Xue Qing-Jie, Li Yun-Qing, Yang Chun-Qing, Chen Ting, Li Xiu-Zhen, Cheng Baohua, Wang Chun-Mei
Department of Pathogenic Biology, Jining Medical University, Shandong 272067, China; Neurobiology Institute, Jining Medical University, Shandong 272067, China.
Department of Pathogenic Biology, Jining Medical University, Shandong 272067, China.
Vaccine. 2017 Mar 14;35(12):1599-1607. doi: 10.1016/j.vaccine.2017.02.024. Epub 2017 Feb 20.
The random primer Oligo(dT) was used in RT-PCR to obtain cDNA from the human granulocyte macrophage colony stimulating factor (hGM-CSF) gene and the Epstein-Barr virus (EBV) gene BZLF1. Then, the sequence splicing overlap extension method was used to obtain a GCBF fusion gene containing a linker sequence that encoded the polypeptide (GlySer). The GCBF fusion gene was inserted into pMV261, which was then transformed into competent E. coli DH5 alpha cells, and positive cells were selected based on kanamycin resistance on LB plates. The recombinant plasmid pMVBZLF1 was extracted from E. coli, and BCG (Bacillus Calmette-Guérin) was transformed into competent cells. According to the RT-PCR results, the target genes hGM-CSF and BZLF1 were 461bp and 788bp in size, which was in agreement with the expected values. Construction of the recombinant plasmid by double enzyme digestion, amplification, sequencing and Western blotting confirmed that the GCBF fusion gene (1204bp) was correctly inserted into pMV261, successfully transformed into BCG competent cells, and properly expressed. After mice were injected with rBCG (recombinant BCG), antibody levels were detected using ELISA, and spleen cells were obtained and the killing rates of specific CTLs by rBCG were detected using a CTL assay kit. Then, the influence of rBCG on tumour cells was analysed in C57BL/6 mice. We found that rBCG-secreting cytokines hybridized with hGM-CSF and BZLF1 antibodies and that the rBCG vaccine stimulated antibody production in C57BL/6 mice. The specific cytotoxic effects of the spleen cells from the rBCG group on EB virus-positive tumour cells was significantly different from the cytotoxic effects of the control group cells (P<0.01). CD8 T and CD4 T lymphocytes were detected in the tumour tissues of the rBCG group mice by flow cytometry, indicating that CD8 T and CD4 T lymphocytes infiltrated into the tumour tissue in the mice. Morphological observations of the tumour sections from the rBCG-immunized mice showed the infiltration of lymphocytes into the tumour tissues. The average rBCG tumour volume was less than the average tumour volume of the control group. Thus, rBCG may inhibit the growth of EB virus-positive tumour cells in mice.
在逆转录聚合酶链反应（RT-PCR）中使用随机引物寡聚脱氧胸苷酸（Oligo(dT)）从人粒细胞巨噬细胞集落刺激因子（hGM-CSF）基因和爱泼斯坦-巴尔病毒（EBV）基因BZLF1中获取互补DNA（cDNA）。然后，采用序列拼接重叠延伸法获得包含编码多肽（甘氨酸-丝氨酸）的接头序列的GCBF融合基因。将GCBF融合基因插入pMV261，随后将其转化到感受态大肠杆菌DH5α细胞中，并基于LB平板上的卡那霉素抗性筛选阳性细胞。从大肠杆菌中提取重组质粒pMVBZLF1，并将卡介苗（Bacillus Calmette-Guérin，BCG）转化到感受态细胞中。根据RT-PCR结果，目标基因hGM-CSF和BZLF1的大小分别为461bp和788bp，与预期值相符。通过双酶切、扩增、测序和蛋白质免疫印迹法构建重组质粒，证实GCBF融合基因（1204bp）正确插入pMV261，成功转化到卡介苗感受态细胞中并正确表达。给小鼠注射重组卡介苗（rBCG）后，使用酶联免疫吸附测定（ELISA）检测抗体水平，并获取脾细胞，使用CTL检测试剂盒检测rBCG对特异性细胞毒性T淋巴细胞（CTL）的杀伤率。然后，在C57BL/6小鼠中分析rBCG对肿瘤细胞的影响。我们发现分泌细胞因子的rBCG与hGM-CSF和BZLF1抗体杂交，并且rBCG疫苗刺激C57BL/6小鼠产生抗体。rBCG组脾细胞对EB病毒阳性肿瘤细胞的特异性细胞毒性作用与对照组细胞的细胞毒性作用有显著差异（P<0.01）。通过流式细胞术检测rBCG组小鼠肿瘤组织中的CD8 T淋巴细胞和CD4 T淋巴细胞，表明CD8 T淋巴细胞和CD4 T淋巴细胞浸润到小鼠的肿瘤组织中。对rBCG免疫小鼠的肿瘤切片进行形态学观察，显示淋巴细胞浸润到肿瘤组织中。rBCG组的平均肿瘤体积小于对照组的平均肿瘤体积。因此，rBCG可能抑制小鼠中EB病毒阳性肿瘤细胞的生长。
