Gp96, a member of HSP90 family, is a versatile molecular chaperone with various newly-discovered functions, for example to serve as a low affinity, high capacity calcium binding protein, a natural adjuvant for therape...Gp96, a member of HSP90 family, is a versatile molecular chaperone with various newly-discovered functions, for example to serve as a low affinity, high capacity calcium binding protein, a natural adjuvant for therapeutic cancer vaccines, a tumor rejection antigen, an immune regulator to pathological cell death. Its multi-functional and structural characteristics make it also an interesting target to develop antibody-based therapeutics. However, its low immunogenicity to mice, because of its high-sequence similarity among different species, is an obstacle to obtain valuable monoclonal antibodies (MAbs). This is a common problem for any low immunogenic proteins, whose sequences share close identity between mice and other species. Here, a new strategy of priming was employed by swine endogenous full-length gp96 and then boosting by E. coli-system heterologously expressed gp96 N-terminal fragment (N-355) to generate MAbs. Twelve different highly-specific MAbs against swine/human endogenous gp96 were successfully obtained. The binding activities of these MAbs were confirmed by enzyme-linked immunosorbent assay (ELISA), Western blot (WB), immunofluorescence and flow cytometry analysis. This provides some important reagents for further research and potential therapeutics. The methods employed can be used for MAb production of any sequence-highly-conserved proteins between mice and swine/human (or any other species).展开更多
Hapten sulfaguanidine （SG） was coupled with carrier protein bovine serum albumin （BSA） to form a full antigen SG- BSA by diazotization and glutaraldehyde methods. Ovalbumin （OVA） was used as protein carrier to c...Hapten sulfaguanidine （SG） was coupled with carrier protein bovine serum albumin （BSA） to form a full antigen SG- BSA by diazotization and glutaraldehyde methods. Ovalbumin （OVA） was used as protein carrier to couple with Hapten SG by glutaraldehyde method. Then, the immunogen and the coating antigen were purified by dialysis and gel exclusion chromatography. The conjugated ratio of SG to BSA in artificial antigen was 5.3 （using diazotization method） and 6.5 （glutaraldehyde method）, and the conjugated ratio of SG to OVA in coating antigen was 2.3 （glutaraldehyde method） by UV-visible spectrophotometer. The coupling was successful according to the analysis of sodium dodecyl sulphate polyacrylamide gel electrophoresis （SDS-PAGE）. BALB/c mice were immunized with the antigen （SG-BSA）, and the titers of antiserum were tested to be 1 ： 6 400 and 1 ： 400 after three periods of immunities by indirect ELISA, which further identified the success of the synthesis of both immunogen SG-BSA and coating antigen SG-OVA.展开更多
基金Project(31030030) supported by the National Natural Science Foundation of China
文摘Gp96, a member of HSP90 family, is a versatile molecular chaperone with various newly-discovered functions, for example to serve as a low affinity, high capacity calcium binding protein, a natural adjuvant for therapeutic cancer vaccines, a tumor rejection antigen, an immune regulator to pathological cell death. Its multi-functional and structural characteristics make it also an interesting target to develop antibody-based therapeutics. However, its low immunogenicity to mice, because of its high-sequence similarity among different species, is an obstacle to obtain valuable monoclonal antibodies (MAbs). This is a common problem for any low immunogenic proteins, whose sequences share close identity between mice and other species. Here, a new strategy of priming was employed by swine endogenous full-length gp96 and then boosting by E. coli-system heterologously expressed gp96 N-terminal fragment (N-355) to generate MAbs. Twelve different highly-specific MAbs against swine/human endogenous gp96 were successfully obtained. The binding activities of these MAbs were confirmed by enzyme-linked immunosorbent assay (ELISA), Western blot (WB), immunofluorescence and flow cytometry analysis. This provides some important reagents for further research and potential therapeutics. The methods employed can be used for MAb production of any sequence-highly-conserved proteins between mice and swine/human (or any other species).
文摘Hapten sulfaguanidine （SG） was coupled with carrier protein bovine serum albumin （BSA） to form a full antigen SG- BSA by diazotization and glutaraldehyde methods. Ovalbumin （OVA） was used as protein carrier to couple with Hapten SG by glutaraldehyde method. Then, the immunogen and the coating antigen were purified by dialysis and gel exclusion chromatography. The conjugated ratio of SG to BSA in artificial antigen was 5.3 （using diazotization method） and 6.5 （glutaraldehyde method）, and the conjugated ratio of SG to OVA in coating antigen was 2.3 （glutaraldehyde method） by UV-visible spectrophotometer. The coupling was successful according to the analysis of sodium dodecyl sulphate polyacrylamide gel electrophoresis （SDS-PAGE）. BALB/c mice were immunized with the antigen （SG-BSA）, and the titers of antiserum were tested to be 1 ： 6 400 and 1 ： 400 after three periods of immunities by indirect ELISA, which further identified the success of the synthesis of both immunogen SG-BSA and coating antigen SG-OVA.
