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目的 :克隆小鼠 4 1BBL基因 ,构建其真核表达载体 ,并观察其在抗肝癌免疫中的作用。方法 :取C5 7BL/6小鼠脾细胞 ,经PHA诱导后 ,以RT PCR克隆 4 1BBLcDNA ,测序 ,构建真核表达质粒 pcDNA3.1(+) m4 1BBL。以重组体转染小鼠肝癌细胞Hepa1 6 ,经G4 18筛选后 ,以RT PCR、间接免疫荧光及流式细胞仪 ,检测m4 1BBL以获得稳定高表达克隆。然后将其制成肿瘤细胞疫苗与同源小鼠脾淋巴细胞混合培养 ,采用MTT比色法测定淋巴细胞的特异性杀伤活性。结果 :从小鼠脾细胞中克隆到m4 1BBLcDNA ,经测序完全正确。所构建的真核表达质粒pcDNA3.1(+) m4 1BBL ,在小鼠肝癌细胞Hepa1 6中获得稳定高效表达。与野生型Hepa1 6细胞相比较 ,m4 1BBL基因转染的Hepa1 6细胞疫苗能较有效地诱导淋巴细胞产生针对野生型Hepa1 6细胞的特异性杀伤活性 (P <0 .0 1)。结论 :将m4 1BBL基因导入肝癌细胞中表达 ,能提高其免疫原性 ,诱导有效地抗肝癌免疫应答
OBJECTIVE: To clone the 4 1BBL gene of mouse and construct its eukaryotic expression vector and observe its function in anti-hepatocellular carcinoma immunity. Methods: The spleen cells of C5 7BL / 6 mice were induced by PHA. The 4 1BBL cDNA was cloned by RT PCR and sequenced. The eukaryotic expression plasmid pcDNA3.1 (+) m4 1BBL was constructed. The HepG6 cells were transfected with the recombinant plasmids. After screened by G418, RT-PCR, indirect immunofluorescence and flow cytometry were used to detect m4 1BBL to obtain stable and highly expressed clones. Then it was made into tumor cell vaccine mixed with spleen lymphocytes of homologous mice, MTT colorimetric determination of lymphocyte specific killing activity. Results: The m4 1BBL cDNA was cloned from mouse spleen cells and sequenced completely. The constructed eukaryotic expression plasmid pcDNA3.1 (+) m4 1BBL, stable and highly efficient expression in mouse hepatoma Hepa1 6. Compared with wild-type Hepa1 6 cells, Hepa1 6 cell vaccine transfected with m4 1BBL gene could induce lymphocyte specific Hepa1 6-specific cytotoxicity (P <0.01). Conclusion: The m4 1BBL gene can be introduced into hepatoma cells to enhance its immunogenicity and induce effective anti-liver cancer immune response