Isolation of Plasmid DNA Encoding the Human 2B4 (CD244) Gene Expressed on Natural Killer Cells

Undergraduate #25
Discipline: Biological Sciences
Subcategory: Cancer Research

Natalie White - Alabama State University
Co-Author(s): Joseph Malaer, Sheila Powers, Stephen Mathew, and Porunelloor Mathew, University of North Texas, Fort Worth

The immune system protects the host from invading microorganisms and cancer. It can be divided into two branches: innate and acquired immunity. Innate immunity is a generalized defense system that is not specific for any infectious agent or cancer. Innate immune responses are largely mediated by immune cells such as natural killer cells (NK cells), granulocytes, and phagocytes; whereas adaptive immunity refers to immune responses mediated by T and B cells that are antigen-specific and result in memory. NK cells are lymphocytes that recognize tumor cells, bacterial, viral, and parasitic infections without antigen stimulation. NK cell function is regulated by specific receptors that, upon interaction with their respective ligands, may send activating or inhibitory signals. One such receptor, 2B4 (CD244), was cloned in our laboratory and is a membrane glycoprotein expressed on NK cells as well as other immune cells. The interaction of 2B4 with its ligand CD48, can regulate killing of target cells and INF- secretion by NK cells. 2B4 dysfunction has been implicated in various diseases, most notably X-linked lymphoproliferative disease (XLPD). 2B4 also plays a major role in other diseases such as rheumatoid arthritis, systemic lupus erythematosus (SLE), HIV, and cancer. The purpose of this project was to isolate and prepare large quantities of the 2B4 gene cloned in pCI-neo plasmid. The pCI-neo plasmid containing the 2B4 gene was transformed into Escherichia coli (E. coli) strain DH5-α, and transformed colonies were selected on LB plates containing ampicillin. A mini prep plasmid isolation kit was utilized to isolate the DNA and 2B4 specific primers were used to amplify the 2B4 gene by PCR. After confirming with the mini prep and PCR amplification, a large scale preparation of plasmid was performed using a plasmid maxi prep kit. The purified plasmid obtained in this study will be utilized to perform future mechanistic studies to understand the role of 2B4 in different diseases.

Funder Acknowledgement(s): This project was funded by the P20CA192976 from the DOD, CBRT.

Faculty Advisor: Porunelloor Mathew, porunelloor.mathew@unthsc.edu

Role: I did the majority of the research such as performing various kits and running PCR. I also did restriction enzyme digestion. I also isolated and prepared a large quantity of 2B4 gene with plasmid. I was helped only with certain things during this project.