA assay, specif-ic immunofluorescence was observed; the TCID50 was low （102.67）; the ful-length genome sequence of the isolated strain was 1 768 bp, sharing 94.1%-96.8% ho-mology with 13 reference strains; to be specific, the isolated strain exhibited the highest homology of 96.8% with AF055392PCV2a; the isolated strain 201105ZJ and reference strain AF055392 belonged to genotype PCV2a, exhibiting a distant genetic relationship with genotype PCV2c. [Conclusion] Characteristics of genetic variation of PCV2 isolate 201105ZJ provided theoretical basis for vaccine development, investi-gation of PCV2 pathogenesis, and prevention and control of porcine circovirus-as-sociated diseases （PCVAD） in East China.展开更多
Porcine circovirus type 2 (PCV2) is closely related to the postweaning multisystemic wasting syndrome (PMWS). In this study, the pig serum and tissue samples collected from different regions of Hangzhou District in Zh...Porcine circovirus type 2 (PCV2) is closely related to the postweaning multisystemic wasting syndrome (PMWS). In this study, the pig serum and tissue samples collected from different regions of Hangzhou District in Zhejiang Province of China between 2003 and 2005 were analyzed by enzyme-linked immunosorbent assay (ELISA) for PCV2 antibody and by polymerase chain reaction (PCR) for ORF2 gene. The results show that out of 1250 randomly collected serum samples, 500 sera (40%) were seropositive for PCV2. PCR results demonstrate that Hangzhou PCV2 with more than 50% Chinese PCV2 strains and French PCV2 formed Cluster A. Only one PCV2 from Hangzhou belonged to Cluster B with some other Chinese PCV2 and Netherlands’s isolates. Cluster C consisted of PCV2 isolates from China, US, Canada, UK and Germany. The results indicate that the PCV2 infection was widespread in Hangzhou.展开更多
基金supported by a grant from the the Key Technology R&D Program of China (2008BADB2B01)
文摘Mature porcine interleukin-2 （pIL-2） gene was amplified by PCR from the plasmid pGEM-T-pIL2 and cloned into the baculovirus pFastBacTM Dual vector of the Bac-to-Bac baculovirus expression system under the control of the PH promoter. Recombinant plL-2 （rpIL-2） expressed in Sf9 insect cells was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunofluorescence assay. Western blot analysis confirmed that the rpIL-2 protein had a molecular mass of 20 kDa, which was larger than the molecular mass of the mature protein predicted based on its peptide sequence. The rpIL-2 protein induced in vitro proliferation of ConA-stimulated porcine splenocytes and enhanced in vivo protective immune responses induced by vaccinating the pigs with inactivated oil emulsion vaccine against swine influenza virus. The results showed that the rpIL-2 expressed in Sf9 insect cells has immunoenhancement effects; the finding lays the foundation for the preparation of a specific recombinant IL-2 protein and the development of a novel immune adjuvant of vaccines against various infectious porcine pathogens to increase the immunoprotective efficacy of vaccines.
基金supported by a grant from the National High Tech R&D Program(863 Program)of China(2001AA213051).
文摘Porcine interleukin-2 and porcine interleukin-6 cDNA sequences were cloned into the expressing vectors pET-28a and pGEX-KG respectively. They were expressed in E. coli BL21(DE3)with high-level production. The gene deleted vaccine of pseudorabies virus Ea strain(TK-/gG-/LacZ+)was mixed with the two different purified recombinant proteins each, or both, with the doses of 2, 5 or 10 μg ml-1. Ten groups of pseudorabies negative antibody swines were immuned twice with tested vaccines with different doses, or control vaccine, respectively. The antibody liters of the test groups were detected by neutralization test, and the daily weight gains of swines were calculated and analyzed statistically. In the study, all the neutralizing antibody ti-ters in test groups were higher than the control group, and the recombinant proteins appeared a dose dependent adjuvant effect. The tested vaccines with 2 μg ml-1 pIL-2 and with 10 μg ml-1 pIL-2/pIL-6 got significant and extremely significant differences, compared with the vaccines without pILs. The difference of the daily weight gain indicated the potential positive influence of pIL-2 and pIL-6 on immune protection.
基金supported by the Korea Research Institute of Bioscience and Biotechnology(KRIBB)Research Initiative Program(KGM4252331,KGM5382322),Republic of Korea.
