The experiment was conducted to study the specific expression of HSP70 caused by heat shock, HSP70 purification and the characteristics of coalescence with antigenic peptide in the formation of the complex. Sixty heal...The experiment was conducted to study the specific expression of HSP70 caused by heat shock, HSP70 purification and the characteristics of coalescence with antigenic peptide in the formation of the complex. Sixty healthy 6-week-old male Wulong geese were selected and randomly divided into three groups. The control group was slaughtered without heat treatment. Treatment group 1 was shocked with an acute heat treatment at （42 ± 1）℃ for 5 h before they were immediately slaughtered. Treatment group 2 was kept for 12 h after the heat treatment under normal conditions in order to recover and was then slaughtered. Cardiac tissue was taken in order to make paraffin sections for the immunohistochemistry experiment and the liver tissue was used to purify HSP70. The geese heart HSP70 expression differences in the three groups were determined and at the same time the experiments of HSP70 purification and appraisal in the liver tissue were carried on. HSP70 purification and synthesis of HBV PreS1 multi-peptides unified the complex, which was determined by bi-specific antibody enzyme-linked immune sandwich assay. The results indicated that widespread HSP70 positive pellets in the cardiac muscle were found under hot shock conditions. HSP70 expression in the treatment group 1 was centered in the karyotheca and its periphery, but in treatment group 2, it was centered in the surrounding cell membrane. The HSP70 purification could be obtained through two sets of purification plans; both the synthesis peptide and the HSP70 purification form the complex under certain conditions. The double antibody sandwich ELISA technique was applied to detect if the complex had been formed. Positive results showed that the complex was formed. The specific expression of HSP70 under heat shock shifted with time, suggesting that HSP70 possibly had some function in cell protection. High-purity HSP70 protein can be obtained under low-pressure chromatography conditions, and in comparison with each other, it was better in the flow of the molecular 展开更多
Hepatitis C (HCV) genome is highly variable, particularly in the hypervariable region 1 (HVR1) of its E2 envelope gene. The variability of HCV genome has been a major obstacle for developing HCV vaccines. Due to B-cel...Hepatitis C (HCV) genome is highly variable, particularly in the hypervariable region 1 (HVR1) of its E2 envelope gene. The variability of HCV genome has been a major obstacle for developing HCV vaccines. Due to B-cell HVR1 mimotopes mimicking the antigenicity of natural HVR1 epitopes; some T-cell epitopes from the consensus sequence of HCV genes conserving among the different HCV genotypes, we synthesized an minigene of HCV-derived multi-epitope peptide antigen (CMEP), which contains 9 B-cell HVR1 mimotopes in E2, 2 conserved CTL epitopes in C, 1 conserved CTL epitope in NS3; 1 conserved Th epitope in NS3. This minigene was cloned into a GST expression vector to generate a fusion protein GST-CMEP. The immunogenic properties of CEMP were characterized by HCV infected patients’ sera,; found that the reactivity frequency reached 75%. The cross reactivity of anti-CEMP antibody with different natural HVR1 variants was up to 90%. Meanwhile, we constructed an HCV DNA vaccine candidate, plasmid pVAX1.0-st-CMEP carrying the recombinant gene (st) of a secretion signal peptide; PADRE universal Th cell epitope sequence in front of the CMEP minigene. Immunization of rabbits with pVAX1.0-st-CMEP resulted in the production of antibody, which was of the same cross reactivity as the fusion protein GST-CMEP. Our findings indicate that the HCV-derived multi-epitope peptide antigen in some degree possessed the characteristics of neutralizing HCV epitopes,; would be of the value as a candidate for the development of HCV vaccines.展开更多
文摘The experiment was conducted to study the specific expression of HSP70 caused by heat shock, HSP70 purification and the characteristics of coalescence with antigenic peptide in the formation of the complex. Sixty healthy 6-week-old male Wulong geese were selected and randomly divided into three groups. The control group was slaughtered without heat treatment. Treatment group 1 was shocked with an acute heat treatment at （42 ± 1）℃ for 5 h before they were immediately slaughtered. Treatment group 2 was kept for 12 h after the heat treatment under normal conditions in order to recover and was then slaughtered. Cardiac tissue was taken in order to make paraffin sections for the immunohistochemistry experiment and the liver tissue was used to purify HSP70. The geese heart HSP70 expression differences in the three groups were determined and at the same time the experiments of HSP70 purification and appraisal in the liver tissue were carried on. HSP70 purification and synthesis of HBV PreS1 multi-peptides unified the complex, which was determined by bi-specific antibody enzyme-linked immune sandwich assay. The results indicated that widespread HSP70 positive pellets in the cardiac muscle were found under hot shock conditions. HSP70 expression in the treatment group 1 was centered in the karyotheca and its periphery, but in treatment group 2, it was centered in the surrounding cell membrane. The HSP70 purification could be obtained through two sets of purification plans; both the synthesis peptide and the HSP70 purification form the complex under certain conditions. The double antibody sandwich ELISA technique was applied to detect if the complex had been formed. Positive results showed that the complex was formed. The specific expression of HSP70 under heat shock shifted with time, suggesting that HSP70 possibly had some function in cell protection. High-purity HSP70 protein can be obtained under low-pressure chromatography conditions, and in comparison with each other, it was better in the flow of the molecular
基金supported by the National Natural Science Foundation of China(Grant No.30471596)
文摘Hepatitis C (HCV) genome is highly variable, particularly in the hypervariable region 1 (HVR1) of its E2 envelope gene. The variability of HCV genome has been a major obstacle for developing HCV vaccines. Due to B-cell HVR1 mimotopes mimicking the antigenicity of natural HVR1 epitopes; some T-cell epitopes from the consensus sequence of HCV genes conserving among the different HCV genotypes, we synthesized an minigene of HCV-derived multi-epitope peptide antigen (CMEP), which contains 9 B-cell HVR1 mimotopes in E2, 2 conserved CTL epitopes in C, 1 conserved CTL epitope in NS3; 1 conserved Th epitope in NS3. This minigene was cloned into a GST expression vector to generate a fusion protein GST-CMEP. The immunogenic properties of CEMP were characterized by HCV infected patients’ sera,; found that the reactivity frequency reached 75%. The cross reactivity of anti-CEMP antibody with different natural HVR1 variants was up to 90%. Meanwhile, we constructed an HCV DNA vaccine candidate, plasmid pVAX1.0-st-CMEP carrying the recombinant gene (st) of a secretion signal peptide; PADRE universal Th cell epitope sequence in front of the CMEP minigene. Immunization of rabbits with pVAX1.0-st-CMEP resulted in the production of antibody, which was of the same cross reactivity as the fusion protein GST-CMEP. Our findings indicate that the HCV-derived multi-epitope peptide antigen in some degree possessed the characteristics of neutralizing HCV epitopes,; would be of the value as a candidate for the development of HCV vaccines.
