| Chronic persistent infection of high-risk HPV is the major etilogical factor of cervical cancer. Thus, the effective vaccination against HPV represents an opportunity to restrain cervical cancer and other important cancers.The genomic organization of HPV is quite conserved among various types of papillomaviruses, HPVs are non-enveloped, double-stranded, circular DNA the genome of the family Papovaviridae viruses encodes six "early"proteins (E1, E2, E4,E5, E6and E7) and two "late" proteins (L1and L2).The virus-like particles (VLPs) of HPV have two capsid proteins, major capsid protein L1and minor capsid protein L2.L1alone or L1and L2co-expression can be Self-assembled into virus-like particles (VLPs) which does not contain virus DNA. The structure of VLPs is similar to natural virus particles. The VLPs have complete virus antigen epitope in animal models and in human body, induce high titers of neutralizing antibodies protecting the organism. L1capsid protein can spontaneously assemble to form empty capsid, virus-like particles (VLPs) in mammals, insects, yeast, or bacterial, can be expressed, it is a method to carry out preventive vaccine’s research.Each year about500000women are diagnosed with cervical cancer,80%of which are from developing countries. The most highest incidence of HPV types in the developing countries are HPV16,18,33,58, The incidence of which are 54.7%,16.3%,7.1%and5.2%，respectively. There are132,000new cases every year in China, about30000women died from cervical cancer. The analysis of HPV serotypes in infed Chinese women showed that the infection of HPV16,18,58and33are most closely related to the occurrence of cervical cancer. Currently, there are two commercial HPV prophylactic vaccines which are VLP hybrid vaccines against the L1protein. The vaccine named Gardasil contains HPV16/18/6/11VLPs, and the vaccine named Cervarix contains HPV16/18VLPs, respectively. Both of them are designed for Europe and North America according to the distribution characteristics of HPV in cervical cancer,but they do not include HPV33and58genotypes. Therefore, these two Hybrid VLP vaccines can not fully meet the Chinese population needs to prevent HPV-related disease.In this study, our expression system of recombinant baculovirus successfully expressed the HPV16/18/33/58major capsid protein LI, and virus-like particles (VLP) can be seen by electron microscopy.In the report, genes encoding the L1proteins of HPV16/18/33/58serotype are successfully cloned, and modified by a variety of strategies. The modified genes were inserted into the pFastBac Dual vector and the recombinant bacmid is generated after the E.coli DH10Bac competent cell is transformed by the recombinant pFastBac Dual vector, recombinant baculovirus are transfected into insect cell by Lipofection method.It obtaines recombinant baculovirus which express the L1proteins of HPV16/18/33/58serotypes.Collect the recombinant baculovirus, and determine the viral titer by Viral Plaque Assay. Then amply viral stock by infectting cells at a multiplicity of infection (MOI) ranging from0.1to0.5to reach a high titer, and infecte Sf9insect cells with high titer virus, Western blotting and SDS-PAGE analysis showed that the L1protein of the HPV strains were successfully expressed when the insect Sf9cells were infected by the recombinant baculovirus, and the L1proteins were found distributed mainly in the cell. We optimized time and MOI of protein expression to obtain the high expression of L1protein.In the electron microscope, the formation of VLP of HPV16/18/33/58L1protein showed a diameter of about55nm virus-like particle, and is Similar to natural HPV virus particle’s size and shape by electron microscopy.High expression of L1protein successfully for China’s independent production of HPV vaccines provides a feasible basis. |
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