文摘Background Oxidative stress,caused by an imbalance in the production and elimination of intracellular reactive oxygen species(ROS),has been recognized for its detrimental effects on mammalian embryonic development.Luteolin(Lut)has been documented for its protective effects against oxidative stress in various studies.However,its specific role in embryonic development remains unexplored.This study aims to investigate the influence of Lut on porcine embryonic development and to elucidate the underlying mechanism.Results After undergoing parthenogenetic activation(PA)or in vitro fertilization,embryos supplemented with 0.5μmol/L Lut displayed a significant enhancement in cleavage and blastocyst formation rates,with an increase in total cell numbers and a decrease in the apoptosis rate compared to the control.Measurements on D2 and D6 revealed that embryos with Lut supplementation had lower ROS levels and higher glutathione levels compared to the control.Moreover,Lut supplementation significantly augmented mitochondrial content and membrane potential.Intriguingly,activation of the Nrf2/Keap1 signaling pathway was observed in embryos supplemented with Lut,leading to the upregulation of antioxidant-related gene transcription levels.To further validate the relationship between the Nrf2/Keap1 signaling pathway and effects of Lut in porcine embryonic development,we cultured PA embryos in a medium supplemented with brusatol,with or without the inclusion of Lut.The positive effects of Lut on developmental competence were negated by brusatol treatment.Conclusions Our findings indicate that Lut-mediated activation of the Nrf2/Keap1 signaling pathway contributes to the enhanced production of porcine embryos with high developmental competence,and offers insight into the mechanisms regulating early embryonic development.
基金National Institutes of Health(NIH):National Heart,Lung,and Blood Institute(NHLBI:R01-HL164772,R01-HL159062,R01-HL146691,T32-HL144456)National Institute of Diabetes and Digestive and Kidney Diseases(NIDDK:R01-DK123259,R01-DK033823)+2 种基金National Center for Advancing Translational Sciences(NCATS:UL1-TR002556-06,UM1-TR004400)(to Gaetano Santulli)Diabetes Action Research and Education Foundation(to Gaetano Santulli)Monique Weill-Caulier and Irma T.Hirschl Trusts(to Gaetano Santulli).
文摘Diabetic cardiomyopathy is a disorder of the cardiac muscle that affects patients with diabetes.The exact mechanisms underlying diabetic cardiomyopathy are mostly unknown,but several factors have been implicated in the pathogenesis of the disease and its progression towards heart failure,including endothelial dysfunction,autonomic neuropathy,metabolic alterations,oxidative stress,and alterations in ion homeostasis,especially calcium transients[1].In Military Medical Research,Jiang et al.[2]sought to determine the functional role of complement factor D(Adipsin)in the pathophysiology of diabetic cardiomyopathy.
文摘BACKGROUND Mycoplasma pneumoniae(MP)frequently causes respiratory infections in children,whereas Epstein-Barr virus(EBV)typically presents subclinical manifestations in immunocompetent pediatric populations.The incidence of MP and EBV coinfections is often overlooked clinically,with the contributory role of EBV in pulmonary infections alongside MP remaining unclear.AIM To evaluate the serum concentrations of interleukin-2(IL-2)and interleukin-12(IL-12)in pediatric patients with MP pneumonia co-infected with EBV and assess their prognostic implications.METHODS We retrospectively analyzed clinical data from patients diagnosed with MP and EBV co-infection,isolated MP infection,and a control group of healthy children,spanning from January 1,2018 to December 31,2021.Serum IL-2 and IL-12 levels were quantified using enzyme-linked immunosorbent assay.Logistic regression was employed to identify factors influencing poor prognosis,while receiver operating characteristic(ROC)curves evaluated the prognostic utility of serum IL-2 and IL-12 levels in co-infected patients.RESULTS The co-infection group exhibited elevated serum IL-2 and C-reactive protein(CRP)levels compared to both the MP-only and control groups,with a reverse trend observed for IL-12(P<0.05).In the poor prognosis cohort,elevated CRP and IL-2 levels,alongside prolonged fever duration,contrasted with reduced IL-12 levels(P<0.05).Logistic regression identified elevated IL-2 as an independent risk factor and high IL-12 as a protective factor for adverse outcomes(P<0.05).ROC analysis indicated that the area under the curves for IL-2,IL-12,and their combination in predicting poor prognosis were 0.815,0.895,and 0.915,respectively.CONCLUSION Elevated serum IL-2 and diminished IL-12 levels in pediatric patients with MP and EBV co-infection correlate with poorer prognosis,with combined IL-2 and IL-12 levels offering enhanced predictive accuracy.
