[ Objective] To develop an indirect ELISA assay for detecting antibodies against envelope glycoprotein （ E protein） of Japanese encephalitis virus （JEV）. [ Method] Specific primers were designed according to JEV s...[ Objective] To develop an indirect ELISA assay for detecting antibodies against envelope glycoprotein （ E protein） of Japanese encephalitis virus （JEV）. [ Method] Specific primers were designed according to JEV sequences published in the GenBank. The cDNA of JEV E gene （about 1 000 10p） was amplified by the RT-PCR with the specific primers. After sequencing analysis, the E gene was cloned into pET30a expression vector and expressed in E. coli BL21 （DE3） with the induction of IPTG. After denaturation, purification and renaturation, the recombinant protein was analyzed by the SDS-PAGE and the westem blotting. An indirect ELISA was developed to detect antibodies against JEV. [ Result] The E protein was mainly expressed in inclusion body. With the purified E protein, the indirect ELISA was developed and displayed good specificity, sensitivity and repeatability, [ Conclusion]The developed ELISA using the truncated E protein as antigen is a simple, convenient and rapid serological method for diagnosis, monitoring antibody level and epidemiological investigation of JEV.展开更多
Japanese encephalitis （JE） is a central nervous system disease caused by Japanese encephalitis virus （JEV）, which can infect human and a variety of animals and cause irreversible nerve damages. NS3 protein plays a...Japanese encephalitis （JE） is a central nervous system disease caused by Japanese encephalitis virus （JEV）, which can infect human and a variety of animals and cause irreversible nerve damages. NS3 protein plays an important role in the process of JEV polyprotein hydrolysis, which is essential for JEV replication. Therefore, NS3 protein may be used as a potential drug target to treat Japanese encephalitis. In this study, the pET-28a-NS3 plasmid was successfully constructed and expressed in E. coli BL21 （ DE3 ） under IPTG induction. The molecular weight of the expressed recombinant protein was 55 ku, which was consistent with the expected result. The positive serum was prepared by immunizing BALB/c mice with NS3 protein and identified by indirect immunofluorescence （IFA）. The results showed that there was a fluorescence reaction between the prepared positive serum of NS3 protein and cells infected with JEV.展开更多
Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus closely related to the human pathogens including yellow fever virus, dengue virus and West Nile virus. There are currently no effective antiviral therap...Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus closely related to the human pathogens including yellow fever virus, dengue virus and West Nile virus. There are currently no effective antiviral therapies for all of the flavivirus and only a few highly effective vaccines are licensed for human use. In this paper, the E protein domain III (DIII) of six heterologous flaviviruses (DENV1-4, WNV and JEV) was expressed in Escherichia coli successfully. The proteins were purified after a solubilization and refolding procedure, characterized by SDS-PAGE and Western blotting. Competitive inhibition showed that all recombinant flavivirus DIII proteins blocked the entry of JEV into BHK-21 cells. Further studies indicated that antibodies induced by the soluble recombinant flavivirus DIII partially protected mice against lethal JEV challenge. These results demonstrated that recombinant flavivirus DIII proteins could inhibit JEV infection competitively, and immunization with proper folding flavivirus DIII induced cross-protection against JEV infection in mice, implying a possible role of DIII for the cross-protection among flavivirus as well as its use in antigens for immunization in animal models.展开更多
The incidence of Japanese encephalitis(JE)has significantly decreased in China due to JE vaccines.In this study,we investigated the post-JE vaccination seroprevalence and protection provided by vaccinated sera against...The incidence of Japanese encephalitis(JE)has significantly decreased in China due to JE vaccines.In this study,we investigated the post-JE vaccination seroprevalence and protection provided by vaccinated sera against Japanese encephalitis virus(JEV)to elucidate the persistence and waning of antibodies to JEV among JE-SA14-14-2-vaccinated children.A total of 300 serum samples were collected from vaccinated children aged 3-10 years in Zhaotong,Yunnan,China.The seroprevalence of anti-JEV antibodies was determined by enzyme-linked immune sorbent assay and plaque reduction neutralization test.The highest seropositivity of 82%was observed in vaccinated children during the first 0.5-1.5 years after booster vaccination.Then,the seropositivity began to decline and remained lower than the original level observed in the 0.5-1.5-year group.An association was found between the waning of seroprevalence and elapsed time of the post-booster vaccination.Similarly,the neutralizing antibody(nAb)titres gradually decreased over time,and the levels showed a positive correlation with the protective efficacy in mice.This finding suggests that nAbs play an important role in the antiviral process and that the nAb titre is an adequately credible parameter for evaluating the protective efficacy induced by the JE vaccine.Our results provide data that clarify the persistence and waning of antibodies to JEV,which may help elucidate the pathogenesis of JE.展开更多
文摘[ Objective] To develop an indirect ELISA assay for detecting antibodies against envelope glycoprotein （ E protein） of Japanese encephalitis virus （JEV）. [ Method] Specific primers were designed according to JEV sequences published in the GenBank. The cDNA of JEV E gene （about 1 000 10p） was amplified by the RT-PCR with the specific primers. After sequencing analysis, the E gene was cloned into pET30a expression vector and expressed in E. coli BL21 （DE3） with the induction of IPTG. After denaturation, purification and renaturation, the recombinant protein was analyzed by the SDS-PAGE and the westem blotting. An indirect ELISA was developed to detect antibodies against JEV. [ Result] The E protein was mainly expressed in inclusion body. With the purified E protein, the indirect ELISA was developed and displayed good specificity, sensitivity and repeatability, [ Conclusion]The developed ELISA using the truncated E protein as antigen is a simple, convenient and rapid serological method for diagnosis, monitoring antibody level and epidemiological investigation of JEV.
