The expressed sequence tag of eukaryotic translation initiation factor 5 （eIF5） from the Schistosoma japonicum adult worm cDNA library through subtractive hybridization between male and female worms was analyzed by ...The expressed sequence tag of eukaryotic translation initiation factor 5 （eIF5） from the Schistosoma japonicum adult worm cDNA library through subtractive hybridization between male and female worms was analyzed by the bioinformatics method. The overlapping sequences were assembled into one that includes the complete open reading frame （GenBank accession number： AY686501）. The full-length cDNA of SjeIF5 was cloned into a pET-28c^（＋） vector, which generated a prokaryotic expression plasmid, and a fusion protein of 18 kDa was induced in Escherichia coll. The recombinant expression of eIF5 protein of Schistosoma japonicum was purified. The immunoprotection test against schistosomiasis demonstrated that the recombinant protein worked to a certain extent, especially in the reduction of eggs in the liver of the host.展开更多
AIM： To promote the development of improved tumor vaccination strategies relying on the intratumoral expression of viral fusogenic membrane proteins, we elucidated whether the size of syncytia or the way tumor cells ...AIM： To promote the development of improved tumor vaccination strategies relying on the intratumoral expression of viral fusogenic membrane proteins, we elucidated whether the size of syncytia or the way tumor cells die has an effect on the therapeutic outcome. METHODS： In two syngeneic subcutaneous murine colon cancer models we assessed the anti-neoplastic effect on vector-treated and contralateral untreated tumors. RESULTS： Intratumoral injection of a replication-defective adenovirus encoding respiratory syncytial virus fusion protein （RSV-F） alone （Ad.RSV-F） or together with the attachment glycoprotein RSV-G （Ad.RSV-F/G） led to a significant growth reduction of the vector-treated and contralateral untreated tumors. The treatment response was associated with a strong tumor-specific CTL response and significantly improved survival with medians of 46 d and 44 d, respectively. Intratumoral injection of Ad,RSV-G or a soluble RSV-F encoding adenovirus （Ad.RSV-Fsol） had no significant anti-neoplastic effect. The median survival of these treabnent groups and of Ad.Null-treated control animals was about 30 d. CONCLUSION： Although in vitro transduction of colon cancer cell lines with Ad.RSV-F/G resulted in about 8-fold larger syncytia than with Ad.RSV-F, the in vivo outcome was not sig nificantly different, Transduction of murinecolon cancer cell lines with Ad.RSVoF or Ad.RSV-F/G caused apoptotic cell death, in contrast to transduction with Ad.RSV-G or Ad.RSV-Fsol, suggesting an importance of the mode of cell death. Overall, these findings provide insight into improved tumor vaccination strategies relying on the intratumoral expression of viral fusogenic membrane proteins.展开更多
To construct and express the fusion protein Stx2B-IntiminC300 of EHEC O157 ： H7, and to further investigate its immunoprophylactic potential, the gene of Stx2B （stx2b） from EHEC O157：H7 chromosome was cloned into ...To construct and express the fusion protein Stx2B-IntiminC300 of EHEC O157 ： H7, and to further investigate its immunoprophylactic potential, the gene of Stx2B （stx2b） from EHEC O157：H7 chromosome was cloned into pMD18-T vector. Thereafter, the amplified gene was cloned into prokary- otic expression plasmid pET-28a （ ＋ ）-eaeC300, which was constructed previously. The recombinant pasmid pET-28a（ ＋ ）-stx2b-eaeC300 was transformed into E. coli BL21 （DE3）. After inducement, the protein Stx2B-IntiminC300 was successfully expressed and analyzed with sodium dodecyl sulfate polyacrylamide gel electrophoresis （SDS-PAGE）, Western blotting and N-terminal amino acid residual sequencing. To evaluate its immunoprophylactic potential, it was primarily purified by ion-exchange chromatography and injected into 30 BALB/c mice with AI（OH）3 in the subscapular region. Ten days after the last booster vaccination, 20 mice were attacked with EHEC O157：H7 lysate and the protective efficacy was observed. In the present study, the gene of Stx2B-intiminC300 was successfully cloned into pET-28a （ ＋ ） vector. The results of SDS-PAGE and Western blotting assay showed that the fusion protein was successfully expressed in the inclusion body form, accounting for 25 % of total expression products, and its molecular weight was about 43 kDa. The result of the N-terminal amino acid residual sequencing showed that it was identical to that of the molecular designed. The purity was about 75 % after primary purification. Animal tests revealed that the fusion protein Stx2B-intiminC300 has elicited high titer of protective antibody relatively. These results demonstrate that the fusion protein Stx2B-IntiminC300 is successfully expressed in prokaryotic expression system and shows certain immunoprophylactic potential.展开更多
文摘The expressed sequence tag of eukaryotic translation initiation factor 5 （eIF5） from the Schistosoma japonicum adult worm cDNA library through subtractive hybridization between male and female worms was analyzed by the bioinformatics method. The overlapping sequences were assembled into one that includes the complete open reading frame （GenBank accession number： AY686501）. The full-length cDNA of SjeIF5 was cloned into a pET-28c^（＋） vector, which generated a prokaryotic expression plasmid, and a fusion protein of 18 kDa was induced in Escherichia coll. The recombinant expression of eIF5 protein of Schistosoma japonicum was purified. The immunoprotection test against schistosomiasis demonstrated that the recombinant protein worked to a certain extent, especially in the reduction of eggs in the liver of the host.
