目的 克隆人 B7.1全长 c DNA及构建相应的逆转录病毒表达载体。 方法 应用逆转录多聚酶链反应从人 B淋巴瘤细胞系 Raji中克隆 B7.1全长 c DNA ,再将 c DNA插入到质粒 p Bluescript中 ,经自动荧光测序证实无误后 ,再通过定向克隆构建...目的 克隆人 B7.1全长 c DNA及构建相应的逆转录病毒表达载体。 方法 应用逆转录多聚酶链反应从人 B淋巴瘤细胞系 Raji中克隆 B7.1全长 c DNA ,再将 c DNA插入到质粒 p Bluescript中 ,经自动荧光测序证实无误后 ,再通过定向克隆构建相应的逆转录病毒表达载体 PL XSNh B7。 结果 逆转录多聚酶链反应扩增产物长度与预期的 889bp一致 ;用 M13正、反向引物进行荧光测序证实 ,克隆出的序列与 Gen Bank的 B7.1c DNA序列完全一致 ;人 B7.1全长 c DNA被成功地插入到质粒 PL XSN中。 结论 人 B7.1全长 c DNA的克隆及相应逆转录病毒表达载体的构建为今后应用 B7.1进行肿瘤免疫基因治疗提供了可能性。展开更多
Objective To express human B7. 2 extracellular domain with prokaryote expression system and to evaluate its biological activity in vitro. Methods PCR was used to amplify the extracellular region of human B7. 2 which c...Objective To express human B7. 2 extracellular domain with prokaryote expression system and to evaluate its biological activity in vitro. Methods PCR was used to amplify the extracellular region of human B7. 2 which contained both the IgV and IgC domains. The recombinant PGEX-4T-3/hB7. 2 (IgV+C) was obtained by cloning the PCR product into a prokaryote expression plasmid PGEX-4T-3 and was transformed into the host strain of DH5-a. Tke fusion protein consisted of GST and hB7. 2(IgV+C) was identified by SDS-PAGE and Western blotting. T cell activation was observed by exposing purified T lymphocytes to the fusion protein and [3H]-TdR incorporation with the presence of the first signal imitated hy anti-CD3 antibody. Results The fusion protein GST-hB7. 2 (IgV+ C) was produced and detected in inclusive body form from engineered bacterial cells. With the first signal existed,T lymphocytes proliferated when it was co-stimulated by the fusion protein. Conclusion These results indicated that the functional human B7. 2(IgV+C) fusion protein can be produced in bacterial cells and the fusion protein displays the co-stimulatory activity in T lymphocytes activation.展开更多
Objective: To construct the human B7. 1 (CD80) eukaryocytic expressing vector and its expression on HL60 cells to investigate the immunotherapeutic and immunoprotective effects of B7. 1 molecule on acute leukemia. Met...Objective: To construct the human B7. 1 (CD80) eukaryocytic expressing vector and its expression on HL60 cells to investigate the immunotherapeutic and immunoprotective effects of B7. 1 molecule on acute leukemia. Methods: ①B7. 1 gene was subcloned from the cloning vector using PCR. Both of tlie PCR products and eukaryocytic expressing vector pHook were digested with Apa I , Sal 1 and linked using T4 DNA ligase. Tlie ligased products were transduced into DH5a. B7. 1 gene containing clones were selected by digestion with Apa I and Sal I which were further conformed by sequencing. ②HL60 cells were transformed by B7. 1 with lipofectamine and detected by FACS. ③Tumor formation, mice survival time and the immunotherapeutic and immunoprotective effects by immunization with B7. 1+ cells were evaluated. Results: ①The PCR products were about 620 bp. Six clones were all digested by Apa I and Sal I to produce 620 bp gene fragment which was in tlie same way as 67. 1. It demonstrated t/iat t/ie recombinant vector had been constructed successfully. Further sequencing confirmed the validity of the construction. There was no nucleotide mutation. ② B7. 1 was availably expressed on HL60 cells. ③ B7. 1+ HL60 cells delayed the growth of tumor and prolonged the survival time of leukemic mice, distinctively. So did tlie immunoprotection of B7. 1 + HL60 cells. Conclusion: The human B7. 1(CD80) eukaryocytic expressing vector can be successfully constructed by molecular cloned methods and can be stably and availably expressed on tlie membrane of B7. 1 negative acute myelocytic leukemia (AML) cell line HL60. Furthermore, the costimulatory molecular vaccine has significant effection of immunotherapy and immunoprotection and gives the rationale for clinical application.展开更多
The data from in vitro and animal experiment study has showed that costimulaory molecule B7 1 plays an important role in antitumor immunity In the present study, B7 1 expression was ob...The data from in vitro and animal experiment study has showed that costimulaory molecule B7 1 plays an important role in antitumor immunity In the present study, B7 1 expression was observed in 130 samples from a veriety of human malignancies by using immunocytochemistry, in situ hybridization and RT PCR combined with dot hybridization and B7 1 specific Mab and probe The results demonstrated B7 1 expression on tumor cells in 76 cases at both protein and mRNA level Forty two specimens were stained with B7 1 HLA ABC and HLA DR Mab and 26 showed that the three antibodies used all were positive Together with the achievement in tumor antigen study, the present findings imply that in most tumors (if not all) the tumor cells have all the requisite element to elicit anti tumor rejection response, the heterogeneous mechanism for tumor escape from immunosurvillance should be emphasized展开更多
基金This work was supported by the National Natural Science Foundation of China(No. 39470293).
