甲胎蛋白DNA疫苗的构建及诱导小鼠抗肿瘤免疫的实验研究  

Construction of DNA vaccine encoding murine AFP and its specific anti-tumor effect on C57BL/6 mice

作　　者：田耕[1] 易继林[1] 熊平[2] 

机构地区：[1]华中科技大学同济医学院附属同济医院普通外科,武汉430030 [2]华中科技大学同济医学院免疫学教研室,武汉430030

出　　处：《第三军医大学学报》2006年第15期1591-1594,共4页Journal of Third Military Medical University

摘　　要：目的克隆小鼠甲胎蛋白(murineα-fetoprote in,mAFP)基因,构建mAFP DNA疫苗并检测其诱导mAFP特异性CTL反应及抗肿瘤免疫的能力。方法利用RT-PCR从Hepa 1-6细胞总RNA中克隆出mAFP基因,亚克隆于pcD-NA3.1中构建DNA疫苗pmAFP,酶切、测序和表达鉴定;pmAFP稳定转染EL-4细胞建立EL-4(mAFP)细胞系;pmAFP免疫小鼠,接种EL-4(mAFP)细胞,观察肿瘤生长情况;E lispot法检测免疫后小鼠脾脏细胞中产生IFN-γ的细胞频数;另外,对免疫的小鼠采血进行肝、肾功能检测。结果RT-PCR成功克隆出小鼠AFP基因;酶切、测序和表达鉴定证实DNA疫苗pmAFP构建成功;RT-PCR证实EL-4(mAFP)细胞中有mAFP mRNA的表达;pmAFP免疫组小鼠的肿瘤体积(1 042.42±123.71)mm3明显小于pcDNA3.1组(P<0.01)和PBS组(P<0.01);pmAFP免疫小鼠的脾脏细胞产生IFN-γ的细胞频数显著高于pcDNA3.1组(P<0.01)和PBS组(P<0.01)。各组肝、肾功能无显著差异。结论mAFP DNA疫苗构建成功,此疫苗能诱导AFP特异性的CTL增殖并诱导小鼠产生明显的抗肿瘤免疫力,对小鼠肝、肾功能不产生影响。Objective To clone the murine α-fetoprotein gene, construct the DNA vaccine of mouse AFP and to observe the specific cellular immunologic responses and the anti-tumor responses in mice induced by this vaccine. Methods The murine AFP gene was amplified by RT-PCR from total RNA extracted from Hepal-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. Mice immunized with DNA vaccine by intramuscular injection were inoculated with EL-4（mAFP） cells at back to observe the inhibitory effect of the immunization on tumor. The frequency of cells producing IFN-T in splenocytes harvested from the immunized mice was measured by ELISPOT. Blood samples were col- lected from the immunized mice to check the functions of liver and kidney. Results The murine AFP gene was successfully cloned by RT-PCR. DNA vaccine was successfully constructed. The expression of mAFP mRNA in EL-4（mAFP） was confirmed by RT-PCR. The tumor volume in pmAFP vaccine group （1 042, 42 ± 123.71 mm3） was significantly smaller than that of other groups （P〈0. 01 ）. The number of IFN-T-producing cells of pmAFP vaccine group was significantly higher than that of other groups （P〈0. 01 ）. The function of mouse liver and kidney in each group was unchanged. Conclusion The DNA vaccine of mAFP was successfully constructed. The vaccine can induce specific cellular immunologic responses and significant anti-tumor responses in mouse and had no impact on the function of mouse liver and kindey.

关 键 词：疫苗 脱氧核糖核酸 甲胎蛋白 小鼠 免疫治疗 

分 类 号：R394-33[医药卫生—医学遗传学] R394.4[医药卫生—基础医学]