哺乳动物细胞表达人细胞珠蛋白

Expressing human cytoglobin by mammalian cells

[目的]构建带His标签的人细胞珠蛋白(cytoglobin,CYGB)哺乳细胞表达载体,转染CHO细胞和HEK293细胞表达CYGB。[方法]采用全基因合成并通过双酶切法将CYGB基因插入到表达载体pcDNA3.4中,经双酶切和测序验证后转染大肠埃希菌DH5α菌株,提取转染级质粒并转染哺乳动物细胞CHO和HEK293,进行瞬时表达,镍柱亲和层析纯化CYGB蛋白。纯化的CYGB蛋白采用Western Blotting法和ELISA法检测蛋白活性。[结果]双酶切和琼脂糖凝胶分析结果表明,pcDNA3.4-His-CYGB真核表达质粒构建成功。CHO细胞瞬时转染pcDNA3.4-His-CYGB真核表达质粒后CYGB表达量较低,HEK293细胞表达量较高,最终采用HEK293细胞进行CYGB表达。SDS-PAGE法检测结果表明CYGB蛋白的分子量约为22 kDa,分子量符合预期。镍柱亲和层析纯化的CYGB的Western Blotting和ELISA检测结果表明HEK293细胞表达的CYGB与抗CYGB单克隆抗体具有生物学结合活性。[结论]HEK293细胞比CHO细胞更适合用于CYGB的哺乳动物细胞表达,其表达的CYGB具有Western Blotting和ELISA生物学活性。

[Objective]To construct the mammalian expression vector of human cytoglobin(CYGB)gene with His tag,and transfected into CHO cells and HEK293 cells to express CYGB.[Method]The CYGB gene was synthesized using whole gene synthesis and inserted into the expression vector pcDNA3.4 through double enzyme digestion.After double enzyme digestion and sequencing verification,the gene was transfected into DH5αclone the strain,extract transfection grade plasmids,and transfected CHO and HEK293 cells for transient expression.Nickel column affinity chromatography to purify the CYGB,and the purified CYGB was detected by Western Blotting and ELISA methods.[Result]Double enzyme digestion and agarose gel analysis showed that the pcDNA3.4-His-CYGB eukaryotic expression plasmid was successfully constructed.CHO cells were transiently transfected with pcDNA3.4-His-CYGB eukaryotic expression plasmid,and the expression level of CYGB was low,while that of HEK293 cells was high.Finally,HEK293 cells were used to express CYGB.The SDS-PAGE detection results showed that the molecular weight of CYGB protein was approximately 22 kDa,which was in line with expectations.The Western Blotting and ELISA detection showed that CYGB expressed in HEK293 cells had biological binding activity with anti CYGB monoclonal antibodies.[Conclusion]HEK293 cells are more suitable for expressing CYGB in mammalian cells than CHO cells,and the expressed CYGB has Western Blotting and ELISA biological activities.