小鼠AFP-CTLA4融合蛋白真核表达载体的构建及鉴定

Construction and identification of the eukaryotic expression vector of murine AFP-CTLA4 fusion protein

目的:克隆小鼠甲胎蛋白(mAFP)基因并构建小鼠甲胎蛋白-细胞毒性T淋巴细胞抗原4(mAFP-CTLA4)融合蛋白真核表达载体.方法:从Hepal-6细胞中提取总RNA进行RT-PCR,扩增出mAFP基因,亚克隆于pcDNA3.1载体.PCR法从质粒pmCTLA4-Ig中克隆出mCTLA4膜外部分基因并通过重叠PCR法添加接头,重组连接于pmAFP质粒中mAFP基因后,转化大肠杆菌DH5α,筛选阳性克隆酶切、测序鉴定.用质粒瞬时转染CHO细胞,Westernblot检测融合蛋白的表达.结果:利用RT-PCR从Hepal-6细胞总RNA中成功克隆出1.8kb的mAFP基因;重组阳性克隆经酶切鉴定证实连有接头的CTLA4膜外部分基因已正确插入pmAFP质粒中,测序结果证实各片段连接方向及阅读框正确.用质粒瞬时转染CHO细胞,Westernblot检测到预计大小分子量蛋白的表达结论:mAFP-CTLA4融合蛋白真核表达载体的构建成功,为进一步研究其在肝癌免疫治疗中的作用奠定了基础.

AIM: To clone the murine α-fetoprotein gene and to construct the eukaryotic expression vector of AFP-CTLA4 fusion protein. METHODS: Total RNAs were extracted from Nepa1-6 cells, then the murine α-fetoprotein gene was amplified by RT-PCR and cloned into the eukaryotic expression vector pcDNA3.1. The extramembrane domain of mouse CTLA4 gene was amplified from plasmid pmCTLA4-Ig, followed by the addition of a linker using overlap PCR. The PCR product was subcloned into pmAFP and fused in frame with the AFP. The recombinant of vector was transformed into Ecoli. DH5α, the positive clones were selected and the plasmid DNA was identified by restriction enzyme analysis and sequencing. After transient transfection of CHO-K1 cells with the recombinant of vector, Western blotting was used to detect the expression of fusion protein. RESULTS: The 1.8 kb murine α-fetoprotein gene was successfully cloned from the total RNA of Hepa1-6 cells. The result obtained from the restriction enzyme analysis showed that the extramembrane domain of mouse CTLA4 gene was successfully inserted into pmAFP. Result of sequencing assertained that the orientation of the ligations and the reading frame were correct, and Western blotting indicated that the recombinant of vector could express murine AFP-CTLA4 fusion protein in CHO-K1 cells. CONCLUSION: We successfully construct eukaryotic expression vector of AFP-CTLA4 fusion protein, which forms an important basis for the research of immunotherapy of hepatocellular carcinoma with pmAFP-CTLA4.