Expression and characterisation of equine interleukin 2 and interleukin 4.

This research paper discusses the study of the expression of interleukins 2 and 4 in horses. The researchers cloned these interleukins and used them to stimulate cell growth. They noticed that Interleukins 2 and 4 induced cell proliferation, whereas only Interleukin 2 supported the proliferation of B cells in horses.

Methodology

• The researchers cloned the cDNA of equine interleukin 2 (IL2) and interleukin 4 (IL4) in a mammalian expression vector. This vector also contained c-terminal myc and six histidines His(6)-epitopes, which served as markers for recognising and purifying these equine cytokines.
• This construct was then used to transfect Chinese Hamster Ovary (CHO) cells, a common lab technique used to insert foreign DNA into the cells.
• Afterward, the expressed equine cytokines were isolated and purified.

Characterisation of Equine Cytokines

• The researchers characterized the purified equine cytokines using western blotting, a technique used to detect specific proteins in a sample.
• Upon characterization, it was found that the interleukin 2 was secreted with a molecular weight of about 17.1kDa.
• On the other hand, Interleukin 4 was expressed in three different sizes—17.1kDa, 19.6kDa, and 22.1kDa. This variance in the sizes can be largely attributed to different glycosylation modifications, which are a type of post-translational modification where carbohydrates are added to proteins that could change the proteins’ eventual size.

Testing the Biological Activities of Equine Cytokines

• Later, the biological activities of both cytokines were tested. This involved performing proliferation assays using Leukoagglutinin (LAG) stimulated equine peripheral blood mononuclear cells (PBMC).
• The results of these proliferation assays showed that both, equine IL2 and IL4, were capable of inducing a dose-dependent proliferation of lymphocytes. This means the rate of lymphocyte growth increased with the increasing dose of these interleukins.
• However, a key difference emerged in the ability of IL2 and IL4 to influence the growth of B cells, a type of white blood cell. While IL4 did not significantly impact B cell proliferation, IL2 supported their proliferation indicating its potential vital role in B cell development and function.