| Human Papillomaviruses(HPV)belong to the family of Papillomaviruses,the genus Papillomaviruses,and is a type of double-stranded DNA viruses with protein capsids.HPV virus was divided into more than 200 subtypes,high-risk HPV infection will greatly increase the risk of cervical cancer in women.Cervical cancer claims to be the second biggest killer of women only to breast cancer.In addition,HPV infection can also lead to genital cancer,oral cancer,throat cancer,and genital warts and other diseases.L1 protein is the main capsid protein on HPV virus,accounting for 80%to 90%of the total proportion of HPV capsid protein.It can self-assemble into virus-like particles and shows good immunogenicity.The HPV vaccines on the market are all based on L1 particles.However,L1 protein is type-specific,which makes it difficult to develop broad-spectrum HPV vaccine.L2 protein is the monor capsid protein on HPV virus and is associated with virus infection,nucleation and assembly after maturation.L2 protein contains the broad spectrum epitope of HPV with less type-specificiy,which has the potential to induce the body to produce cross-type antibodies and provide cross-protection.Therefore,the structure of L2 protein is of great significance for the further study of the infection mechanism of HPV and the development of HPV broad-spectrum vaccine.In order to study the structure and function of L2,this study was tried to focus on the preparation of the L2 VLPs,pentamer and truncated protein.Firstly,we carried out the expression the interacting L1&L2 VLPs in BEVS.L1 protein and L2 protein were expressed through co-infection and co-expression pathways respectively.However,we found there were no apparent interaction between L1 and L2 proteins and L2 protein was largely existed in inclusion bodies.Thus it can be inferred that L1 and L2 proteins fail to form interaction effectively after they were expressed simultaneously in insect cells.Next,this study introduced cysteine point mutations on L1 and L2 to form disulfide bond,respectively,in the expectation that the two cysteine sites mutations enhanced the interaction when L1-Q317C and L2-E338C were expressed simultaneously.Follwing results showed that L2 protein could be detected in the VLPs expressed in the co-infection pathway,but the yield of L2 was extremetly low.Furthermore,we found that the existence of L2 could increase the diameter of VLPs compared to other LI VLPs.The introduction of mutant sites might be substantilly effective.However,the results of immunogenicity evaluation showed that the immunity of this VLPs failed to induce high-titer of L2 neutralizing antibodies in mice.This may be related to the fact that L2 is present in the VLPs lumen and rare neutralization epitopes were exposed.Because of the heterogeneity of VLPs,the expression of pentamer forms by intercting L1 and L2 protein was then carried out.After the identification by SDS-PAGE/WB/HPLC/TEM,it was confirmed that 16L1 protein with double point mutation at 175 and 428 receptively could be expressed in insect cell,and could self-assembly to form HPV-L1 pentamer.We futher introduced new mutation sites on L2 protein:T421C,in expect to strengthen the interaction between L1 and L2 protein.From the subsequent results,it was found that the pentamer didn't contain L2 protein.Finally,we tried to design and prepare the truncated L2 protein.After the first round design of truncation,the truncated L2 protein was firstly purified by Co affinity column.The results showed that the of L2 protein in eluted sample was low-purity and could not be used in structural analysis.In the second round design,six of the truncated proteins were purified by Co column,one of these proteins,L2-140-340,showed high purity in one-step elution,and it might be exist as trimer and monomer with strong interaction inferred from SDS-PAGE.In sum,the L2-140-340 needs more physicochemical property identification and might be a promising candidate to carry out caystalllization for structure determination and futher vaccine research. |
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