AIM:To purify the heat shock protein (HSP) 70-associated tumor peptides and to observe its non-MHC-I molecule restrictive antitumor effect.METHODS:By ConA-sepharose affinity chromatography,ADP-agarose affinity chromat...AIM:To purify the heat shock protein (HSP) 70-associated tumor peptides and to observe its non-MHC-I molecule restrictive antitumor effect.METHODS:By ConA-sepharose affinity chromatography,ADP-agarose affinity chromatography, and DEAE anion exchange chromatography, we were able to purify HSP70-associated peptides from mouse hepatoma (HCaF) cells treated in heat shock at 42℃. Specific active immunization and adoptive cellular immunization assay were adopted to observe the immunoprotective effect elicited by HSP70-associated peptide complexes isolated from HcaF.RESULTS: The finally purified HSP-associated peptides had a very high purity and specificity found by SDS-PAGE and Western blot. Mice immunized with HSP70-associated peptide complexes purified from HCaF cells were protected from HCaF living cell challenge. This effect was dose dependent.Adoptive immunization of immune spleen cells of mice immunized with HSP70-associated peptide complexes could elicit immunity against HCaF challenge, and the tumor-free mice could resist repeated challenges. This effect could be continuously enhanced by repeated challenge with HCaF living cells. The tumor-free mice could tolerate the challenge for as high as 1×10^7 HCaF cells. The mice immunized once with spleen cells pulsed with HSP70-associated peptide complexes in vitro could also result in a certain adoptive immunity against HCaF.CONCLUSION:High purity and specificity of HSP70-associated peptides could be achieved from tumor cells by the low-pressure affinity chromatography method used in this study. HSP70-associated peptide complexes derived from the HCaF can elicit non-MHC-I molecule restrictive immunoprotective effect against HCaF.This effect can be transferred by adoptive immunization to mice and enhanced by repeated challenge with HCaF live cells.展开更多
AM: To produce high-quality potyclonal antibody to lysosomeassociated protein transmembrane 4B-35 and to identify LAPTM4B-35 expression in cancer tissues and its correlation with differentiation status of hepatocellul...AM: To produce high-quality potyclonal antibody to lysosomeassociated protein transmembrane 4B-35 and to identify LAPTM4B-35 expression in cancer tissues and its correlation with differentiation status of hepatocellular carcinoma (HCC). METHODS: The 297 bp 5' end of LAPTM4BcDNA was obtained by PCR and inserted into prokaryotic expression vector pGEX-KG. Then the recombinant pGEX-KG-N_(1-99) was transformed into E. coli JM109 to express GST-fusion protein. The fusion protein was purified by glutathione sepharose^(TM) 4B agarose. The purified GST-LAPTM4B-N_(1-99) was characterized by SDSPAGE, and used to immunize rabbits. The titer and specificity of antisera were detected by ELISA and Western blot, respectively. The correlation between the expression levels of LAPTM4B-35 and the differentiation status of HCC was analyzed via Western blot. The expression of LAPTM4B-35 in HCC and other six cancer tissues was investigated via tissue chip and immunohistochemical analysis. RESULTS: About 6.2 mg of pure GST-LAPTM4B-N_(1-99) was isolated from 1 L of bacteria. The GST-LAPTM4B-N_(1-99) produced high titer antisera in rabbits and showed good immunity. Western blot showed specific reactions for the antibody to the LAPTM4B-35 in the total proteins from HCC tissues and BEL-7402 cells, also to the fusion protein purified or in the transformed bacteria. LAPTM4B-35 was remarkably expressed in several cancers, such as HCC, breast cancer, gastric carcinoma, lung cancer, and colon carcinoma, but not commonly expressed in esophageal cancer and rectum carcinoma. Notably, the expression levels of LAPTM4B-35 were significantly and inversely correlated to the differentiation of HCCs in a 20 case analysis. CONCLUSION: Specific polyclonal antibody (LAPTM4BN_(1-99)-pAb) to LAPTM4B-35 was produced. It identified the expression of LAPTM4B-35 in some cancer tissues originated from single layer cuboidal and columnar epithelial cells and firmly demonstrated that the expression of LAPTM4B-35 in HCC was inversely correlated with the differentiation of HCC.展开更多
