稳定表达Ag85A蛋白细胞系的建立

Establishment of Cell Line for Stable Expression of Ag85A Protein

目的建立稳定表达Ag85A蛋白的K562真核细胞系,为进一步开展以阳离子脂质体为转运载体的口服疫苗的免疫原性研究奠定基础。方法将已构建的重组质粒pCDNA3.1+/Ag85A包裹阳离子脂质体LipofectamineTM 2000后,转染真核细胞K562,SDS-PAGE和Western blot分析重组Ag85A蛋白的表达。用G418筛选稳定表达Ag85A蛋白的细胞株,流式细胞术和间接免疫荧光技术检测Ag85A蛋白的表达及定位。结果重组质粒pCDNA3.1+/Ag85A转染K562细胞时,与脂质体的最适比例为2μg:2μl;转染细胞表达的Ag85A蛋白相对分子质量约为32000,且具有良好的反应原性。稳定转染3个月后,重组质粒转染的细胞有42.37%的细胞显示FITC标记,且细胞胞浆中及细胞膜上均有Ag85A蛋白的表达。结论已获得稳定表达Ag85A蛋白的K562细胞系,可用于Ag85A蛋白的大量制备及抗体方面的研究。

Objective To establish the K562 eukaryotic cell line for stable expression of Ag85A protein and lay a foundation of further study on immunogenicity of oral vaccine using cationic liposome as a delivery carrier.Methods The constructed recombinant plasmid pCDNA3.1＋ /Ag85A was encapsulated with LipofectamineTM 2000,a cationic liposome,and transfected to K562 cells.The expressed Ag85A protein was analyzed by SDS-PAGE and Western blot.The cell strains stably expressing Ag85A gene were screened with G418,and the expression and location of Ag85A protein were determined by flow cytometry and IFA.Results The optimal ratio of recombinant plasmid pCDN A3.1＋ /Ag85A to liposome for transfection of K562 cells was 2 μg：2 μl.The expressed Ag85A protein,with a relative molecular mass of about 32 000,showed good reactogenicity.FITC markers were observed in 42.37% of cells 3 months after stable transfection with recombinant plasmid pCDNA3.1＋ /Ag85A.The expressions of Ag85A were observed in cytoplasm and on cell membrane.Conclusion The K562 cells stably expressing Ag85A protein was established,which might be used for preparation of Ag85A protein in a large quantity and study on antibody against Ag85A.