Proceeding2562

1567 การประชุมวิชาการระดับชาติมหาวิทยาลัยทักษิณ ครั้งที่ 29 ประจ�ำปี 2562 วิจัยและนวัตกรรมเพื่อการพัฒนาที่ยั่งยืน Introduction Persistent infection of High-risk Human papillomavirus (HR-HPV) is the major causative agent of cervical cancer among women worldwide (1). HPV type 16 (HPV16) is the most common HPV type which involves in cervical carcinogenesis approximately 50-70%. Oncoproteins of HPV, E6 and E7 inactivate p53 and retinoblastoma protein (pRb) respectively. E6 incorporates with cellular protein, E6AP and degrades p53 via ubiquitination pathway. Whereas E7 can inactivates pRb and release E2F which can drives cell cycle progression. A tumor suppressor protein, p16 INK4a or cyclin-dependent kinase inhibitor 2A (CDKN2A) involves in cell cycle checkpoint by inhibit complex of cyclin D with CDK4 or CDK6 (CyclinD-CDK4/6 complex). This complex phosphorylates pRb and release free E2F, follow with cell cycle progression(2). Overexpression of p16 INK4a cannot arrest cell cycle from degradation of pRb by E7 (3). Overexpression of p16 INK4a is used as a biomarker of high risk HPV-associated cervical lesion progression. Cellular p16 INK4a in abnormal cells is recognized as tumor antigen. Infiltrated lymphocytes isolated from cervical neoplasia tissues were activated with p16 INK4a epitope and antibodies also detected. These evidences exhibit the p16 INK4a has immunogenicity and stimulate both cellular and humoral immune responses. Anti-p16 INK4a antibodies can be found in patients with cervical lesion with higher cervical severity (HSIL and cervical cancer). Cervical cancer screening with Pap test has low sensitivity and false negative around 6-55 %. Primary screening test is Pap smear with HPV DNA is a more sensitive screening test for cervical cancer (4). These procedures have limitation from sample collection procedure which perform by gynecologist, observe cell morphology with cytologist and higher cost of equipment. The equipment used is very expensive. In this study, we developed a method for detect anti-p16 INK4a antibodies from serum of patients with cervical lesions with enzyme-linked immunosorbent assay (ELISA). This method is high- throughput, gives high sensitivity and specificity. Moreover, the level of p16 INK4a antibodies can be quantified. This can indicate the severity of the disease to the level of the antibody, and the information obtained from this study may be used as a guideline for the development of detect anti-p16 INK4a antibody and used as a biomarker for cervical cancer progression. Materials and methods Histidine-tagged p16 INK4a protein expression Escherichia coli BL-21 strain containing pTrcHisA-p16 INK4a plasmid which previously analyzed by Western blot was grown in LB agar with 100 LB agar with 100 µg/ml of ampicillin. A single colony was picked and inoculated in LB broth 100 µg/ml ampicillin and incubated overnight at 37 °C. Two milliliters of overnight culture LB broth was transferred to 200 ml of fresh LB broth and grown at 37 °C in shaking incubator until the OD 600 of culture broth raise to 0.4-0.6 (mid-log phase). Mid-log phase bacteria were used for protein induction with final concentration of 0.1 mM of isopropyl β -D-1-thiogalactopyranoside (IPTG). Bacteria were further incubated in shaking incubator for 6 hours, collected and wash bacterial cell pellets with sterile phosphate-buffered saline (PBS) pH 7.4 and stored in -20 o C until perform protein expression. Protein extraction was performed by freeze and thaw bacterial cell pellet at -20 °C three times. B- PER ® bacterial protein extraction reagent (Thermo scientific) 5 ml was added per 1 gram of bacteria cell pellet, vortexed homogenously. Cell pellet was incubated at room temperature for 15 min on gently rotator. Crude protein extraction was centrifuged at 4,000 g for 20 min, transferred supernatant to new centrifuge tube. Whole protein extraction was purified with Hispur ® Ni-NTA spin column (Thermo scientific) for