基金Supported by the National Natural Science Foundation of China,No.3973440-Ⅱ
文摘AIM:To purify the heat shock protein (HSP) 70-associated tumor peptides and to observe its non-MHC-I molecule restrictive antitumor effect.METHODS:By ConA-sepharose affinity chromatography,ADP-agarose affinity chromatography, and DEAE anion exchange chromatography, we were able to purify HSP70-associated peptides from mouse hepatoma (HCaF) cells treated in heat shock at 42℃. Specific active immunization and adoptive cellular immunization assay were adopted to observe the immunoprotective effect elicited by HSP70-associated peptide complexes isolated from HcaF.RESULTS: The finally purified HSP-associated peptides had a very high purity and specificity found by SDS-PAGE and Western blot. Mice immunized with HSP70-associated peptide complexes purified from HCaF cells were protected from HCaF living cell challenge. This effect was dose dependent.Adoptive immunization of immune spleen cells of mice immunized with HSP70-associated peptide complexes could elicit immunity against HCaF challenge, and the tumor-free mice could resist repeated challenges. This effect could be continuously enhanced by repeated challenge with HCaF living cells. The tumor-free mice could tolerate the challenge for as high as 1×10^7 HCaF cells. The mice immunized once with spleen cells pulsed with HSP70-associated peptide complexes in vitro could also result in a certain adoptive immunity against HCaF.CONCLUSION:High purity and specificity of HSP70-associated peptides could be achieved from tumor cells by the low-pressure affinity chromatography method used in this study. HSP70-associated peptide complexes derived from the HCaF can elicit non-MHC-I molecule restrictive immunoprotective effect against HCaF.This effect can be transferred by adoptive immunization to mice and enhanced by repeated challenge with HCaF live cells.
基金Supported by the 248 Major R&D Program of Beijing, No. H020220020310, and Special Fund for Promotion of Education, Ministry of Education, China
文摘AM: To produce high-quality potyclonal antibody to lysosomeassociated protein transmembrane 4B-35 and to identify LAPTM4B-35 expression in cancer tissues and its correlation with differentiation status of hepatocellular carcinoma (HCC). METHODS: The 297 bp 5' end of LAPTM4BcDNA was obtained by PCR and inserted into prokaryotic expression vector pGEX-KG. Then the recombinant pGEX-KG-N_(1-99) was transformed into E. coli JM109 to express GST-fusion protein. The fusion protein was purified by glutathione sepharose^(TM) 4B agarose. The purified GST-LAPTM4B-N_(1-99) was characterized by SDSPAGE, and used to immunize rabbits. The titer and specificity of antisera were detected by ELISA and Western blot, respectively. The correlation between the expression levels of LAPTM4B-35 and the differentiation status of HCC was analyzed via Western blot. The expression of LAPTM4B-35 in HCC and other six cancer tissues was investigated via tissue chip and immunohistochemical analysis. RESULTS: About 6.2 mg of pure GST-LAPTM4B-N_(1-99) was isolated from 1 L of bacteria. The GST-LAPTM4B-N_(1-99) produced high titer antisera in rabbits and showed good immunity. Western blot showed specific reactions for the antibody to the LAPTM4B-35 in the total proteins from HCC tissues and BEL-7402 cells, also to the fusion protein purified or in the transformed bacteria. LAPTM4B-35 was remarkably expressed in several cancers, such as HCC, breast cancer, gastric carcinoma, lung cancer, and colon carcinoma, but not commonly expressed in esophageal cancer and rectum carcinoma. Notably, the expression levels of LAPTM4B-35 were significantly and inversely correlated to the differentiation of HCCs in a 20 case analysis. CONCLUSION: Specific polyclonal antibody (LAPTM4BN_(1-99)-pAb) to LAPTM4B-35 was produced. It identified the expression of LAPTM4B-35 in some cancer tissues originated from single layer cuboidal and columnar epithelial cells and firmly demonstrated that the expression of LAPTM4B-35 in HCC was inversely correlated with the differentiation of HCC.
