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
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
PCV2 is considered the main pathogen of porcine circovirus diseases and porcine circovirus-associated diseases(PCVD/PCVAD). However, the exact mechanism underlying PCVD/PCVAD is currently unknown. Mouse models of PCV2...PCV2 is considered the main pathogen of porcine circovirus diseases and porcine circovirus-associated diseases(PCVD/PCVAD). However, the exact mechanism underlying PCVD/PCVAD is currently unknown. Mouse models of PCV2 are valuable experimental tools that can shed light on the pathogenesis of infection and will enable the evaluation of antiviral agents and vaccine candidates. In this review, we discuss the current state of knowledge of mouse models used in PCV2 research that has been performed to date, highlighting their strengths and limitations, as well as prospects for future PCV2 studies.展开更多
In this study, the loop-mediated isothermal amplification (LAMP) method was used to develop a rapid and simple detection system for porcine circovirus type 2 (PCV2). According to the PCV2 sequences published in GenBan...In this study, the loop-mediated isothermal amplification (LAMP) method was used to develop a rapid and simple detection system for porcine circovirus type 2 (PCV2). According to the PCV2 sequences published in GenBank, multiple LAMP primers were designed targeting conserved sequences of PCV2. Using the DNA extracted from PCV2 isolates HUN-09 and SD-09 as the template, LAMP reactions in a PCV2 LAMP system was performed, the amplification products were detected by adding SYBR Green I and could be observed directly by the naked eye. The results showed highly-efficient and specific amplification in 30 min at 63°C with a LAMP real-time turbidimeter. Furthermore, PCV2 DNA templates, with a detection limit of 5.5×10-5 ng of nucleic acid, indicated that this assay was highly sensitive. The results obtained with the naked eye after SYBR Green I staining were consistent with those detected by the real-time turbidimeter, showing the potential simplicity of interpretation of the assay results. The LAMP assay appeared to have greater accuracy than PCR and virus isolation for the analysis of 18 clinical samples. In addition it offers higher specificity and sensitivity, shorter reaction times and simpler procedures than the currently available methods of PCV2 detection. It is therefore a promising tool for the effective and efficient detection of PCV2.展开更多
We characterized the genome sequences of defective-interfering(DI) particle DNA of porcine circovirus type 2(PCV2) by sequencing and bioinformatics analyses. DI particles were both generated by serial passage of PCV2 ...We characterized the genome sequences of defective-interfering(DI) particle DNA of porcine circovirus type 2(PCV2) by sequencing and bioinformatics analyses. DI particles were both generated by serial passage of PCV2 in PK15 cells and obtained from sera of pigs with postweaning multisystemic wasting syndrome(PMWS). These subviral isolates ranged from 358 nt to 1 125 nt genomes. Investigating the complexity and diversity of PCV2 DI in vivo and in vitro can help elucidating the evolutionary history of PCV2.展开更多
In the present study, 89 porcine reproductive and respiratory syndrome virus(PRRSV) isolates in China during 2007 to 2012 were randomly selected from the GenBank genetic sequence database. Evolutionary characteristics...In the present study, 89 porcine reproductive and respiratory syndrome virus(PRRSV) isolates in China during 2007 to 2012 were randomly selected from the GenBank genetic sequence database. Evolutionary characteristics of these isolates were analyzed based on the sequences of non-struc-tural protein 2(Nsp2) and glycoprotein 5(GP5). The genetic variations of the isolates were also compared with six representative strains. The results showed that a high degree of genetic diversity exists among the PRRSV population in China. Highly pathogenic PRRSV isolates, with a discon-tinuous deletion of a 30 amino acid residue in the Nsp2 region, remained the most dominant virus throughout 2007–2012 in China. Owing to the extensive use of representative vaccine strains, natu-ral recombination events occurred between strains. Three isolates – HH08, DY, and YN-2011 – were more closely related to vaccine strains than the other isolates. Both YN-2011 and DY were the evolu-tionary products of recombination events between strains SP and CH-1R. The results of the present study provide useful information for the epidemiology of PRRSV as well as for vaccine development.展开更多
