The double-shelled grass carp reovirus(GCRV) is capable of endogenous RNA transcription and processing. Genome sequence analysis has revealed that the protein VP2, encoded by gene seg-ment 2(S2), is the putative RNA-d...The double-shelled grass carp reovirus(GCRV) is capable of endogenous RNA transcription and processing. Genome sequence analysis has revealed that the protein VP2, encoded by gene seg-ment 2(S2), is the putative RNA-dependent RNA polymerase(RdRp). In previous work, we have ex-pressed the functional region of VP2 that is associated with RNA polymerase activity(denoted as rVP2390-900) in E. coli and have prepared a polyclonal antibody against VP2. To characterize the GCRV RNA polymerase, a recombinant full-length VP2(rVP2) was first constructed and expressed in a baculovirus system, as a fusion protein with an attached His-tag. Immunofluorescence(IF) assays, together with immunoblot(IB) analyses from both expressed cell extracts and purified His-tagged rVP2, showed that rVP2 was successfully expressed in Sf9 cells. Further characterization of the replicase activity showed that purified rVP2 and GCRV particles exhibited poly(C)-dependent poly(G) polymerase activity. The RNA enzymatic activity required the divalent cation Mg2+, and was optimal at 28 °C. The results provide a foundation for further studies on the RNA polymerases of aquareoviruses during viral transcription and replication.展开更多
In this study, the full-length VP2 gene of canine parvovirus type 2 (CPV-2) was cloned into the pBacSC vector which possesses baculovirus transmembrane domain (gp64 TM) gene, baculovirus cytoplasmic domain (gp64 CTD) ...In this study, the full-length VP2 gene of canine parvovirus type 2 (CPV-2) was cloned into the pBacSC vector which possesses baculovirus transmembrane domain (gp64 TM) gene, baculovirus cytoplasmic domain (gp64 CTD) gene, and green florescence protein (GFP) gene. Baculovirus gp64 TM and gp64 CTD in the pBacSC vector were designed to display heterologous proteins on the baculovirus envelope. After cloning the VP2 gene of CPV-2 into pBacSC vector, the recombinant plasmid pBacSC-VP2 was transformed into E. coli DH10Bac competent cells to form recombinant bacmid DNA. One recombinant baculovirus BacSC-VP2 that expresses the VP2 protein of CPV-2 was obtained. Confocal microscopy and immunogold electron microscopy were used to verify whether VP2 expressing on baculovirus envelope or cell membrane. Immunization of BALB/c mice with recombinant baculovirus BacSC-VP2, demonstrated that serum from the BacSC-VP2 treated models had higher levels of virus neutralization titers than the control groups. The results show that the recombinant baculovirus BacSC-VP2 can induce a strong immune response in a mouse model, suggesting that the pseudotyped baculovirus BacSC-VP2 can serve as a potential vaccine against CPV infections.展开更多
将分别编码猪瘟病毒T细胞表位E290多肽和猪细小病毒主要保护性抗原VP2蛋白的重组基因插入干酪乳杆菌分泌型表达载体pPG中,构建了重组表达载体pPG-VP2-E290,将其电转化干酪乳杆菌Lactobacillus casei 393,获得了猪瘟病毒T细胞表位E290多...将分别编码猪瘟病毒T细胞表位E290多肽和猪细小病毒主要保护性抗原VP2蛋白的重组基因插入干酪乳杆菌分泌型表达载体pPG中,构建了重组表达载体pPG-VP2-E290,将其电转化干酪乳杆菌Lactobacillus casei 393,获得了猪瘟病毒T细胞表位E290多肽与猪细小病毒VP2蛋白的乳酸菌共表达系统,经2%乳糖在MRS培养基中的诱导表达,对诱导表达的菌体及培养上清液进行SDS-PAGE检测表明,有约70kDa蛋白得到了表达,表达蛋白的大小与理论值相符。Western blot分析结果表明所表达的蛋白具有与天然病毒蛋白一样的抗原特异性。以诱导表达上清液作为抗原进行的间接ELISA实验也表明,重组的目的蛋白获得了分泌表达。将该重组干酪乳杆菌经口服接种途径免疫BALB/c小鼠,收集粪便样品检测小鼠产生抗PPV的特异性sIgA抗体,采集血液样本检测血清中抗PPV及抗E290的特异性IgG。结果表明分泌型的重组菌pPG-VP2-E290/L.casei 393免疫小鼠能够产生明显的抗体水平,为重组猪瘟与猪细小病毒乳酸菌口服活菌疫苗的研制奠定了重要的物质基础。展开更多
