摘要
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 S7 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 S7 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, Xian 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 reversetranscription. 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 COS7 cell by DEAEDextran method and expression of a secreting fusion protein was identified by sandwich ELISA with antiPTA1 McAb and HRPconjugated antihIg McAb. Results About 1.3mg PTA1/Ig fusion protein could be obtained from 1L COS7 culture supernatants by antiPTA1 affinity chromatography purification. SDSPAGE showed that the molecular weight of PTA1/Ig fusion protein was 83kD and the protein could be recognized by antiIgG polyclonal antibody in Western blot. Conclusions Both PTA1 extracellular domain and IgG Fc domain could be recognized respectively by antiPTA1 and antihIg 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
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
1999年第5期377-381,共5页
Chinese Journal of Microbiology and Immunology
基金
国家自然科学基金