Establishment of leukocyte-associated immunoglobulin like-receptor 2 (CD306) eukaryotic expression vectors and purification and identification of fusion protein

Chunyan WANG; Zhenhua KANG; Xin XIE; Yan LI; Boquan JIN

ABSTRACT

BACKGROUND:

Biological function of leukocyte-associated immunoglobulin like-receptor 1 (LAIR 1) has clearly researched in China and abroad, but the in vivo biological function of LAIR is poorly understood.

OBJECTIVE:

To establish LAIR-2 (CD306) eukaryotic expression vectors and to purify and identify the fusion protein. DESIGN, TIME AND

SETTING:

A single sample observation experiment was performed at the Fourth Military Medical University of Chinese PLA between June 2007 and June 2008.MATERIALS plg/3c vector was offered by Oxford University, pcDNA3.1 vector was provided by Meyaard doctor. Chinese hamster ovary (CHO) cell lines were preserved by the Department of Immunology, Fourth Military Medical University of Chinese PLA.

METHODS:

Two eukaryotic expression vectors plg/3c-LAIR-2 and pcDNA3.1-LAIR-2 were constructed and were transfected into CHO cells. The binding activities of LAIR-2 fusion protein to LAIR-2 mAbs were identified by Western blot, immunocytochemistry and flow cytometry assay.MAIN OUTCOME

MEASURES:

The construction of stably transfected cell lines, and the purification and identification of fusion protein. The activity of LAIR 2 protein combined to corresponding monoclonal antibody.

RESULTS:

Eukaryotic expression vectors were constructed and trasnsfected into CHO cells successfully. Two cells lines CHO/LAIR-2-Fc and CHO/LAIR-2 that steadily expressed LAIR-2-Fc fusion protein and LAIR-2 protein were established. Western blot assay showed that LAIR-2 protein could bind specially to LAIR-2 mAb 1A7, 3H12 and 4A9. Immunocytochemistry and flow cytometry assay demonstrated that 3H12 and 4A9 could bind to LAIR-2 expressed in the transfected CHO cells.

CONCLUSION:

Two ceils lines CHO/LAIR-2-Fc and CHO/LAIR-2 were successfully constructed, which can transfected to CHO cells. The eukaryotic expressed LAIR-2 protein has good binding activity to LAIR-2 mAbs.