Cloning of the E1M gene of human papillomavirus type 16 and production of an antibody to the E1M protein

Since the DNA of human papillomaviruses (HPV) can be found in at least 90% of cervical cancers, it has been implicated that HPV may be the etiology of such tumors. Investigative efforts have focused on analyzing the expression of HPV genes. Two viral genes, E6 and E7, have been shown to be expressed in tumor cells. E1M, which is the 5' portion of the E1 gene, acts to negatively modulate replication of the viral DNA. We are interested in examining whether or not the E1M gene is expressed in the cervical cancer as well. For this purpose, a system for production of an anti-E1M antibody would be advantageous. The E1M gene is thought to be involved in the regulation of HPV replication and could thus be detectable, analyzed biochemically and used in the research of the etiology of cervical cancers. This thesis describes a series of experiments designed to clone the E1M gene into a vector engineered to make the E1M protein as part of a bacterial-viral fusion protein. This fusion protein would then be used as an antigen to raise antibodies to the E1M protein by immunization of rabbits. I have constructed a clone of the E1M gene that produces the viral E1M protein fused to the bacterial trpE protein. These proteins were purified by gel electrophoresis and used to raise antibodies in rabbits. However, no significant antigen-antibody reactivity was detected by using these antibodies against the E1M fusion protein in Western blots. The reason is unknown but could be explained in E1M protein is poorly immunogenic. Also the yield of E1M protein was low and insufficient protein might have been used to immunize rabbits.