Study Of Human Papillomavirus Type 52 Virus-like Particle

Human Papillomavirus（HPV）are small double-stranded DNA virus that infect stratified epithelia,which mainly causes condyloma acuminata and cervical cancer.HPV52is the main type to cause condyloma acuminata.The development of prophylatic HPV52vaccine has prospects and benefits scially and economically.Studies have shown that virus-like particles（VLPs）in vitro reconstitution of HPV L1 protein retained its native conformation and immunogenicity,which is an ideal form of prophylatic HPV vaccine.At present,all prophylatic HPV vaccine on the market are in this form.This study use Escherichia coli to express HPV52 L1 protein in soluble form.It disassembles in vitro,reconstitute into VLPs,and bears with good immunogenicity.Firstly,the complete genome of HPV52 L1 was obtained from GenBank.The codon-optimized by preference of Escherichia coli,and then synthesized the complete genome.The expression vectors pET-30a-HPV52 L1 and pCold-HPV52 L1 were constructed.After double digestion,they were transforred to four competent cells by multilcus enzyme electrophoresis,which were ER2566,Tuner,BL21（DE3）and BL21Star^TM（DE3）.SDS-PAGE confirmed The BL21 Star^TM（DE3）was highly expressed.To explore the optimal conditions of expression,temperature was respectively set to 16°C,20°C,25°C,and IPTG concentration 0.025 mmol/L,0.05 mmol/L and 0.1 mmol/L.The expression time was 16 hours.SDS-PAGE confirmed that the expression condition was25°C,IPTG concentration was 0.1 mmol/L and induced time was 16 hours.Then HPV52 L1 recombinant protein was purified by POROS 50HS ion-exchange chromatography.The target protein was eluted with 1.25 mol/L NaCl elution buffer.SDS-PAGE and Western-blot comfirmed the highly puritied L1 recombinant protein.The purity of recombinant protein in eluent was over 90%by the near-infrared laser imaging system Odyssey.Then uniform and stable VLPs were obtained by in vitro disassembly and reassembly.Female BALB/c mice aged 6-8 weeks were immunized.Animal experiments showed that the VLPs vaccine had good immunogenicity and could induce HPV52neutralizing antibody with high titer.High purity HPV52 L1 protein was expressed in E.coli in this study,and cheap and effective HPV52 L1 VLPs were obtained.The study generated VLPs preparation at laboratory level,which provided a foundation for the research of prophylatic HPV52vaccine.