Cloning Of The Whole Genome Of Bovine Papillomavirus13Hainan Strain And Eukaryotic Expression Of E5Protein

Bovine papillomatosis (BP) is an infectious disease caused by the infection of bovine papillomavirus (BPV), which is characterized by chronic proliferative tumors in the mucosa and surface. This disease is the commonest tumoral disease in cattle, which lead to the death of the animal, and eventually cause serious economic loss of meat, dairy and leather industries. Unfortunately, there is no effective vaccine against bovine papilloma diseases. E5protein is a major oncoprotein of BPV. It functions by interfering the inhibition of cell growth and the regulation of cell cycle, interacting with growth factor receptor and activating cell proliferation. BPV E5protein involves in cell transformation and it is expressed in the initial and the entire infection process, altering normal cellular response to growth factors, inhibiting the expression of cell surface major histocompatibility complex. Because of all these effects, E5is of great significance to the development of DNA vaccines. Therefore, the research about BPV functional proteins will lay a foundation for the future development of BPV vaccine. Moreover, with the increase in the incidence of human condyloma acuminata, cervical cancer and anal cancer, HPV infection has attracted more and more attention. BPV has an enormous value as an in vivo model for the study of HPV infection, its interaction with the host and with environmental co-factors, providing theoretical foundation for the detection and treatment of HPV infection.In this study, we cloned the whole genome of BPV-13Hainan strain. The pEGFP-N1-E5recombinant eukaryotic expression vectors were constructed successfully; E5recombinant protein was expressed in HEK293cells, which was confirmed by fluorescence observation, FCM (Flow Cytometry Method) detection, and Western blot. The results laid a preliminary basis for prophylactic and therapeutic vaccines research of cattle papillomatosis.Here are the methods and results:1. Genotyping and cloning and of the whole genome of bovine papillomavirusWe extracted virus DNA from cattle lesions tissues which were detected by PCR with degenerate primers targeting the conserved region of L1gene. Genotyping of BPVs was conducted by comparing the different DNA sequences. The results showed that the genotype of the virus from the sick cattle belongs to BPV-13type, and it was named BPV-13-HN. Based on the results,6pairs of specific primers of BPV were designed to amplify the whole genome of BPV-13-HN. The genome of BPV-13-HN contains7961bp, including8open reading frame (E1、E2、E4、E5、E6、E7、L1、L2).2. Expression of E5protein of BPV-13-HN in HEK293cellsBPV-13-HN E5gene was amplified and cloned into eukaryotic expressing vector pEGFP-N1. The recombinant vector was transfected into HEK293cells and the expression of E5gene was detected by fluorescence microscope, FCM and Western Blot. The result showed that the recombined eukaryotic expressing vector was constructed successfully, and E5protein was expressed, which lay the foundation for the functional investigation of E5.