基金Supported by Youth Fund of Hubei Academy of Agricultural Sciences(2013NKYJJ12)
文摘Japanese encephalitis （JE） is a central nervous system disease caused by Japanese encephalitis virus （JEV）, which can infect human and a variety of animals and cause irreversible nerve damages. NS3 protein plays an important role in the process of JEV polyprotein hydrolysis, which is essential for JEV replication. Therefore, NS3 protein may be used as a potential drug target to treat Japanese encephalitis. In this study, the pET-28a-NS3 plasmid was successfully constructed and expressed in E. coli BL21 （ DE3 ） under IPTG induction. The molecular weight of the expressed recombinant protein was 55 ku, which was consistent with the expected result. The positive serum was prepared by immunizing BALB/c mice with NS3 protein and identified by indirect immunofluorescence （IFA）. The results showed that there was a fluorescence reaction between the prepared positive serum of NS3 protein and cells infected with JEV.
基金supported by Important National Science & Technology Specific Projects (2012ZX10004403, 2012ZX10004219)National Natural Scientific Fund of China (81072675)
文摘Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus closely related to the human pathogens including yellow fever virus, dengue virus and West Nile virus. There are currently no effective antiviral therapies for all of the flavivirus and only a few highly effective vaccines are licensed for human use. In this paper, the E protein domain III (DIII) of six heterologous flaviviruses (DENV1-4, WNV and JEV) was expressed in Escherichia coli successfully. The proteins were purified after a solubilization and refolding procedure, characterized by SDS-PAGE and Western blotting. Competitive inhibition showed that all recombinant flavivirus DIII proteins blocked the entry of JEV into BHK-21 cells. Further studies indicated that antibodies induced by the soluble recombinant flavivirus DIII partially protected mice against lethal JEV challenge. These results demonstrated that recombinant flavivirus DIII proteins could inhibit JEV infection competitively, and immunization with proper folding flavivirus DIII induced cross-protection against JEV infection in mice, implying a possible role of DIII for the cross-protection among flavivirus as well as its use in antigens for immunization in animal models.
基金supported by grants from the National Natural Science Foundation of China (81471957, 81772172, 81671971, U1602223 and 81871641)a grant from the Open Research Projects of Key Laboratory of Beijing Ditan Hospital, Capital Medical University (DTKF201705)
文摘The incidence of Japanese encephalitis(JE)has significantly decreased in China due to JE vaccines.In this study,we investigated the post-JE vaccination seroprevalence and protection provided by vaccinated sera against Japanese encephalitis virus(JEV)to elucidate the persistence and waning of antibodies to JEV among JE-SA14-14-2-vaccinated children.A total of 300 serum samples were collected from vaccinated children aged 3-10 years in Zhaotong,Yunnan,China.The seroprevalence of anti-JEV antibodies was determined by enzyme-linked immune sorbent assay and plaque reduction neutralization test.The highest seropositivity of 82%was observed in vaccinated children during the first 0.5-1.5 years after booster vaccination.Then,the seropositivity began to decline and remained lower than the original level observed in the 0.5-1.5-year group.An association was found between the waning of seroprevalence and elapsed time of the post-booster vaccination.Similarly,the neutralizing antibody(nAb)titres gradually decreased over time,and the levels showed a positive correlation with the protective efficacy in mice.This finding suggests that nAbs play an important role in the antiviral process and that the nAb titre is an adequately credible parameter for evaluating the protective efficacy induced by the JE vaccine.Our results provide data that clarify the persistence and waning of antibodies to JEV,which may help elucidate the pathogenesis of JE.