文摘Objective To express human B7. 2 extracellular domain with prokaryote expression system and to evaluate its biological activity in vitro. Methods PCR was used to amplify the extracellular region of human B7. 2 which contained both the IgV and IgC domains. The recombinant PGEX-4T-3/hB7. 2 (IgV+C) was obtained by cloning the PCR product into a prokaryote expression plasmid PGEX-4T-3 and was transformed into the host strain of DH5-a. Tke fusion protein consisted of GST and hB7. 2(IgV+C) was identified by SDS-PAGE and Western blotting. T cell activation was observed by exposing purified T lymphocytes to the fusion protein and [3H]-TdR incorporation with the presence of the first signal imitated hy anti-CD3 antibody. Results The fusion protein GST-hB7. 2 (IgV+ C) was produced and detected in inclusive body form from engineered bacterial cells. With the first signal existed,T lymphocytes proliferated when it was co-stimulated by the fusion protein. Conclusion These results indicated that the functional human B7. 2(IgV+C) fusion protein can be produced in bacterial cells and the fusion protein displays the co-stimulatory activity in T lymphocytes activation.
基金Supported by Grant from the Key Clinical Department Development Item of China,Health Ministry(20012131)
文摘Objective: To construct the human B7. 1 (CD80) eukaryocytic expressing vector and its expression on HL60 cells to investigate the immunotherapeutic and immunoprotective effects of B7. 1 molecule on acute leukemia. Methods: ①B7. 1 gene was subcloned from the cloning vector using PCR. Both of tlie PCR products and eukaryocytic expressing vector pHook were digested with Apa I , Sal 1 and linked using T4 DNA ligase. Tlie ligased products were transduced into DH5a. B7. 1 gene containing clones were selected by digestion with Apa I and Sal I which were further conformed by sequencing. ②HL60 cells were transformed by B7. 1 with lipofectamine and detected by FACS. ③Tumor formation, mice survival time and the immunotherapeutic and immunoprotective effects by immunization with B7. 1+ cells were evaluated. Results: ①The PCR products were about 620 bp. Six clones were all digested by Apa I and Sal I to produce 620 bp gene fragment which was in tlie same way as 67. 1. It demonstrated t/iat t/ie recombinant vector had been constructed successfully. Further sequencing confirmed the validity of the construction. There was no nucleotide mutation. ② B7. 1 was availably expressed on HL60 cells. ③ B7. 1+ HL60 cells delayed the growth of tumor and prolonged the survival time of leukemic mice, distinctively. So did tlie immunoprotection of B7. 1 + HL60 cells. Conclusion: The human B7. 1(CD80) eukaryocytic expressing vector can be successfully constructed by molecular cloned methods and can be stably and availably expressed on tlie membrane of B7. 1 negative acute myelocytic leukemia (AML) cell line HL60. Furthermore, the costimulatory molecular vaccine has significant effection of immunotherapy and immunoprotection and gives the rationale for clinical application.
文摘The data from in vitro and animal experiment study has showed that costimulaory molecule B7 1 plays an important role in antitumor immunity In the present study, B7 1 expression was observed in 130 samples from a veriety of human malignancies by using immunocytochemistry, in situ hybridization and RT PCR combined with dot hybridization and B7 1 specific Mab and probe The results demonstrated B7 1 expression on tumor cells in 76 cases at both protein and mRNA level Forty two specimens were stained with B7 1 HLA ABC and HLA DR Mab and 26 showed that the three antibodies used all were positive Together with the achievement in tumor antigen study, the present findings imply that in most tumors (if not all) the tumor cells have all the requisite element to elicit anti tumor rejection response, the heterogeneous mechanism for tumor escape from immunosurvillance should be emphasized
