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
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
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。
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.
