The Recombinant Protein Expression Of Human Papillomavirus Type 16, Type 18 And Monoclonal Antibody Production

Cervical cancer is the second most common cancer in female patients in the world, with high morbidity and mortality. The occurrence and development of cervical cancer in women is due to the infection of high risk type of human papillomavirus (HPV) in the long term. HPV is the most widely spread sexually transmitted virus and there are more than 100 variants of HPV virus. In the world, cervical cancer is caused by a maximum of type 16 and type 18, which two contributing for more than 70%. And the other common low risk type6 and type 11 can cause male genital warts.HPV is a class of dsDNA virus with no capsid. The genome is a ring, consist of about 8000 bp. The genome encodes 8 proteins, including six early proteins, two late proteins. Protein E6 plays a crucial role in the process of HPV infection of cervical squamous epithelial cells, and translating host cells into tumor cells. Protein E6 is expressed in each stage of cervical intraepithelial neoplasia (CIN) and gradually increased with the course of development. Studies have shown that, the positive rate of protein E6 in CIN I, CIN II, CIN III is 75%,88.25% and 100%, respectively. Therefore, the detections of HPV types 16 and 18 E6 gene and gene products can be important indicators for the early detection of cervical cancer and cervical cancer progression. Current methods to detect HPV in the market are molecular biology methods, including nucleic acids hybridization methods, signal amplification based methods as Digene’s HC2 kits, and nucleic acids amplification methods. The disadvantages of Nucleic acids hybridization methods are low sensitivity, requiring a lot of high purity DNA samples, time-consuming procedures, and not for distinguishing different types. While the nucleic acids amplification methods requiring small amount sample, are high sensitivity for distinguishing different types. But the detection result is prone to false positives due to HPV transient infection. Besides, the process of amplifying nucleic acids leads to the detection more environmental requirements, longer time consuming and more testing costs. But methods targeting protein E6 of HPV type 16, and type 18 are not only higher sensitivity, lower cost, better repeatability, but also reflecting the reaction of the virus’ activity, which may be effective in clinical diagnosis of cervical lesions.In this study, firstly we amplified type 16, type 18 E6 protein gene from extracted cervical lesions HPV genomic DNA with the use of designed specific primers. Secondly, protein E6 gene of HPV type 16 and type 18 were constructed into two expression vectors PET28a and PET32a by molecular methods. The proteins were prokaryotic expressed in BL21 (DE3) and purified using a nickel column. Finally, Babl/c mice were immunized with the purified and refolding proteins. By titer testing, we select high titers PET28a-HPV16 E6 and PET28a-HPV18 E6 two proteins immunized mice for next step work, including cell fusion, screening cloning, and preparation of the corresponding monoclonal antibody. Eventually we screened monoclonal antibody HPV18 E6/3-7D cell lines, which can be specific for HPV18 E6 recombinant protein. We found that the antibody does not cross-react with HPV16 E6 protein, protein of expression system and HepG2 cell protein.The prepared specific monoclonal antibodies against HPV 18 E6 protein should be further validated using clinical sample in the next step. Furthermore, we can use the monoclonal antibody for developing a novel immunoassay, combined with chemiluminescence or nano technology.