摘要
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NotI和BamHI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NotI和BamHI双酶切以及测序验证重组质粒的正确性。将重组质粒以Lipo-fectamineTM2000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DC-SIGN基因与原序列一致。pIRES-neo-DC-SIGN转染BHK21细胞后,获得了稳定表达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。
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 sequencing, the latter was then transfected to BHK21 cells by LipofectamineTM 2000. After screening culture by G418, BHK21 cell line stably expressing DC-SIGN was established. The expression of DC-SIGN was identified by flow cytometry, Western blotting and immunofluorescence method. Results The gene sequence of DC-SIGN was consistent with that of design. PCR and double enzyme digestion analysis showed that the recombinant plasmid pIRES-neo-DC-SIGN was constructed successfully. After transfection, positive clones were selected with G418. After limiting dilution assay, BKH21 cell lines stably expressing DC-SIGN were established. The detection result of flow cytometry showed that the expression ratio of DC-SIGN positive clones was close to 90%. The result of immunofluorescence displayed that the expression of DC-SIGN was mostly located on the surface of cell membrane. Western blotting displayed the specific band of DC-SIGN protein. It showed that the BHK21 cells stably expressing DC-SIGN were successfully established. Conclusion DC-SIGN eukaryotic expression vector has been successfully constructed. The successful establishment of BHK21 cell lines which can stably express DC-SIGN provides a substantial foundation for further study on the DC targeting vaccines.
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2010年第3期304-306,共3页
Medical Journal of Chinese People's Liberation Army
基金
国家科技重大专项基金项目(2008ZX10002-003)
国家自然科学基金项目(30772002)
