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目的制备负载RPL8蛋白的DC疫苗,探讨DC疫苗对小鼠乳腺癌生长抑制作用及作用机制。方法用PCR从质粒p UC57-RPL8中扩增RPL8片段,并将该片段插入p ET28α(+)原核表达载体中,实现重组蛋白的表达及纯化。用SDS-PAGE和Western blot对表达产物进行鉴定。分离培养小鼠骨髓细胞来源细胞。RPL8蛋白冲击DC,荧光显微镜下观察DC负载RPL8蛋白情况。MTT法检测淋巴细胞的杀伤效应;将负载RPL8蛋白的DC疫苗注射入乳腺癌荷瘤小鼠体内,观察肿瘤体积变化及小鼠的生存时间。结果成功构建p ET28α(+)-RPL8原核表达载体。经SDS-PAGE和Western blot鉴定,有目的蛋白表达,树突状细胞内有荧光颗粒。负载RPL8蛋白DC疫苗体外杀伤4T1乳腺癌细胞的细胞毒活性与未负载RPL8蛋白的DC及PBS组比较,有显著性差异(P<0.05)。DC疫苗注入荷瘤小鼠体内后,小鼠生存期、肿瘤体积变化较对照组有显著性差异(P<0.05)。结论负载RPL8蛋白的DC疫苗对小鼠乳腺癌具有免疫治疗作用,为乳腺癌的免疫治疗提供理论依据。
Objective To prepare DC vaccine loaded with RPL8 protein and investigate the inhibitory effect and mechanism of DC vaccine on the growth of mouse breast cancer. Methods The RPL8 fragment was amplified from plasmid pUC57-RPL8 by PCR and inserted into pET28α (+) prokaryotic expression vector to express and purify the recombinant protein. The expressed product was identified by SDS-PAGE and Western blot. Mouse bone marrow-derived cells were isolated and cultured. RPL8 protein impact DC, observed under a fluorescence microscope DC load RPL8 protein situation. The killing effect of lymphocytes was detected by MTT assay. The DC vaccine loaded with RPL8 protein was injected into breast cancer-bearing mice to observe the changes of tumor volume and the survival time of mice. Results The prokaryotic expression vector p ET28α (+) - RPL8 was successfully constructed. SDS-PAGE and Western blot identification, the purpose of protein expression, dendritic cells with fluorescent particles. The cytotoxic activity of RPL8 DC vaccine against 4T1 breast cancer cells in vitro was significantly lower than that of DCs and PBS group without RPL8 protein (P <0.05). DC vaccine injected tumor-bearing mice in vivo, mouse survival, tumor volume changes were significantly different from the control group (P <0.05). Conclusion DC vaccine loaded with RPL8 protein has immunotherapy effect on mouse breast cancer and provides a theoretical basis for immunotherapy of breast cancer.