Melissa Hughes and Dr. Eric Wilson, Department of Microbiology and Molecular Biology

Background

I have successfully completed my project of cloning CCL28. CCL28 (MEC) is a protein that acts as a chemical signal in the body to attract antibody-secreting IgA cells that expresses a receptor called CCR10 to the mucosal tissue and to the mammary gland (1). CCL28 plays a vital role in allowing the maternal antibody secreting cells to home to the mammary gland and to pass antibodies to the breast-feeding infant, giving the infant passive immunity. This provides the infant with antibodies to antigens found in the mother’s environment (which is consequently the infant’s environment as well) and decreases mortality rates. The study of CCL28/CCR10 in cows is of interest because cows do not transfer antibodies across the placenta as do humans and mice. They transfer antibodies only through their milk to suckling newborns. Thus, the migration of IgA cells to the CCL28 signal in the mammary glands is imperative for newborn passive immunity.

Interestingly, this protein may have a second function in cows’ milk. One study found that it is antibacterial and so may kill bacteria that would infect the cows’ mammary glands (2). Therefore, the antimicrobial function of CCL28 may also be very important in protecting cows from mastitis, or bacterial infection of the mammary gland. Mastitis costs the dairy industry more than a billion dollars each year (3). When cows become infected, their milk cannot be sold and they must receive treatment that can last several weeks. Thus, mastitis is a serious concern for dairy farmers. Finding out if low levels of CCL28 or CCR10 in cows result in a greater potential of developing mastitis could help lead to preventative treatment and help the dairy industry.

Research

In order to begin studying through assays how CCL28 works in cows against bacteria, Dr. Wilson needed a source of the protein. My project was to clone this protein into a plasmid (bacterial circular DNA) that could give our lab a ready source of protein. This project was very challenging because no one in Dr. Wilson’s lab had ever done cloning. He and I worked through each step of the process starting in Fall 2007. Our first challenge was to find the sequence that coded for CCL28. There was no sequence identified for CCL28 in a cow at the time. By comparing CCL28 sequences in other animals like mouse and human, I was able to predict where the sequence of CCL28 was and what it was. The next step was to amplify this predicted gene sequence using primers and RT-PCR. This portion of the process was the most challenging and required several months of redesigning our RT-PCR protocol and primers. It was very frustrating, but I stuck with it and was finally able to find good primers.

Next, Dr. Wilson and I had to fine-tune all of the procedures that I needed to do before I inserted CCL28 correctly into a plasmid. Once I worked through that and went through several different plasmids (it would not insert correctly into some of the ones I tried), I finally was able to get it into the plasmid. I had the plasmid sequenced and unfortunately, I found that the CCL28 in the plasmid was missing the stop signal to end protein production. I had to go back to the beginning to insert a stop signal and re-do the process. Finally, I was able to successfully insert the gene into the plasmid and put that plasmid into bacteria. Now, I had to make the bacteria produce protein from that plasmid. That was a whole other challenge. When I made the bacteria produce protein and then ran that protein on a gel to visualize it, I could not see anything. Dr. Wilson and I thought that maybe since the protein could possibly be antibacterial, it may have been killing the bacteria I was trying to produce it from after the bacteria had produced it. I tried several different methods to see if I could produce protein without killing the bacteria but to no avail. At this point, Dr. Wilson decided to send the bacteria containing the plasmid to our collaborators at Montana State University who had a graduate student that is experienced in protein biology. He was able to produce protein from my plasmid. And so, I was able to successfully complete my project.

Since the completion of my project, other students have been able to use the cow CCL28 protein in bacterial killing assays and have found that it does indeed kill bacteria. Further experiments are being run and are going to be run that will eventually help our lab answer the question of whether reduced CCL28 levels in cows can make them more susceptible to mastitis. Hopefully, the answer to this question will lead to future therapeutics that could protect cows with low CCL28 levels from mastitis or lead to better dairy cow selection.

Presentation

I presented my research as a poster at the 2007 American Association of Immunologists conference in Miami, Florida. In the future, my work will most likely be included in Dr. Wilson’s research articles on cow CCL28.

References

• Wilson, E., and E. Butcher. 2004. CCL28 controls immunoglobulin (Ig)A plasma cell accumulation in the lactating mammary gland and IgA antibody transfer to the neonate. J. Exp. Med. 200:805-809.
• Hieshima, K., H. Ohtani, M. Shibano, D. Izawa, T. Nakayama,
  Y. Kawasaki, F. Shiba, M. Shiota, F. Katou, T. Saito, and O. Yoshie. 2003. CCL28 has dual roles in mucosal immunity as a chemokine with broad-spectrum antimicrobial activity. J. Immunol. 170:1452–1461.
• Schroeder, J. W. 1997. Mastitis control programs: bovine mastitis and milking management. North Dakota State University and the U. S. Department of Agriculture. Retrieved 25 Jul. 2007 from http://www.ag.ndsu.edu/pubs/ansci/dairy/as1129w.htm.