Objective To construct the eukaryotic expression plasmid of mice cytotoxic T lymphocyte-associated antigen4 ( CTLA4) Fc fusion gene, and detect its expression in the cells of 293T, and lay the foundation for the subsequent study of the antitumor mechanism of CTLA4-Fc in mice.
Methods Gene-specific primers which were used as bridge and template for each other were designed using Gene Runner software. The CTLA4 fusion gene fragment was amplified by overlap PCR with primers; after digesting by restriction endonuclease, the CTLA4 fusion gene fragment was inserted into the eukaryotic expression vector of pcDNA3.1(+ )/Fc (Mouse IgG2a) to construct the expression plasmid of CTLA4-Fc; the clones were confirmed by colony-PCR, restriction enzyme digestion and sequencing. The protein level of the fusion gene was identified by ELISA in 293T cells.
Results The eukaryotic expression vector encoding fusion gene of CTLA4-Fc has been constructed successfully. A 1 200 bp band for the CTLA4-Fc recombinant gene and a 568 bp band for CTLA4 were observed and confirmed by colony PCR and restriction enzyme digestion on agarose gels. The positive recombinant colonies were cultured in liquid LB medium and sent the bacterial solution to the company for sequencing, and the DNA sequence was 100% homologous with in CTLA4-Fc of mouse in GenBank. The expressionof plasmid CTLA4-Fc was examined by ELISA in 293T cells.
Conclusion The eukaryotic expression plasmid of CTLA4-Fc was correctly cloned in pcDNA3.1(+ )/Fc (Mouse IgG2a) plasmid and expressed in 293T cells.In this study, the CTLA4 gene sequence was amplified by overlapping PCR technology, which can quickly obtain the target gene amplification products and improve the success rate of the experiment.
摘要： 目的 构建小鼠细胞毒性T淋巴细胞相关抗原4Fc融合基因 (CTLA4-Fc)的真核表达质粒，并检测其在293T 细胞中的表达，为后续在小鼠体内研究CTLA4-Fc抗肿瘤作用机制奠定基础。 方法 Gene Runner软件设计并合成互 为搭桥、互为模板的CTLA4融合基因引物，利用重叠PCR方法扩增获得CTLA4基因片段，与pcDNA3.1⑴/Fc (Mouse IgG2a)质粒同时进行双酶切，连接合成CTLA4-Fc融合基因表达质粒；菌落PCR扩增、双酶切以及测序鉴定后，在293T 细胞中瞬时转染表达融合基因的质粒，ELISA法检测其在293T细胞内的蛋白表达含量。 结果 成功构建CTLA4-Fc融 合基因。经菌落PCR和双酶切，琼脂糖电泳检测到CTLA4-Fc片段长度为1 200 bp，CTLA4片段长度为568 bp DNA。 将阳性重组克隆菌落挑于液体LB培养基中扩大培养并将菌液送公司测序，测序结果证实DNA序列与GenBank同源性 为100%。通过ELISA方法能够检测到CTLA4-Fc质粒在293T细胞中的表达。 结论 CTLA4-Fc融合基因正确克隆人 pcDNA3.1(+)/Fc (Mouse IgG2a))质粒中，该质粒能够在293T细胞中表达。本研究利用重叠PCR技术扩增获得CTLA4基 因序列，能够快速获得目的基因扩增产物，提高了实验的成功率。