PR-39 is a porcine antimicrobial pep- tide that plays an important role in the innate defense mechanism. We produced monoclonal antibodies （MAbs） against PR-39 by fusing mouse myeloma ceils with lymph node cells fro...PR-39 is a porcine antimicrobial pep- tide that plays an important role in the innate defense mechanism. We produced monoclonal antibodies （MAbs） against PR-39 by fusing mouse myeloma ceils with lymph node cells from BALB/c mice immu- nized with the reactive site sequence PR-11 of PR-39. Furthermore, we investigated the effect of lactoferrin on PR-39 gene levels and peptide concentrations in cultural supernatants of bone marrow granulocytes by RT-PCR and the competitive inhibition ELISA meth- od established in this study. Two hybridomas 3H5 and 5H7 were selected for developing ascites and con- tained MAbs against PR-11, which were used for screening the specificity to PR-39 by competitive inhi- bition ELISA. We found that MAbs were successfully produced against PR-11 and the MAbs had a strong reaction with PR-39 excreted by porcine marrow gran-ulocytes. The ascites had a relatively high titer and the purified MAbs were determined as IgG1. Incubation with 10 or 100 ixg/mL lactoferrin significantly in- creased （ P 〈 0.05 ） PR-39 mRNA levels in bone mar- row granulocytes after 3 h and 6 h, but 1000 pog/mL lactoferrin reduced （ P 〈 0.05） PR-39 mRNA expres- sion after 3 h and 6 h compared with control （ no lact- oferrin added）. The relative concentrations of PR-39 in supernatant secreted by marrow granulocytes were significantly increased by 1000 p,g/mL lactoferrin af- ter both 3 h and 6 h. These findings suggest a regula- tory role of lactoferrin for the antimicrobial peptide PR-39 in marrow cells in vitro. It is possible that the regulation of antimicrobial peptide PR-39 expression may be one of the protective mechanisms of lacto- ferrin in pigs.展开更多
96头36日龄杜长嘉(杜洛克×♀长嘉)断奶仔猪,按体质量相近原则随机分成4个处理组,每个组设3个重复,每个重复8头猪(公母各半)。对照组(处理1)饲喂不添加任何锌源的基础饲料(DE 13.25 M J/kg,CP 18.5%),处理2、处理3、处理4分别饲喂...96头36日龄杜长嘉(杜洛克×♀长嘉)断奶仔猪,按体质量相近原则随机分成4个处理组,每个组设3个重复,每个重复8头猪(公母各半)。对照组(处理1)饲喂不添加任何锌源的基础饲料(DE 13.25 M J/kg,CP 18.5%),处理2、处理3、处理4分别饲喂添加硫酸锌(Zn 100 m g/kg)、氧化锌(Zn 3 000 m g/kg)和纳米氧化锌(Zn 100 m g/kg)饲粮。根据已报道的猪抗菌肽PR-39和看家基因β-肌动蛋白(-βactin)基因序列,分别设计PR-39和-βactin的引物,探讨了PCR体系中适宜的M gC l2浓度、循环次数,以及2对引物间竞争情况。以此构建一优化的半定量RT-PCR法。以β-actin为内标,研究了不同锌源对仔猪抗菌肽PR-39基因表达的影响。结果显示,与对照组相比,高氧化锌组(Zn 3 000m g/kg)显著提高了抗菌肽PR-39基因的表达量,提高量为378.26%(P<0.01),硫酸锌组(Zn 100 m g/kg)和纳米氧化锌组(Zn 100 m g/kg)也分别使抗菌肽PR-39基因的表达提高了38.4%和23.91%,但统计结果差异不显著。展开更多
Human thioredoxin and antibacterial peptide, PR39, have been shown to have potent antioxidant effects that may prolong survival of cells during hypoxia. The pSSCMV/human thioredoxin-PR39 vector was successfully constr...Human thioredoxin and antibacterial peptide, PR39, have been shown to have potent antioxidant effects that may prolong survival of cells during hypoxia. The pSSCMV/human thioredoxin-PR39 vector was successfully constructed in this study and used to infect ECV304 cells. Transfected ECV304 cells were incubated at 1%, 5% hypoxic, and normal oxygen conditions. We found that the number of apoptotic cells after transfection with recombinant adeno-associated virus-human thioredoxin -PR39 was significantly lower than controls, suggesting a protective effect of the recombinant human thioredoxin-PR39 protein on hypoxic cells.展开更多
