The Replication Of HPV-16 In HEK293T Cells And The Immunological Effects Of HPV-16 L1 Recombinant Virus Vaccine

Human papillomaviruses (HPVs) are nonencapsulated, small DNA viruses that induce epithelial neoplasias or malignant tumors. The circular HPV DNA genomes are approximately 8 kb in size and consist of early and late coding regions as well as sequences that regulate transcription and replication,the major regulatory region is referred to as the upstream regulatory region. HPVs have obvious species-specific, only infected the human being, and is closely linked to the differentiation program of the host epithelial tissue. The study of the papillomavirus life cycle has been hampered by the inability of these viruses to replicate to high titer. In this work,pTXJHPV-16 which contains Xinjiang HPV-16 genome was transfected into the HEK293T cells to discuss the replication of HPV-16 in vitro.The plasmid pTXJHPV-16 was transfected solely (or co- transfected with pcDNA3.1- L1L2 ) into HEK293T cells, or lined pTXJHPV-16 was transfected solely (or co- transfected with pcDNA3.1-L1L2) into HEK293T cells respectively, the VLPs of HPV-16 were find in the cells through electron microscope entirely, and the cells transfected with pTXJHPV-16 produced more VLPs. So, pTXJHPV-16 was transfected into HEK293T cells to investigate the replication of HPV-16 in vitro. PCR was performed to detect the statement of the virus DNAs; to confirm the stability and replication of and HPV-16 DNAs, southern blotting was used to analyze the total DNA extracted from cells transfected with pTXJHPV-16 in 2h、20h、40h、60h respectively; to detect the fresh HPV-16 DNAs replicated in the cell, DpnⅠdigested the total DNAs after transfecting 2h、20h、40h respectively, and then southern blotting analyzed the DNAs; and RT-PCR was used to detect HPV-16 E1、L1 and E1^E4 transcripts in the cells.The results showed E1 and E2 gene were detected by PCR, so HPV-16 existed in the cells as episome. HPV-16 DNAs in the cells reduced from 20h to 40h, which indicated some HPV-16 DNAs were digested in the cells. However, the quantity of HPV-16 DNAs began to increase after 60h, which suggested that HPV-16 DNAs can replicate in HEK293T cells. And the unmethylation HPV-16 DNAs had been detected with southern blotting after transfected 20 h, which further confirmed that HPV-16 DNAs can replicate briefly in HEK293T cells. The trincranscripts of E1, L1 and E1^E4 also were detected by RT-PCR, so HPV-16 genes can be expressed in HEK293T cells. All the results show that HPV-16 can replicate in HEK293T cells.HPV-16 L1 protein possesses a strong immunogenicity, and can automatically assemble to the VLPs, so it is an ideal target antigen for prophylactic cervical cancer vaccine. HSV-1 is an encapsulated DNA virus, and is excellent vector due to accommodate big foreign DNA fragment, infected broad host cells including dividing cells and nondividing cells efficiently. In this work, HPV-16 L1 gene was recombined into the HSV-1 vector to develop a HPV-16 L1 vaccine.In previous studies, we have obtained the recombinant HSV-1/BN which has deleted the inverted repeat sequences within the HSV-1 genome. In this work, HPV-16 L1 gene was cloned into the shuttle vector pKO5-B/N. Then the shuttle vector was electroporated into the E.coli cells containing the HSV-1 BAC (Bacterial artificial chromosome), the recombinant bacterium strains were screened through the properties of antibiotic resistance and biochemistry, and we got the recombinant DNA BAC-HSV-L1. Then BAC-HSV-L1 was transfected into Vero cells, the recombinant virus HSV-1/L1 was purified through plaque picking, and the titer of the virus was detecting. Then the BALB/C mice were immunized three times (interval 14d) through eyelid、intramuscular injection and intradermal injection with 1×10~5、1×10~6 and 1×10~6 pfu HSV-1/L1 respectively, and the BALB/C mice immunized with HSV-1/WT as control. The blood serums of the mice were collected at 14 d, 28 d and 42 d respectively, and then ELISA tested the level of IgG against L1.The results show that recombinant DNA HSV-BAC- L1 was correct by PCR and Southern blotting, HSV-1/L1 can infect and replicate in vero cells quickly, the titer of HSV-1/L1 come to 2.2×10~7 pfu/mL. The results of ELISA showed that the level of antibody against L1 from the mice immunized with HSV-1/L1 through eyelid is extremely higher than HSV-1/WT at 14 d (P < 0.01)、28 d (P < 0.001) and 42 d (P < 0.001), which suggested that HSV-1/L1 can stimulate humoral immune response through eyelid. However, both of intramuscular injection and intradermal injection with HSV-1/L1 didn’t stimulate humoral immune response. The results provide the basement to develop the HSV-1 vaccine vector.