To study the expression characteristic of Japanese encephalitis virus (JEV) prME and E proteins and the efficacy of DNA immunization by different recombinant plasmids containing JEV prME (2001 bp) and E (1500 bp) gene...To study the expression characteristic of Japanese encephalitis virus (JEV) prME and E proteins and the efficacy of DNA immunization by different recombinant plasmids containing JEV prME (2001 bp) and E (1500 bp) genes, two recombinants (pJME and pJE) containing JEV prME and E genes fused with FLAG were constructed and then transfected into HepG2 and COS-1 cells by liposome fusion. The expression feature of FLAG-prME (about 72 kDa) and FLAG-E (about 54 kDa) proteins in transfected cells were analyzed by Western blot and two antibody systems (anti-FLAG and anti-E). BALB/c mice were immunized with 100 μg of two kinds of recombinants by intramuscular injection, and JEV JaGAr-01 strains (10 5 PFU/100 μl)were given to BALB/c mice by intraperioneal injection 3 wk after twice DNA immunization by a lethal virus challenge. BALB/c mice were observed for 21 days after challenge. 80% plaque reduction neutralization test was performed to titrate neutralization antibody before and after viral challenge. It was found that the expression of proteins associated with pJME and pJE was determined in transfected cells with anti-FLAG and a new protein of 11 kDa was detected in HepG2 and COS-1 cells transfected with pJME. Only E (53 kDa) protein was identified as transfected with pJME using anti-E. Higher level of neutralization antibodies and the efficacy of protective immunity were induced with pJME immunization, and were similar to those induced by inactivated Japanese encephalitis vaccine, but were better than those induced with pJE. It concludes that the expression level from prM to E proteins of JEV is different in vitro, and the in vitro expression efficiency of pJME was better than that of pJE. FLAG-prME protein expressed by pJME could be cleaved by peptidase from host. The efficacy of DNA immunization is correlated to the expression characterization of related proteins expressed in vitro.展开更多
The specific recombinant proteins rOspA B. afzelii, rOspA B. garinii a rOspA B. burgdorferi sensu stricto were used as antigens for construction of ELISA sets. ELISA examination enables determination of specific post-...The specific recombinant proteins rOspA B. afzelii, rOspA B. garinii a rOspA B. burgdorferi sensu stricto were used as antigens for construction of ELISA sets. ELISA examination enables determination of specific post-vaccination antibodies against OspA B. afzelii, B. garinii a B. burgdorferi sensu stricto.Using recombinant DNA technology, genes from B. afzelii, B. garinii and B. burgdorferi sensu stricto were inserted into E. coli-expression vectors and the rOspA proteins were produced. These proteins were used for the construction of ELISA kits for the determination of post-vaccination antibodies against individual Borrelia serovars contained in the vaccine. The antibody response of dogs vaccinated with whole-cell vaccine BORRELYM 3 and non-vaccinated dogs was monitored and compared. The ELISA method proved as highly sensitive for the determination of post-vaccination antibodies against individual Borrelia serovars in vaccinated animals. Detection of these antibodies and their quantification may be used for evaluation of efficiency of vaccines against Lyme borreliosis in dogs caused by Borrelia burgdorferi sensu lato. Prospectively, it will be necessary to establish a correlation between post-vaccination antibody levels and protective immunity of vaccinated dogs.展开更多
基金This research was supported by a grant for project research from high Technology center of Kanazawa Medical University(H2000 2)
文摘To study the expression characteristic of Japanese encephalitis virus (JEV) prME and E proteins and the efficacy of DNA immunization by different recombinant plasmids containing JEV prME (2001 bp) and E (1500 bp) genes, two recombinants (pJME and pJE) containing JEV prME and E genes fused with FLAG were constructed and then transfected into HepG2 and COS-1 cells by liposome fusion. The expression feature of FLAG-prME (about 72 kDa) and FLAG-E (about 54 kDa) proteins in transfected cells were analyzed by Western blot and two antibody systems (anti-FLAG and anti-E). BALB/c mice were immunized with 100 μg of two kinds of recombinants by intramuscular injection, and JEV JaGAr-01 strains (10 5 PFU/100 μl)were given to BALB/c mice by intraperioneal injection 3 wk after twice DNA immunization by a lethal virus challenge. BALB/c mice were observed for 21 days after challenge. 80% plaque reduction neutralization test was performed to titrate neutralization antibody before and after viral challenge. It was found that the expression of proteins associated with pJME and pJE was determined in transfected cells with anti-FLAG and a new protein of 11 kDa was detected in HepG2 and COS-1 cells transfected with pJME. Only E (53 kDa) protein was identified as transfected with pJME using anti-E. Higher level of neutralization antibodies and the efficacy of protective immunity were induced with pJME immunization, and were similar to those induced by inactivated Japanese encephalitis vaccine, but were better than those induced with pJE. It concludes that the expression level from prM to E proteins of JEV is different in vitro, and the in vitro expression efficiency of pJME was better than that of pJE. FLAG-prME protein expressed by pJME could be cleaved by peptidase from host. The efficacy of DNA immunization is correlated to the expression characterization of related proteins expressed in vitro.
文摘The specific recombinant proteins rOspA B. afzelii, rOspA B. garinii a rOspA B. burgdorferi sensu stricto were used as antigens for construction of ELISA sets. ELISA examination enables determination of specific post-vaccination antibodies against OspA B. afzelii, B. garinii a B. burgdorferi sensu stricto.Using recombinant DNA technology, genes from B. afzelii, B. garinii and B. burgdorferi sensu stricto were inserted into E. coli-expression vectors and the rOspA proteins were produced. These proteins were used for the construction of ELISA kits for the determination of post-vaccination antibodies against individual Borrelia serovars contained in the vaccine. The antibody response of dogs vaccinated with whole-cell vaccine BORRELYM 3 and non-vaccinated dogs was monitored and compared. The ELISA method proved as highly sensitive for the determination of post-vaccination antibodies against individual Borrelia serovars in vaccinated animals. Detection of these antibodies and their quantification may be used for evaluation of efficiency of vaccines against Lyme borreliosis in dogs caused by Borrelia burgdorferi sensu lato. Prospectively, it will be necessary to establish a correlation between post-vaccination antibody levels and protective immunity of vaccinated dogs.
