摘要
目的：获得人血小板／Ｔ细胞活化抗原１（ｐｌａｔｅｌｅｔａｎｄＴｃｅｌａｃｔｉｖａｔｉｏｎａｎｔｉｇｅｎ１，ＰＴＡ１）胞膜外区与人ＩｇＧ１Ｆｃ段融合蛋白，为ＰＴＡ１配体的鉴定及其功能的研究提供有力的工具。方法：针对人ＰＴＡ１ｃＤＮＡ序列设计３段引物，通过反转录、半套式ＰＣＲ方法从活化Ｊｕｒｋａｔ细胞中扩增出ＰＴＡ１胞膜外区基因片段，并将其克隆入融合蛋白表达载体ｐＩＧ中，通过酶切、ＰＣＲ及序列测定鉴定重组表达载体。重组载体通过ＤＥＡＥｄｅｘｔｒａｎ法转染ＣＯＳ７细胞，经夹心ＥＬＩＳＡ及亲和层析、ＳＤＳＰＡＧＥ鉴定融合蛋白的表达及免疫学活性。结果：经序列测定后证实重组表达载体含有正确的ＰＴＡ１胞膜外区序列及剪接供体序列，转染ＣＯＳ７细胞后，通过亲和层析纯化证实融合蛋白的Ｍｒ为８３０００，并能被抗ＰＴＡ１胞膜外区单抗及抗人ＩｇＧＦｃ的单抗同时识别。结论：ｐＰＴＡ１／Ｉｇ融合表达载体的构建及表达成功，为ＰＴＡ１的功能及配体（ＰＴＡ１Ｌ）的研究打下了良好的基础。
Aim: Platelet and T cell activation antigen 1 (PTA1) is a novel member of immunoglobulin superfamily. Its ligand and function is still not clear. The aim of this paper is to express and purify the PTA1 IgGFc fusion protein for further research on PTA1 ligand and its function. Methods: we amplified the 800bp extracellular region of PTA1 cDNA by RT PCR from activated Jurkat cell, and cloned this fragment into fusion expression vector pIG. The recombinant vector pPTA1/Ig was obtained after sequencing and PCR identification. After transfection of pPTA1/Ig into COS 7 cells by DEAE dextran, expression of a secreting fusion protein was identified by affinity chromatography and sandwich ELISA assay with anti PTA1 mAb and HRP anti hIgFc mAb. Results: The correct insertion of PTA1 extracellular region and splicer donor sequence into pIG vector was identified. After transfection of pPTA1/Ig into COS 7 cells, a M r83 000 secreting protein was obtained by anti PTA1 mAb affinity chromatography, and this protein could also be recognized by both anti PTA1 and anti hIgFc mAb. Conclusion: This fusion protein could mimic the nature PTA1 protein and could be used as a potential tool in the investigation of PTA1 ligand and function.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
1998年第1期1-4,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金
