高级搜索
人生存素基因的克隆、表达与抗血清制备[J]. 肿瘤防治研究, 2008, 35(04): 240-243. DOI: 10.3971/j.issn.1000-8578.2204
引用本文: 人生存素基因的克隆、表达与抗血清制备[J]. 肿瘤防治研究, 2008, 35(04): 240-243. DOI: 10.3971/j.issn.1000-8578.2204
Cloning and Expression of survivin Gene in E.coli and Preparation of Its Antiserum[J]. Cancer Research on Prevention and Treatment, 2008, 35(04): 240-243. DOI: 10.3971/j.issn.1000-8578.2204
Citation: Cloning and Expression of survivin Gene in E.coli and Preparation of Its Antiserum[J]. Cancer Research on Prevention and Treatment, 2008, 35(04): 240-243. DOI: 10.3971/j.issn.1000-8578.2204

人生存素基因的克隆、表达与抗血清制备

Cloning and Expression of survivin Gene in E.coli and Preparation of Its Antiserum

  • 摘要: 目的克隆表达重组人生存素,制备其抗血清并检测其免疫识别活性。方法用RT-PCR方法从培养的人白血病细胞株HL-60中克隆人生存素cDNA,克隆到原核表达载体pET32a(+),重组质粒转化大肠杆菌BL21(DE3)进行表达,目的蛋白经Ni-NTA树脂亲和纯化后免疫小鼠制备抗血清,ELISA检测抗血清的效价,Western blot和免疫细胞化学法鉴定抗血清的特异性。结果所克隆生存素cDNA序列与Genbank中序列完全一致,将其读码框插入原核表达载体pET32a(+)诱导表达后,SDS-PAGE电泳分析证实重组蛋白为分子量约37KD的融合蛋白,表达量占总菌体的30%,纯化后融合蛋白的纯度为95%,所制备的鼠抗血清的效价达1:1600,Western blotting证实其有较好的特异性,可以检测肿瘤细胞生存素的表达。结论我们已克隆并表达了人生存素基因,所制备的抗人生存素血清可用于相关肿瘤检测。

     

    Abstract: Objective To clone and express the human survivin gene in E.coli and prepare its antiserum. Methods The survivin cDNA was amplified from the total RNA of HL-60 cells by RT-PCR.the open reading form of survivin gene was inserted to the expression plasmid pET32 a(+),transfected into E.coil BL21(DE3)and induced with IPTG,the expressed protein was analyzed with SDS-PAGE.Then,the Balb/c mice were immunized with the purified expression protein,the antiserum titer was detected with ELISA,and the specificity was identified with Western blot and immunocytochemist ry techniques. Results  DNA sequencing result s showed that survivin gene was exactly consistent with the sequence reported in Genbank, the survivin expressed in E. coli, the protein amounted to 30 % of total bacteria protein and the molecular weight was 37 KD. The titer of antiserum against recombinant surviving was 1∶1600, identified the recombinant protein in E. coli and the surviving in tumor cell specifically. Conclusion  The survivin gene was cloned and expressed in E. coli, the antiserum against recombinant survivin was prepared and could be detect the target protein in tumor cell.

     

/

返回文章
返回