Department of Pathogenic Biology, Jining Medical University, Shandong, 272067, China.
Department of Infectious Disease, Yantai Yuhuangding Hospital, Shandong, 264000, China.
Appl Microbiol Biotechnol. 2022 Jan;106(1):185-195. doi: 10.1007/s00253-021-11682-4. Epub 2021 Dec 2.
At present, studies have found that latent Epstein-Barr virus (EBV) infection is associated with a variety of human tumours, and a vaccine is not available in this field. In this research, RT-PCR was used to obtain BZLF1 (immediately expressed early antigen Z) and LMP2 (latent membrane protein 2) cDNA from EBV. A ZLP2 fusion gene containing a linker sequence that encoded the polypeptide (GlySer) was obtained using the sequence splicing overlap extension method. Then, ZLP2 was inserted into pMV261 cells, and the recombinant plasmid pMV-ZLP2 was transformed into BCG competent cells. After EB virus-positive tumour cell (NPRC18) cancer models were established with C57BL/6 J mice, tumour weight, tumour formation time and mouse survival conditions were analyzed, and flow cytometry was used to analyze the quantities of CD8 + and CD4 + T cells. HE staining was used to detect and analyze lymphocyte infiltration, and statistical analysis was used to analyze the immunological effect of recombinant BCG (rBCG). Compared with the control group, rBCG could significantly prolong the survival time of mice, slow tumour growth and delay tumour formation time. Recombinant BCG exhibits an obvious immune effect in mice and an inhibitory effect on EBV-positive cancer.Key points• AZLP2 fusion gene with BZLF1 and LMP2 of EB virus was constructed.• ZLP2 fusion gene was expressed with rBCG.• rBCG with ZLP2 has an obvious effect on EBV-positive cancer.
目前研究发现潜伏性 EBV(Epstein-Barr virus)感染与多种人类肿瘤有关,而该领域尚无疫苗可用。本研究采用 RT-PCR 从 EBV 中获得 BZLF1(立即早期抗原 Z)和 LMP2(潜伏膜蛋白 2)cDNA,应用序列拼接重叠延伸法获得含连接肽序列编码多肽(GlySer)的 ZLP2 融合基因,将 ZLP2 插入 pMV261 细胞中,将重组质粒 pMV-ZLP2 转化至 BCG 感受态细胞,构建携带 ZLP2 融合基因的 rBCG,建立 C57BL/6J 小鼠 EBV 阳性肿瘤细胞(NPRC18)肿瘤模型,分析肿瘤重量、肿瘤形成时间和小鼠生存状况,流式细胞术分析 CD8+、CD4+T 细胞数量,HE 染色检测分析淋巴细胞浸润,统计分析重组 BCG(rBCG)的免疫效果。rBCG 组较对照组小鼠生存时间明显延长,肿瘤生长缓慢,肿瘤形成时间延迟,rBCG 在小鼠体内表现出明显的免疫效果,对 EBV 阳性肿瘤有抑制作用。
• 构建含有 EBV 的 BZLF1 和 LMP2 的 AZLP2 融合基因。
• 用 rBCG 表达 ZLP2 融合基因。
• 带有 ZLP2 的 rBCG 对 EBV 阳性癌症有明显作用。