将编码猪细小病毒主要免疫保护性抗原VP2基因插入干酪乳杆菌细胞表面表达载体pPG中,构建了重组表达载体pPG-VP2,将其电转化干酪乳杆菌Lactobacillus casei 393,获得了表达猪细小病毒VP2蛋白的重组干酪乳杆菌系统,经2%乳糖在MRS培养基中...将编码猪细小病毒主要免疫保护性抗原VP2基因插入干酪乳杆菌细胞表面表达载体pPG中,构建了重组表达载体pPG-VP2,将其电转化干酪乳杆菌Lactobacillus casei 393,获得了表达猪细小病毒VP2蛋白的重组干酪乳杆菌系统,经2%乳糖在MRS培养基中的诱导表达,SDS-PAGE检测表明,有约74kD蛋白得到了表达,表达蛋白的大小与理论值相符。Western-blot结果分析表明,表达的蛋白可被鼠源PPV抗血清所识别,间接免疫荧光实验结果表明,所表达的蛋白能够在干酪乳杆菌菌体表面检测到。展开更多
基金supported by funding from the National Natural Science Foundation of China (grants: 31172434, 31372565)
文摘The double-shelled grass carp reovirus(GCRV) is capable of endogenous RNA transcription and processing. Genome sequence analysis has revealed that the protein VP2, encoded by gene seg-ment 2(S2), is the putative RNA-dependent RNA polymerase(RdRp). In previous work, we have ex-pressed the functional region of VP2 that is associated with RNA polymerase activity(denoted as rVP2390-900) in E. coli and have prepared a polyclonal antibody against VP2. To characterize the GCRV RNA polymerase, a recombinant full-length VP2(rVP2) was first constructed and expressed in a baculovirus system, as a fusion protein with an attached His-tag. Immunofluorescence(IF) assays, together with immunoblot(IB) analyses from both expressed cell extracts and purified His-tagged rVP2, showed that rVP2 was successfully expressed in Sf9 cells. Further characterization of the replicase activity showed that purified rVP2 and GCRV particles exhibited poly(C)-dependent poly(G) polymerase activity. The RNA enzymatic activity required the divalent cation Mg2+, and was optimal at 28 °C. The results provide a foundation for further studies on the RNA polymerases of aquareoviruses during viral transcription and replication.
文摘In this study, the full-length VP2 gene of canine parvovirus type 2 (CPV-2) was cloned into the pBacSC vector which possesses baculovirus transmembrane domain (gp64 TM) gene, baculovirus cytoplasmic domain (gp64 CTD) gene, and green florescence protein (GFP) gene. Baculovirus gp64 TM and gp64 CTD in the pBacSC vector were designed to display heterologous proteins on the baculovirus envelope. After cloning the VP2 gene of CPV-2 into pBacSC vector, the recombinant plasmid pBacSC-VP2 was transformed into E. coli DH10Bac competent cells to form recombinant bacmid DNA. One recombinant baculovirus BacSC-VP2 that expresses the VP2 protein of CPV-2 was obtained. Confocal microscopy and immunogold electron microscopy were used to verify whether VP2 expressing on baculovirus envelope or cell membrane. Immunization of BALB/c mice with recombinant baculovirus BacSC-VP2, demonstrated that serum from the BacSC-VP2 treated models had higher levels of virus neutralization titers than the control groups. The results show that the recombinant baculovirus BacSC-VP2 can induce a strong immune response in a mouse model, suggesting that the pseudotyped baculovirus BacSC-VP2 can serve as a potential vaccine against CPV infections.
文摘将分别编码猪瘟病毒T细胞表位E290多肽和猪细小病毒主要保护性抗原VP2蛋白的重组基因插入干酪乳杆菌分泌型表达载体pPG中,构建了重组表达载体pPG-VP2-E290,将其电转化干酪乳杆菌Lactobacillus casei 393,获得了猪瘟病毒T细胞表位E290多肽与猪细小病毒VP2蛋白的乳酸菌共表达系统,经2%乳糖在MRS培养基中的诱导表达,对诱导表达的菌体及培养上清液进行SDS-PAGE检测表明,有约70kDa蛋白得到了表达,表达蛋白的大小与理论值相符。Western blot分析结果表明所表达的蛋白具有与天然病毒蛋白一样的抗原特异性。以诱导表达上清液作为抗原进行的间接ELISA实验也表明,重组的目的蛋白获得了分泌表达。将该重组干酪乳杆菌经口服接种途径免疫BALB/c小鼠,收集粪便样品检测小鼠产生抗PPV的特异性sIgA抗体,采集血液样本检测血清中抗PPV及抗E290的特异性IgG。结果表明分泌型的重组菌pPG-VP2-E290/L.casei 393免疫小鼠能够产生明显的抗体水平,为重组猪瘟与猪细小病毒乳酸菌口服活菌疫苗的研制奠定了重要的物质基础。
文摘将编码猪细小病毒主要免疫保护性抗原VP2基因插入干酪乳杆菌细胞表面表达载体pPG中,构建了重组表达载体pPG-VP2,将其电转化干酪乳杆菌Lactobacillus casei 393,获得了表达猪细小病毒VP2蛋白的重组干酪乳杆菌系统,经2%乳糖在MRS培养基中的诱导表达,SDS-PAGE检测表明,有约74kD蛋白得到了表达,表达蛋白的大小与理论值相符。Western-blot结果分析表明,表达的蛋白可被鼠源PPV抗血清所识别,间接免疫荧光实验结果表明,所表达的蛋白能够在干酪乳杆菌菌体表面检测到。
