Tian Geng, Yi Ji-Lin, Xiong Ping
Departmentof General Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China.
World J Gastroenterol. 2004 Jan 15;10(2):200-4. doi: 10.3748/wjg.v10.i2.200.
To develop a tumor DNA vaccine encoding a fusion protein of murine AFP and CTLA4, and to study its ability to induce specific CTL response and its protective effect against AFP-producing tumor.
Murine alpha-fetoprotein (mAFP) gene was cloned from total RNA of Hepa1-6 cells by RT-PCR. A DNA vaccine was constructed by fusion murine alpha-fetoprotein gene and extramembrane domain of murine CTLA4 gene. The DNA vaccine was identified by restriction enzyme analysis, sequencing and expression. EL-4 (mAFP) was developed by stable transfection of EL-4 cells with pmAFP. The frequency of cells producing IFN-gamma in splenocytes harvested from the immunized mice was measured by ELISPOT. Mice immunized with DNA vaccine were inoculated with EL-4 (mAFP) cells in back to observe the protective effect of immunization on tumor. On the other hand, blood samples were collected from the immunized mice to check the functions of liver and kidney.
1.8 kb mAFP cDNA was cloned from total RNA of Hepa1-6 cells by RT-PCR. The DNA vaccine encoding a fusion protein of mAFP-CTLA4 was constructed and confirmed by restriction enzyme analysis, sequencing and expression. The expression of mAFP mRNA in EL-4 (mAFP) was confirmed by RT-PCR. The ELISPOT results showed that the number of IFN-gamma-producing cells in pmAFP-CTLA4 group was significantly higher than that in pmAFP, pcDNA3.1 and PBS group. The tumor volume in pmAFP-CTLA4 group was significantly smaller than that in pmAFP, pcDNA3.1 and PBS group, respectively. The hepatic and kidney functions in each group were not altered.
AFP-CTLA4 DNA vaccine can stimulate potent specific CTL responses and has distinctive antitumor effect on AFP-producing tumor. The vaccine has no impact on the function of mouse liver and kidney.
构建一种编码小鼠甲胎蛋白（AFP）与细胞毒性T淋巴细胞相关抗原4（CTLA4）融合蛋白的肿瘤DNA疫苗，并研究其诱导特异性细胞毒性T淋巴细胞（CTL）反应的能力及其对产生AFP肿瘤的保护作用。
通过逆转录聚合酶链反应（RT-PCR）从Hepa1-6细胞的总RNA中克隆小鼠甲胎蛋白（mAFP）基因。将小鼠甲胎蛋白基因与小鼠CTLA4基因的胞外结构域融合构建DNA疫苗。通过限制性内切酶分析、测序和表达对DNA疫苗进行鉴定。用pmAFP稳定转染EL-4细胞构建EL-4（mAFP）细胞。用酶联免疫斑点法（ELISPOT）检测免疫小鼠脾细胞中产生γ干扰素的细胞频率。用DNA疫苗免疫的小鼠背部接种EL-4（mAFP）细胞，观察免疫对肿瘤的保护作用。另一方面，采集免疫小鼠的血样检测肝肾功能。
通过RT-PCR从Hepa1-6细胞的总RNA中克隆出1.8 kb的mAFP cDNA。构建了编码mAFP-CTLA4融合蛋白的DNA疫苗，并通过限制性内切酶分析、测序和表达得到证实。通过RT-PCR证实EL-4（mAFP）中mAFP mRNA的表达。ELISPOT结果显示，pmAFP-CTLA4组中产生γ干扰素的细胞数量显著高于pmAFP组、pcDNA3.1组和磷酸盐缓冲液（PBS）组。pmAFP-CTLA4组的肿瘤体积分别显著小于pmAFP组、pcDNA3.1组和PBS组。各组的肝肾功能均未改变。
AFP-CTLA4 DNA疫苗可刺激产生强效特异性CTL反应，对产生AFP的肿瘤具有显著的抗肿瘤作用。该疫苗对小鼠肝肾功能无影响。
