Abstract：
Mycoplasma hyopneumoniae, the primary pathogen responsible for porcine enzootic pneumonia, reduces average daily weight gain and causes substantial economic losses to the pig industry worldwide. Vaccination is the most common strategy to control this disease but offers partial protection. Therefore, developing next-generation vaccines by screening protective antigens is crucial.
The aim of this study was to evaluate the antibody response to 33 recombinant proteins in pigs naturally infected with M. hyopneumoniae.
The genes encoding 33 (hypothetical) membrane proteins or secretory proteins were ligated into pGEX-6P-1, pGEX-6P-2, pGEX-5X-3 or pGEX-4T-3 vectors and transformed into Escherichia coli BL21(DE3) or E. coli XL-1 Blue to construct recombinant bacteria and to express the recombinant proteins. The recombinant bacteria expressing the target proteins reacted with porcine convalescent sera and negative sera to screen immunodominant proteins by ELISA. Then, recombinant bacteria expressing immunodominant proteins were used to identify the discriminating immunodominant proteins that were recognised by convalescent sera nut not hyperimmune sera.
All recombinant bacteria could express the target recombinant proteins in soluble form. Twenty-one proteins were shown to present immunodominant antigens, and four proteins were not recognised by convalescent sera. Moreover, six proteins were considered discriminating and reacted with convalescent sera but not with hyperimmune sera.
The identified immunodominant proteins were antigenic and expressed during bacterial infection, suggesting that these proteins, especially those capable of discriminating between sera, can be used to identify protective antigens with the view to develop more effective vaccines against M. hyopneumoniae infection.
The aim of this study was to evaluate the antibody response to 33 recombinant proteins in pigs naturally infected with M. hyopneumoniae.
The genes encoding 33 (hypothetical) membrane proteins or secretory proteins were ligated into pGEX-6P-1, pGEX-6P-2, pGEX-5X-3 or pGEX-4T-3 vectors and transformed into Escherichia coli BL21(DE3) or E. coli XL-1 Blue to construct recombinant bacteria and to express the recombinant proteins. The recombinant bacteria expressing the target proteins reacted with porcine convalescent sera and negative sera to screen immunodominant proteins by ELISA. Then, recombinant bacteria expressing immunodominant proteins were used to identify the discriminating immunodominant proteins that were recognised by convalescent sera nut not hyperimmune sera.
All recombinant bacteria could express the target recombinant proteins in soluble form. Twenty-one proteins were shown to present immunodominant antigens, and four proteins were not recognised by convalescent sera. Moreover, six proteins were considered discriminating and reacted with convalescent sera but not with hyperimmune sera.
The identified immunodominant proteins were antigenic and expressed during bacterial infection, suggesting that these proteins, especially those capable of discriminating between sera, can be used to identify protective antigens with the view to develop more effective vaccines against M. hyopneumoniae infection.
摘要：
背景：猪肺炎支原体，导致猪地方性肺炎的主要病原体，降低了平均每日体重增加，并对全球养猪业造成了巨大的经济损失。疫苗接种是控制这种疾病的最常见策略，但可提供部分保护。因此,通过筛选保护性抗原来开发下一代疫苗至关重要。
目的：本研究的目的是评估自然感染猪肺炎支原体的猪对33种重组蛋白的抗体反应。
方法：将编码33种（假设的）膜蛋白或分泌蛋白的基因连接到pGEX-6P-1，pGEX-6P-2，pGEX-5X-3或pGEX-4T-3载体中，并转化到大肠杆菌BL21（DE3）或大肠杆菌XL-1Blue中，以构建重组细菌并表达重组蛋白。表达靶蛋白的重组细菌与猪恢复期血清和阴性血清反应，通过ELISA筛选免疫显性蛋白。然后，表达免疫显性蛋白的重组细菌用于鉴定由恢复期血清坚果而不是超免疫血清识别的区分性免疫显性蛋白。
结果：所有重组菌都能以可溶性形式表达目标重组蛋白。21种蛋白质被证明提供免疫显性抗原，四种蛋白质未被恢复期血清识别。此外，6种蛋白质被认为是有区别的，并与恢复期血清反应，但与超免疫血清不反应。
结论：鉴定的免疫显性蛋白是抗原性的，在细菌感染期间表达，表明这些蛋白质，尤其是那些能够区分血清的，可用于鉴定保护性抗原,以期开发针对猪肺炎支原体感染的更有效的疫苗。
目的：本研究的目的是评估自然感染猪肺炎支原体的猪对33种重组蛋白的抗体反应。
方法：将编码33种（假设的）膜蛋白或分泌蛋白的基因连接到pGEX-6P-1，pGEX-6P-2，pGEX-5X-3或pGEX-4T-3载体中，并转化到大肠杆菌BL21（DE3）或大肠杆菌XL-1Blue中，以构建重组细菌并表达重组蛋白。表达靶蛋白的重组细菌与猪恢复期血清和阴性血清反应，通过ELISA筛选免疫显性蛋白。然后，表达免疫显性蛋白的重组细菌用于鉴定由恢复期血清坚果而不是超免疫血清识别的区分性免疫显性蛋白。
结果：所有重组菌都能以可溶性形式表达目标重组蛋白。21种蛋白质被证明提供免疫显性抗原，四种蛋白质未被恢复期血清识别。此外，6种蛋白质被认为是有区别的，并与恢复期血清反应，但与超免疫血清不反应。
结论：鉴定的免疫显性蛋白是抗原性的，在细菌感染期间表达，表明这些蛋白质，尤其是那些能够区分血清的，可用于鉴定保护性抗原,以期开发针对猪肺炎支原体感染的更有效的疫苗。
