【摘要】 目的构建能够表达AFP和CTLA4融合蛋白的DNA疫苗并检测其诱导特异性CTL反应及抗产生AFP肿瘤的能力。方法利用RT-PCR从Hepa1-6细胞总RNA中克隆mAFP基因。连接mAFP基因和编码小鼠CTLA4膜外部分的基因构建质粒DNA疫苗。对此疫苗进行酶切、测序和表达鉴定。用pmAFP稳定转染EL-4细胞建立EL-4(mAFP)细胞系。以此DNA疫苗免疫小鼠,用ELISPOT检测免疫后小鼠脾脏细胞中产生IFN-γ的细胞频数。以EL-4(mAFP)肿瘤细胞攻击免疫后小鼠,观察肿瘤的生长情况。另外,对免疫的小鼠采血进行肝、肾功能检测。结果利用RT-PCR从Hepa1-6细胞总RNA中成功克隆出1．8 kb的mAFP基因。酶切、测序和表达鉴定证实编码mAFP-CTLA4融合蛋白的DNA疫苗构建成功。RT-PCR证实EL-4(mAFP)细胞中有mAFP mRNA的表达。ELISPOT检测结果显示:pmAFP-CTLA4免疫组产生IFN-γ的细胞频数显著高于pmAFP组、pcDNA3．1组和PBS组。pmAFP- CTLA4免疫组小鼠的肿瘤体积为(385．93±52．9)mm3,明显小于pmAFP组(1042．42±123．71)mm3、pcD- NA3．1组(2292．38±276．94)mm3和PBS组(2303．5±233．13)mm3,P均<0．01。各组肝、肾功能差异无统计学意义。结论编码mAFP-CTLA4融合蛋白的DNA疫苗能诱导产生AFP特异性的CTL增殖并诱导小鼠产生明显的抗肿瘤免疫力,此疫苗对小鼠肝、肾功能不产生影响。更多还原

【Abstract】 Objective To develop a antitumor DNA vaccine encoding a fusion protein of murine AFP and CTLA4, and to study its efficacy to induce specific CTL response. Methods Murineα-fetoprotein( mAFP) gene was cloned from total RNA of Hepa1-6 cells by RT-PCR. A DNA vaccine was constructed by fusion murineα-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-γin splenocytes harvested from the immunized mice were measured by ELISPOT. Mice immunized with DNA vaccine by intramuscular injection were inoculated with EL-4 (mAFP) cells to observe the protective effect of the immunization on tumor. On the other hand, blood samples were collected from the immunized mice to check the functions of liver and kidney. Results 1.8 kb mAFP cDNA was cloned from total RNA of Hepal-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 results of ELISPOT show that the number of IFN-γ-producing cells of pmAFP-CTLA4 group was significantly higher than that of 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. The function of murine liver and kidney in each group did not change. Concluaon AFP-CTLA4 DNA vaccine can stimulate potent specific antitumor effect on AFP-producing tumor. The vaccine had no impact on the function of murine liver and kidney.更多还原