| Human Papillomavirus (HPV) is human double-stranded DNA tumor virus. It is replicated and assembled in the nuclear of cells. HPV infect mainly human cutaneous and mucosa in a strict speies-and tissue-specific manner and cause proliferative lesions. They are divided to two parts : high-risk and low-risk HPV accounding the different lesions caused by them . Epidemiological data and laboratory research demonstrated the relationship between HPV and occurrence,development of human malignancy tumor sufficiently, especially high relationship between HPV 16,58 and cervical cancer. Human Papillomavirus Type 16 (HPV16) and Type (HPV58) are very common in China. In order to decrease the occurrence of HPV infecting and cervical cancer, the vaccine against HPV infection and HPV associated tumor is needed. Because virions from natural lesions are scarce and it is not impossible to propagate HPV by animal infection or to obain abundant virions by in vitro culture systems, the availability of virus particles for vaccination studies is limited. This problem has been overcome by genitic engineering production of HPV subunit. Molecular biological studies demonstrated that the major capsid protein L1 or major protein L1 plus minor capsid protein L2 expressed in eukaryotic cells can ressamble into virus like particles (VLP). VLPs are structurally and antigenically similar to naturally occurring virious but without virus DNA. VLP preserves comformational epitopes from authentic virions , is highly immunogenitic and can induce preventative neutralizig antibodies in animals. VLP is considered to be the most prospective vaccine associated with HPV. But the antibody of one type can only inhibit the same type of HPV infection, different type cannot be inhibited. Therefore, it is necessary to make the vaccine that can prevent all of HPV types. There is no detailed report about VLP of HPV58 now, so we selected HPV58 to investigate the behavior of HPV58 VLP and discover the condition of making HPV58 VLP,immunological activity. Try to find out whether it can be used to prevent infection of HPV58. The L1 proteins, which expressed in E.coli, are not able to form VLPs. Even if some little VLPs are formed by complicated operations such as denaturalization, naturalization, re-denaturalization, re-naturalization, the re-naturalization rate is poorly low(0.02%-0.04). It is obviously unsuitable for vaccine development and research; also it is difficult to appropriately evaluate assembling efficiency of L1-VLP, molecular properties and immunogenicity of L1 and VLP. On the contrary, proteins expressed in eukaryotic expression system folded to natural conformation correctly and have biological activities, A large number of VLPs can be obtained during protein preparation. So we choose the eukaryotic expression system –baculovirus insect cell system to research.1. Construction of recombinant HPV58L1-and HPV58L1L2-baculovirus vectors. Using PCR technique, major capsid protein(L1) and minor capsid protein(L2) genes were separately amplified from the plasmid containing all of HPV58 sequence, and were inserting into pGEM-T easy vector and sequencing. It is coincidence with the sequence reported in gene bank NC-001443. We cut out 72 base from the C-end of HPV58 L1. The cloned HPV58L1 gene were subcloned into baculovirus transfer vector pFastBac-1 downstream of polyhedron(PPH ) promoter. The other, L1 and L2 were separately subcloned into the other baculovirus transfer vector pFastBacTM Dual downstream of PPH and PP10 promoter. The resulting recombinant baculovirus transfer vector were used to transform DH10Bac.through transposonmediated site-specific in vivo transposition foreign gene expression cassette was integrated into a baculovirus shuttle vector(bacmid) genome under help of transposase encoded by a helper plasmid in DH10Bac. The recombinant baculovirus DNA was isolated and used for transfection of Sf9 cells. The transfection was successful as judged by cell phenotype. After transfecting, the cells and the supernants were harvested. The baculovirus DNA were extracted and were used to confirm successful transfection and replication of baculovirus DNA. Finally,the recombinant baculovirus vectors expressing HPV58L1 protein and HPV58L1 plus L2 protein were created successfully. 2. Expression of HPV58 L1 and L1L2 in Sf9 cells and the VLP assembly Recombinant baculovirus Bac58L1 and Bac58L1L2 were used to infect Sf9 cells. When harvesting the cells after 72h post-infectionthat were infected at multiplicity of infection(MOI) of 10, SDS-PAGE and Western blot showed that L1 protein were mass expressed and can specially bind with anti-L1 ,with apparent molecular weight of 55kD. The cells infected by Bac58L1L2 can also express L2 protein with apparent molecular weight of 70kD. Western blot showed this protein can bind with anti-L2(made by 108-120 aa of L2 protein). This protein could be purified with CsCl and sucrose density gradient ultracentrifugation. The purified HPV58L1 alone and L1 + L2 protein could form VLPs , the VLPs could be observed with electron microscope and the VLPs were similar to the natural virus. Their density were 1.27g/ml in CsCl solutions. 3.The immunogenicity of recombinant HPV-58 VLP To evaluate VLP immunogenicity, purified VLPs were used to vaccinate 6 weeks BALB/c mice that were randomly grouped. Mice were vaccinated subcutaneously with 75ug and ajuvant twice. Negative control group were also treated similarly. Sera were prepared 7 days after the last vaccination. The titers of serum anti-L1 in L1-VLP and L1+L2-VLP vaccinated groups were above 1:1000 as detected by ELISA. 4 . Construction of HPV58L1L2 pseudovirions and neutralizing reactivity of immunized sera. HPV58L1L2 VLPs that had been dissembled with the reducing agent 2-ME were allowed to reassemble by the removal of 2-ME in the presence of closed circular plasmid DNA of pSV/?-gal, an expression plasmid for ?-galactosidase, purified by the ethidium bromide-CsCl buoyant density method. It could be observed with electron microscope and...
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