Rat complement factor H: molecular cloning, sequencing and quantification with a newly established ELISA.

Area of Science:

• Immunology and Molecular Biology
• Complement System Regulation

Background:

• Factor H (FH) is the primary soluble regulator of the complement system, crucial for preventing host tissue damage.
• FH functions as a cofactor for Factor I, promoting C3b cleavage, and exhibits decay-accelerating activity on the C3bBb complex.

Purpose of the Study:

• To clone, sequence, and characterize the rat Factor H (FH) gene and protein.
• To establish a quantitative assay for measuring FH in rat biological samples.
• To investigate the regulation of FH production by hepatocytes.

Main Methods:

• Screening of a rat liver cDNA library using PCR-generated hybridization probes derived from conserved human and murine FH sequences.
• Nucleotide sequencing and comparative analysis of the rat FH cDNA sequence against human and mouse orthologs.
• Development and validation of a sandwich-enzyme-linked immunosorbent assay (ELISA) for FH quantification.

Main Results:

• The complete rat FH cDNA sequence is 4240 nucleotides, with an open reading frame encoding a 1236 amino acid protein.
• Rat FH shares significant nucleotide (74% human, 87% mouse) and amino acid (63% human, 81.5% mouse) identity with orthologs.
• Rat serum contains FH at a concentration of 238 ± 21 μg/ml, and interferon-gamma significantly upregulates FH production in cultured rat hepatocytes.

Conclusions:

• The characterization of rat FH provides a valuable tool for studying complement regulation in a mammalian model.
• The developed ELISA is a sensitive method for quantifying FH in rat serum and cell culture supernatants.
• Interferon-gamma is identified as a potent inducer of FH production in rat hepatocytes, suggesting a role in immune modulation.