采用绿色荧光蛋白腺病毒载体表达HBsAgCTL优势表位肽MHC单链三聚体

Expression of a single-chain trimer of MHC restricted HBsAg CTL epitope using adenovirus vector containing GFP- report gene

目的采用携带GFP报告基因腺病毒载体表达HBsAg细胞毒性T细胞（CTL）表位MHC单链三聚体，为增强HBV特异性免疫提供新方法。方法构建小鼠MHCⅠ类分子（H-2L^d）限制的HBsAg优势CTL表位肽与MHCⅠ类分子重链（Ld）和β2M链（β2-microglobulin，β2微球蛋白）的单链三聚体（HBsAg—SCT）。并将HBsAg-SCT亚克隆到GFP标记腺病毒载体，以HBsAg和OVA—SCT作为对照，转染293A细胞，包装产生携带HBsAg—SCT，HBsAg和OVA—SCT的重组腺病毒。结果经双酶切和克隆测序鉴定，HBsAg—SCT能成功克隆到编码GFP标记的腺病毒载体，通过荧光显微镜观察到转染的293A细胞含有绿色荧光素蛋白。通过Western Blot进一步证实表达的重组蛋白HBsAg—SCT能与抗-Flag抗体发生反应。结论编码HBsAg—SCT的荧光蛋白腺病毒载体成功构建，并能在293A细胞中完整包装成重组腺病毒颗粒。

Objective To generate a recombinant Adenovirus encoding a GFP （green fluorescent protein）-report gene and a single-chain trimer of MHC restricted HBsAg CTL epitope. Methods An oligonueleotidc encoding H-2L^d restricted HBsAg CTL epitope was synthesized and fused with H-2L^d DNA molecule to construct the eukaryotic expression vector carrying the HBsAg-SCT gene. The HBsAg-SCT gene was subcloned into a GFP adenovirus expression vector,which was transfected into Ad293 cells for packaging and amplification of recombinant adenovirus encoding HBsAg-SCT. Results HBsAg-SCT has been cloned into an adenovirus vector encoding GFP report gene successfully as confirmed by double enzyme digestion and direct sequencing. HBsAg- SCT was expressed by infected Ad293 cells demonstrated by western blot assay. Conclusion A recombinant adenovirus expressing HBsAg-SCT and green fluorescent protein report gene has been generated.