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
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.