文摘PR-39 is a porcine antimicrobial pep- tide that plays an important role in the innate defense mechanism. We produced monoclonal antibodies （MAbs） against PR-39 by fusing mouse myeloma ceils with lymph node cells from BALB/c mice immu- nized with the reactive site sequence PR-11 of PR-39. Furthermore, we investigated the effect of lactoferrin on PR-39 gene levels and peptide concentrations in cultural supernatants of bone marrow granulocytes by RT-PCR and the competitive inhibition ELISA meth- od established in this study. Two hybridomas 3H5 and 5H7 were selected for developing ascites and con- tained MAbs against PR-11, which were used for screening the specificity to PR-39 by competitive inhi- bition ELISA. We found that MAbs were successfully produced against PR-11 and the MAbs had a strong reaction with PR-39 excreted by porcine marrow gran-ulocytes. The ascites had a relatively high titer and the purified MAbs were determined as IgG1. Incubation with 10 or 100 ixg/mL lactoferrin significantly in- creased （ P 〈 0.05 ） PR-39 mRNA levels in bone mar- row granulocytes after 3 h and 6 h, but 1000 pog/mL lactoferrin reduced （ P 〈 0.05） PR-39 mRNA expres- sion after 3 h and 6 h compared with control （ no lact- oferrin added）. The relative concentrations of PR-39 in supernatant secreted by marrow granulocytes were significantly increased by 1000 p,g/mL lactoferrin af- ter both 3 h and 6 h. These findings suggest a regula- tory role of lactoferrin for the antimicrobial peptide PR-39 in marrow cells in vitro. It is possible that the regulation of antimicrobial peptide PR-39 expression may be one of the protective mechanisms of lacto- ferrin in pigs.
文摘96头36日龄杜长嘉(杜洛克×♀长嘉)断奶仔猪,按体质量相近原则随机分成4个处理组,每个组设3个重复,每个重复8头猪(公母各半)。对照组(处理1)饲喂不添加任何锌源的基础饲料(DE 13.25 M J/kg,CP 18.5%),处理2、处理3、处理4分别饲喂添加硫酸锌(Zn 100 m g/kg)、氧化锌(Zn 3 000 m g/kg)和纳米氧化锌(Zn 100 m g/kg)饲粮。根据已报道的猪抗菌肽PR-39和看家基因β-肌动蛋白(-βactin)基因序列,分别设计PR-39和-βactin的引物,探讨了PCR体系中适宜的M gC l2浓度、循环次数,以及2对引物间竞争情况。以此构建一优化的半定量RT-PCR法。以β-actin为内标,研究了不同锌源对仔猪抗菌肽PR-39基因表达的影响。结果显示,与对照组相比,高氧化锌组(Zn 3 000m g/kg)显著提高了抗菌肽PR-39基因的表达量,提高量为378.26%(P<0.01),硫酸锌组(Zn 100 m g/kg)和纳米氧化锌组(Zn 100 m g/kg)也分别使抗菌肽PR-39基因的表达提高了38.4%和23.91%,但统计结果差异不显著。
基金sponsored by the National Natural Science Foundation of China,No.30970992
文摘Human thioredoxin and antibacterial peptide, PR39, have been shown to have potent antioxidant effects that may prolong survival of cells during hypoxia. The pSSCMV/human thioredoxin-PR39 vector was successfully constructed in this study and used to infect ECV304 cells. Transfected ECV304 cells were incubated at 1%, 5% hypoxic, and normal oxygen conditions. We found that the number of apoptotic cells after transfection with recombinant adeno-associated virus-human thioredoxin -PR39 was significantly lower than controls, suggesting a protective effect of the recombinant human thioredoxin-PR39 protein on hypoxic cells.
