DC-SIGN真核表达载体的构建及其稳定转染BHK21细胞系的建立

目的 构建DC-SIGN分子真核表达载体,获得可稳定表达DC-SIGN分子的BHK21细胞系.方法 以pUNO-hDC-SIGN1Aa质粒为模板,通过PCR方法扩增人DC-SIGN基因,经过Not Ⅰ和BamH Ⅰ双酶切之后,将其克隆入真核表达载体pIRES-neo,构建真核表达载体pIRES-neo-DC-SIGN,以Not Ⅰ和BamH Ⅰ双酶切以及测序验证重组质粒的正确性.将重组质粒以Lipo-fectamine~(KM) 2000转染到BHK21细胞,通过G418及有限稀释法进行阳性克隆的筛选,建立稳定表达DC-SIGN分子的BHK21细胞系,利用流式细胞仪、Western blotting及免疫荧光法检测DC-SIGN分子的表达.结果 双酶切鉴定证明DC-SIGN基因已经成功克隆入真核表达载体pIRES-neo中,测序结果表明EC-SIGN基因与原序列一致.pIRES-neo-DC-SIGN转染BHK21细胞后,获得了急定表达DC-SIGN分子的细胞系.流式细胞仪检测结果显示,阳性克隆中DC-SIGN分子的表达率为85.42%.免疫荧光结果显示,DC-SIGN分子主要表达于细胞膜表面.Western blotting能检测到DC-SIGN蛋白表达的特异条带.结论 成功构建了稳定、高效表达DC-SIGN分子的BHK21细胞系,为后续DC靶向性疫苗研究奠定了基础.

Construction of DC-SIGN eukaryotic expression vector and establishment of BHK21 cell line stably transfected by the recombinant vector

Objective To construct a eukaryotic expressing vector harboring human DC-SIGN, and establish a BHK21 cell line stably and highly expressing DC-SIGN. Methods The DC-SIGN gene fragment which contained Not I and BamH I sites was amplified by PCR from pUNO-hDCSIGN1Aa plasmid, digested with Not I and BamH I, and then cloned into an eukaryotic expression vector pIRES-neo to construct eukaryotic expression vector pIRES-neo-DC-SIGN. The recombined plasmid was identified with Not I and BamH I enzyme digestion and se...