Abstract
Cervical cancer is the second death cause of all cancers in women. One screening result showed that HPV infection was 9 percent in Taiwan. The major cause of cervical cancer is human papillomavirus (HPVs) type 16 and 18, which consist of oncogenes E5, E6, and E7. E6 protein interacts with tumor suppressor p53 and promotes its degradation through an ubiquitin-dependent pathway. Therefore, E6 protein plays a significant role for malignant transformation. The HPV16E6 protein is about 18 kDa, and has been fully sequenced previously. Because of the absence of commercial available anti-E6 antibodies, it is reasonable to prepare E6 antigen from regular molecular cloning and produce anti-E6 antibodies after immunize E6 in animals to fulfill the purpose. Sense- and antisense- primers containing BamHI and SalI restriction sites in 5’-end and E6 gene containing plasmid-p1436 were used as PCR template. After E6 DNA fragments were obtained from PCR, they were treated by CIP (Calf-Intestinal Phosphatase) to remove 5’-phosphate groups. HPV16 E6 DNA fragment was subcloned into commercially available pET32a expression vector (from Novagen, contain thioredoxin and 6xhis fusion gene sequence). The pET32a-E6 recombinant vector were transformed into BL21 E. coli, and screened for suitable E6 containing candidates in using BamHI and SalI restriction digestion and PCR with E6 primers. Thirty clones were picked up on antibiotic-selective plates from three transformation experiments. Finally, there were 2 candidates (No.2 and No.6) with right base sequences and 2 candidates with wrong base sequences. E6 containing vector in No.2 BL21 E. coli showed most E6 containing fusion protein expression in IPTG- and antibiotic-containing LB medium. Large amount of purified E6 fusion protein from No.2 was collected from Ni-NTA resin elution in this study to serve as antigen in continuous immunization study in rabbits. Expected E6-antiserum was collected from rabbits after several E6 antigen injections into rabbits. The results in from Western blot and Cy3-fluorescent-immunoassay analysis showed that 5 ng and 1 ng E6 antigen could be detected with 1:1000 or 1:3000 dilution of antibodies-containing serum, respectively. From this study, it is expected that E6 antibodies can be used to examine E6 expression in various cells and tissues samples. Since the strong association of HPV16 E6 antibodies in patient’s sera with cervical carcinoma had been reported, it is also expected that E6 fusion protein from this study might contribute to the immunological monitoring of E6-specific antibodies with cervical cancer.