基金Supported by The Wilhelm Sander-Stiftung, No. 204.036.1
文摘AIM： To promote the development of improved tumor vaccination strategies relying on the intratumoral expression of viral fusogenic membrane proteins, we elucidated whether the size of syncytia or the way tumor cells die has an effect on the therapeutic outcome. METHODS： In two syngeneic subcutaneous murine colon cancer models we assessed the anti-neoplastic effect on vector-treated and contralateral untreated tumors. RESULTS： Intratumoral injection of a replication-defective adenovirus encoding respiratory syncytial virus fusion protein （RSV-F） alone （Ad.RSV-F） or together with the attachment glycoprotein RSV-G （Ad.RSV-F/G） led to a significant growth reduction of the vector-treated and contralateral untreated tumors. The treatment response was associated with a strong tumor-specific CTL response and significantly improved survival with medians of 46 d and 44 d, respectively. Intratumoral injection of Ad,RSV-G or a soluble RSV-F encoding adenovirus （Ad.RSV-Fsol） had no significant anti-neoplastic effect. The median survival of these treabnent groups and of Ad.Null-treated control animals was about 30 d. CONCLUSION： Although in vitro transduction of colon cancer cell lines with Ad.RSV-F/G resulted in about 8-fold larger syncytia than with Ad.RSV-F, the in vivo outcome was not sig nificantly different, Transduction of murinecolon cancer cell lines with Ad.RSVoF or Ad.RSV-F/G caused apoptotic cell death, in contrast to transduction with Ad.RSV-G or Ad.RSV-Fsol, suggesting an importance of the mode of cell death. Overall, these findings provide insight into improved tumor vaccination strategies relying on the intratumoral expression of viral fusogenic membrane proteins.
文摘To construct and express the fusion protein Stx2B-IntiminC300 of EHEC O157 ： H7, and to further investigate its immunoprophylactic potential, the gene of Stx2B （stx2b） from EHEC O157：H7 chromosome was cloned into pMD18-T vector. Thereafter, the amplified gene was cloned into prokary- otic expression plasmid pET-28a （ ＋ ）-eaeC300, which was constructed previously. The recombinant pasmid pET-28a（ ＋ ）-stx2b-eaeC300 was transformed into E. coli BL21 （DE3）. After inducement, the protein Stx2B-IntiminC300 was successfully expressed and analyzed with sodium dodecyl sulfate polyacrylamide gel electrophoresis （SDS-PAGE）, Western blotting and N-terminal amino acid residual sequencing. To evaluate its immunoprophylactic potential, it was primarily purified by ion-exchange chromatography and injected into 30 BALB/c mice with AI（OH）3 in the subscapular region. Ten days after the last booster vaccination, 20 mice were attacked with EHEC O157：H7 lysate and the protective efficacy was observed. In the present study, the gene of Stx2B-intiminC300 was successfully cloned into pET-28a （ ＋ ） vector. The results of SDS-PAGE and Western blotting assay showed that the fusion protein was successfully expressed in the inclusion body form, accounting for 25 % of total expression products, and its molecular weight was about 43 kDa. The result of the N-terminal amino acid residual sequencing showed that it was identical to that of the molecular designed. The purity was about 75 % after primary purification. Animal tests revealed that the fusion protein Stx2B-intiminC300 has elicited high titer of protective antibody relatively. These results demonstrate that the fusion protein Stx2B-IntiminC300 is successfully expressed in prokaryotic expression system and shows certain immunoprophylactic potential.
