African swine fever virus(ASFV)poses a significant threat to the global swine industry.Currently,there are no effective vaccines or treatments available to combat ASFV infection in pigs.The primary means of controllin...African swine fever virus(ASFV)poses a significant threat to the global swine industry.Currently,there are no effective vaccines or treatments available to combat ASFV infection in pigs.The primary means of controlling the spread of the disease is through rapid detection and subsequent elimination of infected pig.Recently,a lower virulent ASFV isolate with a deleted EP402R gene(CD2v-deleted)has been reported in China,which further complicates the control of ASFV infection in pig farms.Furthermore,an EP402R-deleted ASFV variant has been developed as a potential live attenuated vaccine candidate strain.Therefore,it is crucial to develop detection methods that can distinguish wild-type and EP402R-deleted ASFV infections.In this study,two recombinant ASFV-p72 and-CD2v proteins were expressed using a prokaryotic system and used to immunize Bactrian camels.Subsequently,eight nanobodies against ASFV-p72 and ten nanobodies against ASFV-CD2v were screened.Following the production of these nanobodies with horse radish peroxidase(HRP)fusion proteins,the ASFV-p72-Nb2-HRP and ASFV-CD2v-Nb22-HRP fusions were selected for the development of two competitive ELISAs(cELISAs)to detect anti-ASFV antibodies.The two cELISAs exhibited high sensitivity,good specificity,repeatability,and stability.The coincidence rate between the two cELISAs and commercial ELISA kits was 98.6%and 97.6%,respectively.Collectively,the two cELISA for detecting antibodies against ASFV demonstrated ease of operation,a low cost,and a simple production process.The two cELISAs could determine whether pigs were infected with wild-type or CD2v-deleted ASFV,and could play an important role in monitoring ASFV infections in pig farms.展开更多
本研究旨在通过在Bac to Bac系统中表达非洲猪瘟病毒(ASFV)的P72基因,为进一步研究P72基因的结构、功能和免疫学特性奠定基础。本研究从含P72全长基因的菌株pGEX-6p-1-P72中扩增出P72基因全序列1941bp,将扩增的基因连接于pFastBac HTa...本研究旨在通过在Bac to Bac系统中表达非洲猪瘟病毒(ASFV)的P72基因,为进一步研究P72基因的结构、功能和免疫学特性奠定基础。本研究从含P72全长基因的菌株pGEX-6p-1-P72中扩增出P72基因全序列1941bp,将扩增的基因连接于pFastBac HTa杆状病毒载体中,构建重组质粒pFastBac HTa-P72,转化至感受态DH10Bac中,获得重组杆粒rBac-mid-P72,再将其转染至Sf9昆虫细胞,获得重组杆状病毒。经Western blotting和夹心ELISA分析,结果表明,P72在昆虫细胞Sf9中获得正确表达,重组P72蛋白可被特异性兔抗ASFV P72血清、P72单抗识别。结果提示,Bac to Bac系统表达的P72重组蛋白特异性强、活性稳定,具有良好的抗原性,为快速诊断ASF提供良好的候选抗原。展开更多
基金supported by the Natural Science Foundation of China(grant no.32273041)the Natural Science Foundation of Shaanxi Province of China(grant no.2022JC-12)+1 种基金the Key R&D Program of Shaanxi Province(grant no.S2022-YF-YBNY-0673)the Central Public-interest Scientific Institution Basal Research Fund.
文摘African swine fever virus(ASFV)poses a significant threat to the global swine industry.Currently,there are no effective vaccines or treatments available to combat ASFV infection in pigs.The primary means of controlling the spread of the disease is through rapid detection and subsequent elimination of infected pig.Recently,a lower virulent ASFV isolate with a deleted EP402R gene(CD2v-deleted)has been reported in China,which further complicates the control of ASFV infection in pig farms.Furthermore,an EP402R-deleted ASFV variant has been developed as a potential live attenuated vaccine candidate strain.Therefore,it is crucial to develop detection methods that can distinguish wild-type and EP402R-deleted ASFV infections.In this study,two recombinant ASFV-p72 and-CD2v proteins were expressed using a prokaryotic system and used to immunize Bactrian camels.Subsequently,eight nanobodies against ASFV-p72 and ten nanobodies against ASFV-CD2v were screened.Following the production of these nanobodies with horse radish peroxidase(HRP)fusion proteins,the ASFV-p72-Nb2-HRP and ASFV-CD2v-Nb22-HRP fusions were selected for the development of two competitive ELISAs(cELISAs)to detect anti-ASFV antibodies.The two cELISAs exhibited high sensitivity,good specificity,repeatability,and stability.The coincidence rate between the two cELISAs and commercial ELISA kits was 98.6%and 97.6%,respectively.Collectively,the two cELISA for detecting antibodies against ASFV demonstrated ease of operation,a low cost,and a simple production process.The two cELISAs could determine whether pigs were infected with wild-type or CD2v-deleted ASFV,and could play an important role in monitoring ASFV infections in pig farms.
文摘本研究旨在通过在Bac to Bac系统中表达非洲猪瘟病毒(ASFV)的P72基因,为进一步研究P72基因的结构、功能和免疫学特性奠定基础。本研究从含P72全长基因的菌株pGEX-6p-1-P72中扩增出P72基因全序列1941bp,将扩增的基因连接于pFastBac HTa杆状病毒载体中,构建重组质粒pFastBac HTa-P72,转化至感受态DH10Bac中,获得重组杆粒rBac-mid-P72,再将其转染至Sf9昆虫细胞,获得重组杆状病毒。经Western blotting和夹心ELISA分析,结果表明,P72在昆虫细胞Sf9中获得正确表达,重组P72蛋白可被特异性兔抗ASFV P72血清、P72单抗识别。结果提示,Bac to Bac系统表达的P72重组蛋白特异性强、活性稳定,具有良好的抗原性,为快速诊断ASF提供良好的候选抗原。
