Huang Shuo, Shao Shuyang, Fan Liding, Wang Junying, Meng Furen, Guan Siyuan, Wang Changhao, Liu Ang, Wang Yuanhui, Xue Qingjie
School of Basic Medical, Jining Medical University, Jining, 272067, Shandong, China.
Department of Biological Sciences, Xi'an Jiaotong-Liverpool University (XJTLU), Suzhou, 215123, Jiangsu, China.
Appl Microbiol Biotechnol. 2025 Jun 3;109(1):134. doi: 10.1007/s00253-025-13528-9.
To obtain recombinant Bacillus Calmette-Guérin (rBCG) containing an Epstein-Barr virus (EBV) fusion gene that can inhibit EBV-positive cancer, we obtained BZLF1 and EBNA1 cDNA and overlapped these sequences to assemble the fusion gene BFNA. Then, BFNA was inserted into pMV261, and pMV-BFNA was transfected into BCG-competent cells. Western blotting was performed to detect the target fusion protein, specific antibodies were detected in the serum via enzyme-linked immunosorbent assays (ELISAs), and spleen cell-specific cytokines were detected via enzyme-linked immunospot (ELISPOT) assays. Cytotoxic T lymphocyte (CTL) activity, tumor weight, tumor formation time, and mouse survival were determined in EBV-positive tumor cell (NPRC18) cancer models, and flow cytometry was performed to analyze the quantities of CD8 and CD4 T cells in C57BL/6 J mice. Single-factor analysis of variance was performed with SPSS 19.0 to evaluate rBCG inhibition. The molecular weight of the fusion protein was approximately 55.5 kDa. The titer of the antibody in the rBCG group was highly significant (P ≤ 0.01), which delayed tumorigenesis, and the specific killing ability of rBCG targeting the recombinant target protein was increased. Compared with BCG-EBNA1 and BCG-BZLF1, the rBCG with the BFNA fusion gene presented a greater effect on tumors. Flow cytometric analysis revealed that the numbers of CD4 T and CD8 T cells in the blood of the rBCG group were significantly greater than those in the blood of the control group (P < 0.01). The mice injected with rBCG had greater lymphocyte infiltration in the tumor area. Thus, rBCG exerts a notable immune effect in mice and an inhibitory effect on EBV-positive tumor cell cancer models. KEY POINTS: • rBCG with the BFNA fusion gene induced strong immune responses and delayed EBV tumor growth. • rBCG promoted CD4+ and CD8+ T cell infiltration in EBV-positive tumor models. • The BFNA fusion protein effectively increased CTL activity and prevented tumor progression.
为了获得含有能抑制EBV阳性癌症的爱泼斯坦-巴尔病毒（EBV）融合基因的重组卡介苗（rBCG），我们获取了BZLF1和EBNA1 cDNA，并将这些序列重叠以组装融合基因BFNA。然后，将BFNA插入pMV261中，并将pMV-BFNA转染到卡介苗感受态细胞中。通过蛋白质免疫印迹法检测目标融合蛋白，通过酶联免疫吸附测定（ELISA）检测血清中的特异性抗体，并通过酶联免疫斑点（ELISPOT）测定检测脾细胞特异性细胞因子。在EBV阳性肿瘤细胞（NPRC18）癌症模型中测定细胞毒性T淋巴细胞（CTL）活性、肿瘤重量、肿瘤形成时间和小鼠存活率，并通过流式细胞术分析C57BL / 6 J小鼠中CD8和CD4 T细胞的数量。使用SPSS 19.0进行单因素方差分析以评估rBCG的抑制作用。融合蛋白的分子量约为55.5 kDa。rBCG组中抗体的效价非常显著（P≤0.01），这延迟了肿瘤发生，并且rBCG靶向重组靶蛋白的特异性杀伤能力增强。与BCG-EBNA1和BCG-BZLF1相比，具有BFNA融合基因的rBCG对肿瘤的作用更大。流式细胞术分析显示，rBCG组血液中CD4 T细胞和CD8 T细胞的数量显著高于对照组血液中的数量（P <0.01）。注射rBCG的小鼠在肿瘤区域有更大的淋巴细胞浸润。因此，rBCG在小鼠中发挥显著的免疫作用，并对EBV阳性肿瘤细胞癌症模型具有抑制作用。要点：• 具有BFNA融合基因的rBCG诱导强烈的免疫反应并延迟EBV肿瘤生长。• rBCG促进EBV阳性肿瘤模型中CD4 +和CD8 + T细胞浸润。• BFNA融合蛋白有效提高CTL活性并阻止肿瘤进展。
