人AFP全长基因编码序列的克隆及表达

Construction and Expression of Human α-Fetoprotein Recombinant Plasmid

目的 构建人 AFP全长基因编码序列的真核表达质粒 ,并在 CHO及小鼠肌肉组织中表达。方法 从人胎肝组织中提取 AFP基因 ,以全长基因序列为目的基因片段 ,真核表达质粒 pc DNA3.1(+)为载体 ,构建AFP重组质粒 ph AFP,经酶切分析和 DNA测序对 ph AFP进行鉴定。重组质粒 ph AFP经脂质体转染真核细胞CHO以及直接注射小鼠胫前肌 ,并采用免疫荧光染色及免疫组化对其表达产物进行检测。结果 获取的 AFP基因片段与 Gen Bank报道的序列一致 ,确认为人 AFP基因的全长编码序列 ,构建的重组质粒 ph AFP能在体外真核细胞 CHO、体内肌细胞中有效表达。结论 成功构建了能在真核细胞中表达的重组质粒 ph AFP,为 AFP

Objective To construct AFP-expressing plasmid and study the expression in eukaryotic cells. Methods Total RNA was isolated from fetal liver tissue. Full-length human AFP cDNA was obtained by RT-PCR amplification and then recombined into eukaryotic expression plasmid pcDNA3.1. The AFP sequence of the recombinant plasmid phAFP was determined. The AFP expressions in CHO transfected with phAFP and in muscle after injection phAFP were investigated by immunohistological methods. Results The restriction endonuclease mapping of recombinant plasmid showed 1.8 kb fragment as well as full-length human AFP cDNA, and the sequence is consistent with that from GenBank. The phAFP was successfully expressed in CHO and in muscle tissues. Conclusion These results suggested that AFP can be used as a target gene of DNA vaccine in heptocellular carcinoma therapy.