血小板/T 细胞活化抗原1(PTA1)胞膜外区基因片段的克隆及其融合蛋白的表达

Preparation of a novel human platelet/T cell activation antigen 1 (PTA1) IgG Fc fusion protein, a potential tools for identification of PTA1 ligand

目的 克隆血小板／ Ｔ 细胞活化抗原１ （ｐｌａｔｅｌｅｔ ａｎｄ Ｔｃｅｌｌ ａｃｔｉｖａｔｉｏｎ ａｎｔｉｇｅｎ １ ， Ｐ Ｔ Ａ１）胞膜外区基因片段，构建、表达、纯化 Ｐ Ｔ Ａ１ 胞膜外区与 Ｉｇ Ｇ Ｆｃ 融合蛋白，用于 Ｐ Ｔ Ａ１ 配体鉴定的研究。方法 针对人 Ｐ Ｔ Ａ１ ｃ Ｄ Ｎ Ａ 设计引物，通过反转录、半套式 Ｐ Ｃ Ｒ 从 Ｔ Ｐ Ａ 活化的 Ｊｕｒｋａｔ 细胞中扩增 Ｐ Ｔ Ａ１ 胞膜外区基因片段，并进行序列测定，而后将其进一步克隆入融合蛋白表达载体ｐ Ｉ Ｇ 中，构建重组表达载体ｐ Ｐ Ｔ Ａ１／ Ｉｇ ，经 Ｄ Ｅ Ａ Ｅ Ｄｅｘｔｒａｎ 法转染 Ｃ Ｏ Ｓ７ 细胞后通过亲和层析纯化，融合蛋白经 Ｓ Ｄ Ｓ Ｐ Ａ Ｇ Ｅ、夹心 Ｅ Ｌ Ｉ Ｓ Ａ 及 Ｗｅｓｔｅｒｎ ｂｌｏｔ 鉴定。结果 获得７９３ｂｐ Ｐ Ｔ Ａ１ 胞膜外区基因片段，构建成功 Ｐ Ｔ Ａ１／ Ｉｇ 融合蛋白表达载体，制备了可用于纯化 Ｐ Ｔ Ａ１／ Ｉｇ 的亲和层析柱，建立了检测融合蛋白的夹心 Ｅ Ｌ Ｉ Ｓ Ａ 方法。经 Ｓ Ｄ Ｓ Ｐ Ａ Ｇ Ｅ、夹心 Ｅ Ｌ Ｉ Ｓ Ａ 及 Ｗｅｓｔｅｒｎ ｂｌｏｔ 证实融合蛋白在 Ｃ Ｏ Ｓ７ 细胞中获得表达，相对分子质量为８３ ×１０３ ，表达效率约为１ ．３ ｍｇ／ ?

Preparation of a novel human platelet/T cell activation antigen 1 (PTA1)IgG Fc fusion protein, a potential tools for identification of PTA1 ligand$$$$ SUN Kai, JIN Boquan, FENG Qi, et al. Department of Immunology, The Fourth Military Medical University, Xian 710032 【 Abstract 】 Objective To express and purify the PTA1/IgG Fc fusion protein as a tool for further research on PTA1 ligand and its function. Methods The mRNA of PTA1 was extracted from TPA activated Jurkat cells and used as a template for reversetranscription. After PCR amplification, a 793bp fragment including extracellular region and the splice donor sequence “ACTTACCTGT” was obtained and cloned into fusion expression vector pIG. The recombinant vector was named pPTA1/Ig. The pPTA1/Ig vector was transfected in COS7 cell by DEAEDextran method and expression of a secreting fusion protein was identified by sandwich ELISA with antiPTA1 McAb and HRPconjugated antihIg McAb. Results About 1.3mg PTA1/Ig fusion protein could be obtained from 1L COS7 culture supernatants by antiPTA1 affinity chromatography purification. SDSPAGE showed that the molecular weight of PTA1/Ig fusion protein was 83kD and the protein could be recognized by antiIgG polyclonal antibody in Western blot. Conclusions Both PTA1 extracellular domain and IgG Fc domain could be recognized respectively by antiPTA1 and antihIg Fc McAb, suggesting that PTA1/Ig could mimic the nature PTA1 molecule and be a potential tool in the research of PTA1 ligand and its functions. 【 Subject words 】 Platelet/T cell activation antigen Fusion protein Gene expression