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Description
Human papillomavirus (HPV) is the causative agent of cervical cancer. Persistent infection with high-risk HPV 16, 18 or 45 species is associated with the development and progression of cervical cancer. HPV genotyping and Pap smear tests are the regular methods used to detect pre-invasive cervical lesions, but there is a need for developing a rapid biomarker to profile immunity to these viruses. The viral E7 oncogene is expressed in most HPV-associated cancers and anti-E7 antibodies can be detected in the blood of patients with cervical cancer. This research was focused on viral E7 oncogene expression to be used in development of low-cost point of care tests, enabling patients from low resource settings to detect the asymptotic stage of cervical cancer and be able to seek treatment early. In order to produce the E7 protein in vitro to measure antibody levels, GST tagged E7 genes from HPV 16, 18 and 45 species were inserted into the pDEST15 vector and expressed in E. coli BL21DE3 cells that were induced with 1mM of IPTG. The E7-GST fused expressed protein was then purified using glutathione beads and resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein expression was 5.8 \u03bcg/ml for HPV 16E7 in 500 ml culture and for the 500 ml culture of HPV 18 E7 and 45 E7 were 10.5 \u03bcg/ml and 10.5 \u03bcg/ml for HPV 18E7 and 45E7 respectively. High yield values are showing high expression levels of GST-tagged E7 recombinant protein which can be used for serotyping a number of individuals. This shows that HPV E7 can be produced in large quantities that can potentially be used in point of care tests that can help identify women at risk of cervical cancer. In conclusion, the E7 protein produced in this study can potentially be used to induce humoral responses in patients\u2019 sera for understanding the immune response of cervical cancer.
ContributorsMakuyana, Ntombizodwa (Author) / Anderson, Karen (Thesis director) / Ewaisha, Radwa (Committee member) / Varsani, Arvind (Committee member) / Hou, Ching-Wen (Committee member) / School of Mathematical and Statistical Sciences (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-12
Description
Recent studies have shown that human papillomavirus (HPV) plays a role in development of cancers, one of which is head and neck cancer. There is strong and consistent molecular evidence demonstrating that human papillomavirus (HPV) is an etiological cause of these oropharyngeal cancers. Despite the introduction of HPV vaccines, there is still an increase in human papillomavirus associated OPC (HPVOPC) and it is expected that the incidence of head and neck cancer, specifically oropharyngeal cancer (OPC) will increase. The aim of this study is to utilize human papillomavirus (HPV) seropositivity for rapid detection of HPV early specific antigen-antibodies using a lateral flow assay.
Human papillomavirus (HPV) 16 proteins of interest, E7, E6 and CE2 were expressed and purified in E. coli for detection of specific antibodies using lateral flow assay because viral and host factors impact the serologic responses to HPV early antigens in HPV-positive oropharyngeal cancer. 17 samples and 5 controls with already known antibody reactivity from ELISA analysis were selected for HPV serologic responses. The lateral flow strip was evaluated for its color band intensity using Image J software. Peak area was used to quantify the color intensity of the lateral flow strip. Out of the 17 samples, 11 (64.7%) showed high antibody levels to E7, 12 (70.6%) showed high Ab levels to E6 and 6 (35.3%) showed high Ab levels to CE2. Correlation coefficient between antibody detection by sight and ELISA for E7, CE2 and E6 were 0.6614, 0.4845 and 0.2372 respectively and correlation coefficient between lateral flow assay and ELISA for E7, CE2 and E6 were 0.3480, 0.1716 and 0.1644 respectively. This further proves patients or samples with HPV 16 oropharyngeal cancer have detectable antibodies to early E7, E6 and E2 proteins, which are potential biomarkers for HPV-associated oropharyngeal cancer.
Human papillomavirus (HPV) 16 proteins of interest, E7, E6 and CE2 were expressed and purified in E. coli for detection of specific antibodies using lateral flow assay because viral and host factors impact the serologic responses to HPV early antigens in HPV-positive oropharyngeal cancer. 17 samples and 5 controls with already known antibody reactivity from ELISA analysis were selected for HPV serologic responses. The lateral flow strip was evaluated for its color band intensity using Image J software. Peak area was used to quantify the color intensity of the lateral flow strip. Out of the 17 samples, 11 (64.7%) showed high antibody levels to E7, 12 (70.6%) showed high Ab levels to E6 and 6 (35.3%) showed high Ab levels to CE2. Correlation coefficient between antibody detection by sight and ELISA for E7, CE2 and E6 were 0.6614, 0.4845 and 0.2372 respectively and correlation coefficient between lateral flow assay and ELISA for E7, CE2 and E6 were 0.3480, 0.1716 and 0.1644 respectively. This further proves patients or samples with HPV 16 oropharyngeal cancer have detectable antibodies to early E7, E6 and E2 proteins, which are potential biomarkers for HPV-associated oropharyngeal cancer.
ContributorsLadipo, Evelyn (Author) / Anderson, Karen S (Thesis advisor) / Hogue, Brenda G (Committee member) / Hou, Ching-Wen (Committee member) / Arizona State University (Publisher)
Created2019