| Human papillomavirus(HPV),one of the oldest known families of viruses,can cause epithelial hyperplasia and a variety of cancers,including cervical and head and neck cancers.HPV virus particles have a conservative icosahedral structure.Studies have shown that exogenously expressed HPV major capsid protein(L1)can be spontaneously assembled into virus-like particle(VLP),with the main neutralization epitopes of HPV on its surface and a high degree of immunogenicity.Currently,HPV prophylactic vaccines use HPV L1 VLP as their main antigenic component.Since the high cost and high price of the vaccine is the main factor limiting its application in developing countries,it is of great significance to develop a low-cost and polyvalent HPV prophylactic vaccines.In addition,adjuvants are also a key factor affecting vaccine immunity.Therefore,this paper intends to optimize the prokaryotic expression conditions of HPV6 L1 protein to improve its soluble expression,and carry out adjuvant screening to improve the immunogenicity of the vaccine,so as to provide experimental data for the development of HPV VLP prophylactic vaccines.In this study,we first constructed a prokaryotic expression plasmid pET30a(+)-6L1.In order to determine the best optimal soluble expression conditions,we optimized the amount of inducer,the inducing temperature and the co-expression molecular chaperone.Then three adjuvants(Alum-Phosphate,Alum-Hydroxide and Nano-Alum)were immunized with HPV6 L1 VLP,and subsequently subjected to immunological evaluation by using ELISA and neutralization assay.The results showed that:(1)Suitably reducing the rate of protein synthesis could increase the soluble expression of the target protein.When the concentration of IPTG was 0.1mM and the induced temperature was 25?,the soluble expression of HPV6 L1 protein was higher.(2)Theco-expression of molecular chaperone TF significantly increased the soluble expression of HPV 6L1 protein.(3)All vaccines provided long-lasting protection,among which Alum-Hydroxide adjuvant group significantly displayed higher antigen-specific immunoglobulin G(IgG)titers and neutralizing antibody titers.Immunogenicity evaluation is an important part of HPV vaccine clinical research,but the traditional neutralization assay is complicated.Currently,HPV pseudovirusbased neutralization assay utilized at home and abroad makes use of pseudovirions encapsidated reporter gene(EGFP).When it comes to detecting nine-valent immune serum,it'll pass nine separate tests by using this assay,which is complicated and time-consuming.In this study,three kinds of fluorescence proteins(EGFP,mTagBFP2 and mRFP)with different excitation and emission wavelengths were selected as reporter genes,co-transfected with three types of HPV structural genes(p6shell,p16 shell and p52shell)to prepare tricolor fluorescent pseudoviruses(PsVs).In order to evaluate the accuracy of the tricolor mixed fluorescent pseudovirus detection system,we used the single-and triple-color fluorescent PsVs(PsV6G,PsV16 B,and PsV52R)to detect the serum neutralizing antibody titers of mice immunized with HPV 9-valent vaccine.The results showed that:(1)The coefficient of variation(CV)within the plate was less than 10% and the CV among the plates were less than 20%,indicating good repeatability of the tests.(2)There was no statistical significance(P>0.05)in the neutralizing antibody titers of single-and triple-color fluorescent PsVs detecting assays,indicating that the two methods were consistent,and there was no interference in the detection serum after using the mixture of tricolor fluorescent pseudovirus.In conclusion,the tricolor mixed fluorescent pseudoviral neutralization can be applied to the detection of HPV nine-valent immune serum neutralizing antibodies,which paves a new way for the establishment of a high-throughput HPV pseudoviral neutralizing antibody detection method,laying a solid foundation for the efficient and rapid detection of HPV nine-valent vaccine clinical serum. |
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