摘要
目的构建含OVA-Fc融合基因的真核表达质粒,证明其能在真核细胞CHO细胞表达。方法通过PCR从OVA-pcDNA3.1质粒中扩增出全长的小鼠OVAcDNA,酶切后克隆到含有小鼠IgG2aFccDNA的真核载体pMIgV,然后将OVA-Fc酶切后亚克隆入pcDNA3.1,构建真核表达载体OVA-Fc-pcDNA3.1;脂质体转染法将其导入CHO细胞,流式细胞仪、Westernblot、ELISA法检测融合蛋白OVA-Fc的表达。结果酶切鉴定和序列测定证明真核表达载体OVA-Fc-pcDNA3.1构建正确;流式细胞术、Westernblot、ELISA法均检测到转染的CHO细胞能表达OVA-Fc融合蛋白。结论OVA-Fc-pcDNA3.1真核表达质粒构建正确并能在真核细胞中表达,该质粒的成功构建与表达为进一步将其作为DNA疫苗治疗肿瘤、哮喘等疾病奠定了基础。
Objective To construct the co-expression plasmid of ovalbumin (OVA) and Fc, and to detect its expression in CHO cells. Methods Complete murine OVA cDNA, which was amplified from OVA-pcDNA3.1 plasmid by polymerase chain reaction (PCR), was spliced and then cloned into pMIgV containing murine IgG2a Fc cDNA. OVA-Fc-pcDNA3.1 plasmid was finally constructed after sub-cloning spliced OVA-Fc into pcDNA3.1 plasmid. OVA-Fc-pcDNA3.1 plasmids were then transfected into CHO cells with lipofectamine. The expression of OVA-Fc was determined by flow cytometry, Western blotting, and enzyme-linked immunosorbent assay (ELISA). Results DNA sequencing and restriction endonuclease digestion analysis indicated that the eukaryotic expression vector OVA-Fc-pcDNA3.1 had been con- structed successfully. OVA-Fc expression could be detected in CHO cells by Western blotting, ELISA, and flow cytometry. Conclusion Success of this plasmid construction and expression lays the foundation for future design of DNA vaccine targeting dendritic cells (DC) in the treatment of diseases, such as tumor and allergic asthma.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2005年第3期163-165,169,共4页
Immunological Journal
基金
国家自然科学基(30200120
30300150)
国家"973"计划(2001CB510001)资助项目
