[通过重链中预先形成的二硫键提高HLA - A2 - 肽复合物的体外复性效率]

[Improvement of refolding efficiency of HLA-A2-peptide complex in vitro by preformed disulfide bonds in heavy chain].

作者信息

Fei Shi-jiang, Wu Xiong-wen, Wong Xiu-fang, Cai Lei, Liang Zhi-hui, Han Jun-yan, Gong Fei-li

机构信息

Department of Immunology, Tongji Medical College, Huazhong University of Sciences and Technology, Wuhan 430030, China.

出版信息

Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2005 Mar;21(2):167-70.

PMID:15766400

Abstract

AIM

To improve the refolding efficiency of soluble HLA-A2-peptide complex in vitro.

METHODS

The heavy chain (HC) of MHC class I was extracted from bacteria under denaturing and non-reducing conditions. Anion-exchange and (NH4)2SO4 precipitation were applied to purify the HC. Then the purified HC, beta2m and an antigenic peptide (N-YMDGTMSQV-COOH of Try(369-377)) were refolded to form an HLA-A2-peptide complex by dilution method in the buffer of pH 6.6. The refolded products were detected by Western blot and ELISA with W6/32 and anti-human beta2m antibody.

RESULTS

The refolded products consisted of HLA-A2-peptide complex, beta2m, and a little amount of HC polymer. The refolding efficiency was 2.5 fold higher than that of the conventional method.

CONCLUSION

This study confirmed that the refolding efficiency of the method reported in this paper is higher as compared with the conventional method, which is of importance to the preparation of HLA-peptide tetramers and artificial antigen presenting cells.

摘要

目的

提高可溶性HLA - A2 - 肽复合物的体外复性效率。

方法

在变性和非还原条件下从细菌中提取MHC I类重链（HC）。采用阴离子交换和硫酸铵沉淀法纯化HC。然后将纯化的HC、β2m和一种抗原肽（来自Try(369 - 377)的N - YMDGTMSQV - COOH）在pH 6.6的缓冲液中通过稀释法复性形成HLA - A2 - 肽复合物。用W6/32和抗人β2m抗体通过蛋白质印迹法和酶联免疫吸附测定法检测复性产物。