Tian Geng, Yi Ji-Lin, Xiong Ping
Department of Oncology, Shenzhen Second Hospital, Shenzhen 518035, China.
Hepatobiliary Pancreat Dis Int. 2004 Aug;3(3):440-3.
The inoculation of plasmid DNA encoding tumor-associated antigens is a novel and powerful strategy for antitumor vaccination. This study was designed to construct the DNA vaccine of mouse AFP and to observe the specific cellular immunologic responses and the antitumor responses in mice induced by this vaccine.
The murine AFP gene was amplified by RT-PCR from total RNA extracted from Hepa1-6 cells and cloned into the vector pcDNA3.1 to construct pmAFP. 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 mice immunized with the DNA vaccine by intramuscular injection was measured by enzyme linked immunospot (ELISPOT). The mice immunized with the DNA vaccine were inoculated with EL-4 (mAFP) cells in back to observe the inhibitory effect of the immunization on tumor. On the other hand, blood samples were collected from the immunized mice to check the functions of the liver and kidney.
The murine AFP gene was successfully cloned by RT-PCR. Results from restriction enzyme analysis, sequencing and expression showed that the DNA vaccine was successfully constructed. The expression of mAFP mRNA in EL-4 (mAFP) was confirmed by RT-PCR. The results of ELISPOT showed that the number of IFN-gamma- producing cells of the pmAFP vaccine group was significantly higher than that of other groups (P<0.01). The tumor volume in the pmAFP vaccine group (1042.42+/-123.71 mm(3)) was significantly smaller than that in other groups (P<0.01). The function of mouse liver and kidney in each group was unchanged.
The successfully constructed DNA vaccine of AFP can induce specific cellular immunologic responses and significant antitumor responses in mouse and has no impact on the function of mouse liver and kidney.
接种编码肿瘤相关抗原的质粒DNA是一种新型且强大的抗肿瘤疫苗策略。本研究旨在构建小鼠甲胎蛋白（AFP）DNA疫苗，并观察该疫苗诱导的小鼠特异性细胞免疫反应和抗肿瘤反应。
通过RT-PCR从Hepa1-6细胞提取的总RNA中扩增小鼠AFP基因，并克隆到载体pcDNA3.1中构建pmAFP。通过限制性内切酶分析、测序和表达对DNA疫苗进行鉴定。通过用pmAFP稳定转染EL-4细胞构建EL-4（mAFP）。通过酶联免疫斑点法（ELISPOT）测量经肌肉注射DNA疫苗免疫的小鼠脾细胞中产生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，观察免疫对肿瘤的抑制作用。另一方面，从免疫小鼠采集血样检查肝肾功能。
通过RT-PCR成功克隆了小鼠AFP基因。限制性内切酶分析、测序和表达结果表明成功构建了DNA疫苗。通过RT-PCR证实了EL-4（mAFP）中mAFP mRNA的表达。ELISPOT结果显示，pmAFP疫苗组产生γ干扰素的细胞数量显著高于其他组（P<0.01）。pmAFP疫苗组的肿瘤体积（1042.42±123.71mm³）显著小于其他组（P<0.01）。各组小鼠的肝肾功能未改变。
成功构建的AFP DNA疫苗可诱导小鼠产生特异性细胞免疫反应和显著的抗肿瘤反应，且对小鼠肝肾功能无影响。
