论文部分内容阅读
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。
Objective To construct a DNA vaccine capable of expressing AFP and CTLA4 fusion proteins and to detect its ability to induce specific CTL responses and anti-AFP tumors. Methods The mAFP gene was cloned from total RNA of Hepa1-6 cells by RT-PCR. Constructs plasmid DNA vaccine that links the mAFP gene and the gene encoding the extracellular portion of mouse CTLA4. The vaccine digestion, sequencing and expression identification. EL-4 (mAFP) cell line was established by stably transfecting EL-4 cells with pmAFP. Mice were immunized with this DNA vaccine and the frequency of IFN-γ-producing cells in spleen cells of mice immunized with ELISPOT was measured. Immunized mice were challenged with EL-4 (mAFP) tumor cells to observe tumor growth. In addition, the immunized mice blood for liver and kidney function tests. Results 1.8 kb mAFP gene was cloned successfully from total RNA of Hepa1-6 cells by RT-PCR. Digestion, sequencing and expression identification confirmed that the DNA vaccine encoding the mAFP-CTLA4 fusion protein was successfully constructed. RT-PCR confirmed the expression of mAFP mRNA in EL-4 (mAFP) cells. The ELISPOT results showed that the frequency of IFN-γ production in pmAFP-CTLA4 group was significantly higher than that in pmAFP group, pcDNA3.1 group and PBS group. The tumor volume of pmAFP-CTLA4 immunized mice was (385.93 ± 52.9) mm3, which was significantly lower than that of pmAFP group (1042.42 ± 123.71) mm3 and pcD-NA3.1 group (2292.38 ± 276). 94) mm3 and PBS group (2303.5 ± 233.13) mm3, P <0.01. There was no significant difference in liver and kidney function between groups. Conclusion The DNA vaccine encoding mAFP-CTLA4 fusion protein can induce AFP-specific CTL proliferation and induce significant antitumor immunity in mice. The vaccine does not affect the liver and kidney function of mice.