Molecular cloning, sequencing, and expression of equine interleukin-6.

The research study focuses on the molecular cloning, sequencing, and expression of equine interleukin-6 (IL-6), exploring its amplification, translation, sequence similarities with other mammalian IL-6 and its functional impact when expressed in Chinese hamster ovary cells.

Procedure and Findings:

• The study involved the amplification of equine interleukin-6 (IL-6) cDNA, using consensus sequence primers, from mitogen-stimulated equine peripheral blood mononuclear cells (PBMC).
• The amplified cDNA was 727bp long, encompassing the complete coding region for equine IL-6 and also included 118 bases in the 3′ non-translated region.
• The coding sequence could be translated into a protein consisting of 208 amino acids, including a predicted leader sequence of 28 amino acids.

Resultant IL-6 Protein Characteristics:

• The mature IL-6 protein, comprising 180 amino acids, had a predicted molecular mass of 20,471Da without any post-translational modifications.
• The experimental study resulted in highlighting the similarity between the amino acid sequence of equine IL-6 and other mammalian IL-6 sequences, with a similarity percentage ranging between 46 and 84%.

Functional Impact of Equine IL-6:

• The expression of equine IL-6 in Chinese hamster ovary (CHO) cells resulted in a supernatant that boosted the proliferation of B9 cells in a dose-dependent manner.
• A treatment procedure was carried out on B9 cells with an anti-IL-6 receptor antibody. The result showcased a termination of the response to the recombinant equine IL-6, validating the functional importance of the IL-6 receptor in mediating its effects.