基金Supported by Natural Science Foundation of Beijing "Effect of porcine skin-derived dendritic cells on PCV infection" (6062006)Beijing Organization Department Project"Influence of PCV infection on bone marrow cell differentiation" (20061D0502100282)~~
文摘[Objective] This study aimed to investigate the changes of the transcriptional levels of molecules associated with endogenous antigen processing and presenta- tion in porcine skin-derived dendritic cells infected with PCV2 in vivo. [Method] Healthy 40-day-old Landrace piglets were infected with porcine circovirus type 2 （PCV2） and euthanized on the 34, 7rd, 14th, 21st and 35th d post inoculation （DPI）. The porcine skin-derived dendritic cells （DCs） were collected to analyze the transcrip- tional levels of molecules （LMP7, UBP, MHC-I, calreticulin） associated with endogenous antigen processing and presentation by using real-time fluorescent quantitative PCR （real-time FQ-PCR）. [Result] The results showed that the level of LMP7 mR- NAs was reduced significantly on the 3DPI （P〈0.05）; the level of UBP mRNAs was consistently up-regulated, which increased significantly on the 21DPI and 35DPI （P〈 0.05）; the level of MHC-I mRNAs was significantly down-regulated on the 7DPI （P〈 0.05）; the level of calreticulin mRNAs was up-regulated slightly without significant dif- ference. [Conclusion] PCV2 can inhibit the endogenous antigen processing and presentation ability of porcine skin-derived DCs at early stages of infection.
基金Supported by National Natural Science Foundation of China(31302071)AgriculturalScience and Technology Independent Innovation Fund of Jiangsu Province(TechnicalInnovation)[CX(13)3065]~~
文摘BALB/c mice were immunized using synthetic tandem polypeptide of Cap protein epitope of porcine circovirus type 2 （PCV2） as the antigen. By using lym-phocyte hybridoma technique, a hybridoma cellline stably secreting monoclonal an-tibody against PCV2-rCap protein was successful y obtained and named as 670#. The ascites titer of the obtained monoclonal antibody was 1∶100 000. Western blot results showed that the monoclonal antibody could react with prokaryotical y ex-pressed PET32a-ORF2 recombinant protein, eukaryotical y expressed ORF1-ORF2 tandem protein and PCV2 whole virus celllysate. Indirect EILSA demonstrated that the monoclonal antibody could bind with ORF1-ORF2 tandem protein. Indirect im-munofluorescence assay （IFA） indicated that the monoclonal antibody could identify native PCV2 virus. The preparation of this monoclonal antibody provided technical tools for epitope analysis and molecular diagnosis of PCV2 virus.
基金Supported by National Natural Science Foundation of China(31302071)Special Fund for Agro-scientific Research in the Public Interest(201303046)+1 种基金Independent Innovation Project of Jiangsu Province[CX(13)3065]Project of the Fourth Period of "333" High-level Personnel Training Program of Jiangsu Province(BRA2012194)~~
文摘[Objective] This study aimed to investigate the genetic variation of porcine circovirus type 2 （PCV2） in China. [Method] The strain was isolated from infected samples by cel passage and preliminarily identified by PCR and IFA. Ful-length genome of the isolated strain was obtained by specific amplification for homology and phylogenetic analysis. [Result] A PCV2 strain was successful y isolated and named 201105ZJ, which could proliferate in PK15 cel lines. Specific fragments could be amplified by specific PCR assay. According to results of IFA assay, specif-ic immunofluorescence was observed; the TCID50 was low （102.67）; the ful-length genome sequence of the isolated strain was 1 768 bp, sharing 94.1%-96.8% ho-mology with 13 reference strains; to be specific, the isolated strain exhibited the highest homology of 96.8% with AF055392PCV2a; the isolated strain 201105ZJ and reference strain AF055392 belonged to genotype PCV2a, exhibiting a distant genetic relationship with genotype PCV2c. [Conclusion] Characteristics of genetic variation of PCV2 isolate 201105ZJ provided theoretical basis for vaccine development, investi-gation of PCV2 pathogenesis, and prevention and control of porcine circovirus-as-sociated diseases （PCVAD） in East China.
基金Project supported by the Foundation of Agricultural Development of Hangzhou City, China
文摘Porcine circovirus type 2 (PCV2) is closely related to the postweaning multisystemic wasting syndrome (PMWS). In this study, the pig serum and tissue samples collected from different regions of Hangzhou District in Zhejiang Province of China between 2003 and 2005 were analyzed by enzyme-linked immunosorbent assay (ELISA) for PCV2 antibody and by polymerase chain reaction (PCR) for ORF2 gene. The results show that out of 1250 randomly collected serum samples, 500 sera (40%) were seropositive for PCV2. PCR results demonstrate that Hangzhou PCV2 with more than 50% Chinese PCV2 strains and French PCV2 formed Cluster A. Only one PCV2 from Hangzhou belonged to Cluster B with some other Chinese PCV2 and Netherlands’s isolates. Cluster C consisted of PCV2 isolates from China, US, Canada, UK and Germany. The results indicate that the PCV2 infection was widespread in Hangzhou.