[ Objective] The study aimed to analyze the prevalence of Porcine Parvovirus （PPV） and Porcine Circovirus Type 2 （ PCV2 ） and the mixed infection in Sichuan Province to lay the foundation for further predicting th...[ Objective] The study aimed to analyze the prevalence of Porcine Parvovirus （PPV） and Porcine Circovirus Type 2 （ PCV2 ） and the mixed infection in Sichuan Province to lay the foundation for further predicting the tendency of the plague and formulating appropriate prevention and control strategies. [ Method] Two hundred and seventy -three samples were collected from 21 large pig farms in Sichuan province, and epidemiology of PPV and PCV2 were investigated by PCR detecting. [ Result] The positive rate of PPV was 17.22% （47/273）, and positive rate of pig farms was 38.1% （8/21）, meanwhile it also displayed the feature that infection rate of boar was higher than that of piglets; The positive rate of PCV2 was 52.38% （143/273）, and positive rate of pig farms was 85.7% （18/21）, and it showed the trend that the infection rate of PCV2 was rising with the growth of the age. The co-infection rate of PPV and PCV2 was 10.62% （29/273）, and co-infection rate of pig farms was 28.7% （6/21）, which mainly concentrated in the sow and nursery piglets. Only 14.3% （3/21） pig farms was epidemiologically negative of PPV and PCV2. [ Conclusion] PPV and PCV2 and co-infection was widely popular in Sichuan province, and it did more serious harm to the pig-raising industry.展开更多
Porcine cimovirus type 2 (PCV2) infection causes huge economic losses, but no serological method is available for batch detection of field samples. The aim of the study was to develop a method for large-batch detectio...Porcine cimovirus type 2 (PCV2) infection causes huge economic losses, but no serological method is available for batch detection of field samples. The aim of the study was to develop a method for large-batch detection of PCV2 infection. Colloidal gold-labeled staphylococcal protein A (SPA) was sprayed on glass fibers to prepare a conjugate pad. The recombinant ORF2 protein of PCV2 was blotted on the test line of the nitrocellulose (NC) membrane, and pig IgG was streaked on the control line of the NC membrane. Then, the immunochromatographic strip was used for detection of antibodies against PCV2. The results show that the strip test was simple and the results could be determined within 10 min with naked eyes. The test strip was highly specific for pig serum against PCV2 and no cross-reaction with pig serum against other pathogens was observed. The test strip had close similarity with ELISA. Storage at RT for 6 months did not affect the specificity and sensitivity obviously. A total of 324 clinical pig sera were detected by both ELISA and the developed test strip, and the coincidence was 98.77%. Therefore, the developed immunochromatographic strip is specific, sensitive, stable, fast and simple, and it is suitable for on-site detection of antibodies against PCV2.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.
基金National Key Research and Development Program of China,Grant/Award Number:2017YFD0500103National Natural Science Foundation of China,Grant/Award Number:31772747,31272385+3 种基金Jilin Province Science and Technology Development Projects,Grant/Award Number:20150204077NYGraduate Innovation Fund of Jilin Universitythe Program for Changjiang Scholarsthe University Innovative Research Team,Grant/Award Number:IRT1248
文摘PCV2 is considered the main pathogen of porcine circovirus diseases and porcine circovirus-associated diseases(PCVD/PCVAD). However, the exact mechanism underlying PCVD/PCVAD is currently unknown. Mouse models of PCV2 are valuable experimental tools that can shed light on the pathogenesis of infection and will enable the evaluation of antiviral agents and vaccine candidates. In this review, we discuss the current state of knowledge of mouse models used in PCV2 research that has been performed to date, highlighting their strengths and limitations, as well as prospects for future PCV2 studies.
基金Fifteenth National Science and Technology Support Program of China(2007BAD86B04-4)
文摘In this study, the loop-mediated isothermal amplification (LAMP) method was used to develop a rapid and simple detection system for porcine circovirus type 2 (PCV2). According to the PCV2 sequences published in GenBank, multiple LAMP primers were designed targeting conserved sequences of PCV2. Using the DNA extracted from PCV2 isolates HUN-09 and SD-09 as the template, LAMP reactions in a PCV2 LAMP system was performed, the amplification products were detected by adding SYBR Green I and could be observed directly by the naked eye. The results showed highly-efficient and specific amplification in 30 min at 63°C with a LAMP real-time turbidimeter. Furthermore, PCV2 DNA templates, with a detection limit of 5.5×10-5 ng of nucleic acid, indicated that this assay was highly sensitive. The results obtained with the naked eye after SYBR Green I staining were consistent with those detected by the real-time turbidimeter, showing the potential simplicity of interpretation of the assay results. The LAMP assay appeared to have greater accuracy than PCR and virus isolation for the analysis of 18 clinical samples. In addition it offers higher specificity and sensitivity, shorter reaction times and simpler procedures than the currently available methods of PCV2 detection. It is therefore a promising tool for the effective and efficient detection of PCV2.
