[Clone and expression of human soluble CD14 and study of its function]

Area of Science:

• Molecular Biology
• Immunology
• Biochemistry

Background:

• Soluble CD14 (sCD14) plays a crucial role in the innate immune response.
• Understanding sCD14's function requires reliable methods for its production and purification.

Purpose of the Study:

• To construct a recombinant expression plasmid for human soluble CD14 (sCD14).
• To express and purify functional sCD14 in eukaryotic cells.
• To validate the biological activity of the purified sCD14.

Main Methods:

• RNA extraction from U937 cells and RT-PCR for sCD14 cDNA amplification.
• Construction of the recombinant expression plasmid pEF1/HisC/sCD14 348aa.
• Liposome-mediated transfection for eukaryotic cell expression.
• Immunoaffinity chromatography for protein purification.
• LPS stimulation assay to confirm functional binding.

Main Results:

• High-level expression of sCD14 was achieved in eukaryotic cells.
• Purified sCD14 protein reached a purity level exceeding 90%.
• The expressed sCD14 protein demonstrated functional binding to LPS, as evidenced by changes in U937 cells.

Conclusions:

• A method for high-yield, high-purity production of functional human sCD14 was established.
• The recombinant sCD14 protein is suitable for further functional studies.
• This work provides a valuable tool for research in innate immunity and LPS interactions.