目的:构建人B7.1(CD80)重组真核表达载体,并在HL60细胞上表达,探讨其在急性白血病免疫治疗及免疫保护中的重要作用。方法:①应用分子克隆方法,采用PCR法扩增人B7.1基因片段,ApaⅠ、SalⅠ分别双酶切PCR产物和表达载体pHook,纯化后的酶切产物由T4 DNA连接酶连接,转化大肠杆菌DH5α,应用ApaⅠ、SalⅠ双酶切和DNA序列分析技术进一步鉴定阳性克隆。②应用脂质体转化技术将B7.1转化HL60细胞,并以FACS检测其表达。③将B7.1~+细胞免疫小鼠,通过成瘤时间及存活时间等指标观察其免疫治疗和免疫保护作用。结果:①PCR法扩增出一大小约620 bp的基因片段;共筛选出6个阳性克隆,均可ApaⅠ、SalⅠ双酶切出大小约620 bp的基因片段,与人B7.1基因片段大小相符,提示重组载体构建成功。进一步DNA序列分析证明人B7.1基因序列和读码框架正确,与文献报道人B7.1cDNA序列一致,无基因突变。②应用FACS在转化HL60上检测到B7.1的高效表达。③B7.1~+ HL60细胞能明显抑制肿瘤生长,延长白血病鼠存活时间,且其免疫预防时成瘤时间较野生型HL60(B7.1-)显著延迟,小鼠存活时间明显延长。结论:应用分子克隆方法可成功构建人B7.1(CD80)重组真核表达载体,并稳定、高效表达于B7.1之急性髓性白血病(AML)细胞系HL60胞膜上,这种共刺激分子瘤苗具有显著 OBJECTIVE: To construct a recombinant eukaryotic expression vector of human B7.1 (CD80) and express it on HL60 cells, and to explore its important role in the immunotherapy and immune protection of acute leukemia. Methods: (1) Human B7.1 gene fragment was amplified by PCR using molecular cloning method. PCR product and pHook were digested with ApaⅠand SalⅠ, respectively. The purified products were ligated with T4 DNA ligase and transformed into E.coli DH5α, using Apa Ⅰ, Sal Ⅰ double digestion and DNA sequence analysis technology to further identify positive clones. ② B7.1 was transformed into HL60 cells by lipofectamine and the expression of B7.1 was detected by FACS. ③ B7.1 ~ + cells were immunized mice, through the tumorigenic time and survival time and other indicators to observe the immunotherapy and immune protection. Results: (1) A gene fragment of about 620 bp in length was amplified by PCR. Six positive clones were screened out. The gene fragment of about 620 bp in length was digested with ApaI and SalI, which was consistent with the size of human B7.1 gene fragment , Suggesting that the recombinant vector was successfully constructed. Further DNA sequence analysis confirmed that the human B7.1 gene sequence and the reading frame were correct, consistent with the reported human B7.1 cDNA sequence, gene-free mutation. ② The high expression of B7.1 was detected by FACS on transformed HL60. ③B7.1 ~ + HL60 cells can significantly inhibit tumor growth, prolong the survival time of leukemia mice, and the time of tumorigenesis in immunoprophylaxis is significantly delayed than that of wild-type HL60 (B7.1-), and the survival time of mice is obviously prolonged. CONCLUSION: The recombinant eukaryotic expression vector of human B7.1 (CD80) can be successfully constructed by molecular cloning method and stably and efficiently expressed on the cell membrane of acute myeloid leukemia (AML) cell line B7.1. This costimulation Molecular tumor vaccine is significant