基金Supported by National Natural Science Foundation of China(31272574,30972184)Fund for Independent Innovation of Agricultural Sciences in Jiangsu Province[CX(11)2060]
文摘We characterized the genome sequences of defective-interfering(DI) particle DNA of porcine circovirus type 2(PCV2) by sequencing and bioinformatics analyses. DI particles were both generated by serial passage of PCV2 in PK15 cells and obtained from sera of pigs with postweaning multisystemic wasting syndrome(PMWS). These subviral isolates ranged from 358 nt to 1 125 nt genomes. Investigating the complexity and diversity of PCV2 DI in vivo and in vitro can help elucidating the evolutionary history of PCV2.
基金supported by the Jilin Province Science and Technology Development Project(No.20140101123JC)the Fundamental Research Fund of Jilin Universitythe Program for Changjiang Scholars and Innovative Research Team in University(No.IRT1248)
文摘In the present study, 89 porcine reproductive and respiratory syndrome virus(PRRSV) isolates in China during 2007 to 2012 were randomly selected from the GenBank genetic sequence database. Evolutionary characteristics of these isolates were analyzed based on the sequences of non-struc-tural protein 2(Nsp2) and glycoprotein 5(GP5). The genetic variations of the isolates were also compared with six representative strains. The results showed that a high degree of genetic diversity exists among the PRRSV population in China. Highly pathogenic PRRSV isolates, with a discon-tinuous deletion of a 30 amino acid residue in the Nsp2 region, remained the most dominant virus throughout 2007–2012 in China. Owing to the extensive use of representative vaccine strains, natu-ral recombination events occurred between strains. Three isolates – HH08, DY, and YN-2011 – were more closely related to vaccine strains than the other isolates. Both YN-2011 and DY were the evolu-tionary products of recombination events between strains SP and CH-1R. The results of the present study provide useful information for the epidemiology of PRRSV as well as for vaccine development.
基金supported by the Chengdu Science and Technology Bureau
文摘[ Objective] The study aimed to analyze the prevalence of Porcine Parvovirus （PPV） and Porcine Circovirus Type 2 （ PCV2 ） and the mixed infection in Sichuan Province to lay the foundation for further predicting the tendency of the plague and formulating appropriate prevention and control strategies. [ Method] Two hundred and seventy -three samples were collected from 21 large pig farms in Sichuan province, and epidemiology of PPV and PCV2 were investigated by PCR detecting. [ Result] The positive rate of PPV was 17.22% （47/273）, and positive rate of pig farms was 38.1% （8/21）, meanwhile it also displayed the feature that infection rate of boar was higher than that of piglets; The positive rate of PCV2 was 52.38% （143/273）, and positive rate of pig farms was 85.7% （18/21）, and it showed the trend that the infection rate of PCV2 was rising with the growth of the age. The co-infection rate of PPV and PCV2 was 10.62% （29/273）, and co-infection rate of pig farms was 28.7% （6/21）, which mainly concentrated in the sow and nursery piglets. Only 14.3% （3/21） pig farms was epidemiologically negative of PPV and PCV2. [ Conclusion] PPV and PCV2 and co-infection was widely popular in Sichuan province, and it did more serious harm to the pig-raising industry.
文摘Porcine cimovirus type 2 (PCV2) infection causes huge economic losses, but no serological method is available for batch detection of field samples. The aim of the study was to develop a method for large-batch detection of PCV2 infection. Colloidal gold-labeled staphylococcal protein A (SPA) was sprayed on glass fibers to prepare a conjugate pad. The recombinant ORF2 protein of PCV2 was blotted on the test line of the nitrocellulose (NC) membrane, and pig IgG was streaked on the control line of the NC membrane. Then, the immunochromatographic strip was used for detection of antibodies against PCV2. The results show that the strip test was simple and the results could be determined within 10 min with naked eyes. The test strip was highly specific for pig serum against PCV2 and no cross-reaction with pig serum against other pathogens was observed. The test strip had close similarity with ELISA. Storage at RT for 6 months did not affect the specificity and sensitivity obviously. A total of 324 clinical pig sera were detected by both ELISA and the developed test strip, and the coincidence was 98.77%. Therefore, the developed immunochromatographic strip is specific, sensitive, stable, fast and simple, and it is suitable for on-site detection of antibodies against PCV2.
