高效表达肝癌相关抗原HAb18G的CHO细胞株的建立

ESTABLISHMENT OF CHO CELL STRAIN THAT CAN HIGHLY EFFICIENTLY EXPRESS HEPATOMA ASSOCIATE ANTIGEN HAb18G

目的 获得高效表达肝癌相关抗原 HAb18G的CHO细胞株。方法 构建含有肝癌相关抗原 HAb18G基因的真核表达载体并经限制性酶切及部分序列分析证明插入是否正确。用阳离子脂质体介导转染 CHO细胞经 G418筛选 ,稳定表达 ,间接免疫荧光染色证实蛋白表达情况。用有限稀释法将筛选的细胞进行单克隆化 ,通过流式细胞仪筛选高效表达株。结果 成功构建了真核表达载体 pc DNA- 3/HAb18G并经限制性酶切及部分序列分析证明基因插入正确。间接免疫荧光染色证实 :HAb18G高效表达于转染细胞的胞膜上。流式细胞仪筛选获得了 1株高效表达的 CHO细胞。结论 实验结果为

Objective To establish CHO cell strain that can highly efficiently express hepatoma associated antigen HAb18G.Methods An eukaryotic expression vector pc DNA 3/HAb18G for expressing hepatoma associated antigen HAb18G was constructed. It was checked whether the gene was inserted correctly or not by partial nucleotide sequencing and restriction endonuclease digestion. Then vector pcDNA 3/HAb18G was transfected into CHO cell by mixing with LIPOFECTAMINE 2000 reagent. After transfected cell was screened by G418 in order to obtain stable expression. The expression of HAb18G was detected by indirect immunofluorescence staining. The obtained positive cells were then single clonized by limited dilution and were classified and screened by fluorocytometry assay. Results The result showed that HAb18G was expressed highly efficiently on the membranes of transfected cells by indirect immunofluorescence staining. In addition, one such strain had been obtained. Conclusion These results lay the foundation for getting expression of the protein and studying its biological functions.