Expression of Genetically Modified Human Papillomavirus Type 58 L 1 Gene and Production of Antisera Against Resultant VLP

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1 ISSN CN ΠQ Chinese Journal of Biochemistry and Molecular Biology (7) : HPV 58 L1 VLP,,,,,, 3 (, ) (human papillomavirus, HPV). HPV (L1) (virus2like particle, VLP),,,. HPV 58, HPV 58. HPV 58 L1, HPV 58 L1, HPV 58 ml1, 2 HPV 58 ml1,cscl HPV 58 ml1,, 55 nm VLP.,ELISA, HPV 58 ml1 VLP,, VLP. HPV 58 ml1 VLP, HPV 58 ml1 VLP, HPV ; ; ; R37319 ;Q786 Expression of Genetically Modified Human Papillomavirus Type 58 L 1 Gene and Production of Antisera Against Resultant VLP FAN Dong2Sheng, ZHANG Ting, PENG Qing2Lin, YANGLi2Jun, XU Yu2Fei, ZHANG Ran, XU Xue2Mei 3 ( Department of Biophysics and Structural Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and School of Basic Medicine, Peking Union Medical College, Beijing , China) Abstract Persistent infection with high2risk types of human papillomavirus has been proved as an important etiological factor for cervical cancer. HPV major capsid protein (L1) can self2assemble into VLPs when expressed in a recombinant expression system. VLP can induce high levels of type2specific neutralizing antibodies to efficiently prevent the infection of the same genotype HPV and eventually prevent cervical cancer and other infection related diseases. HPV type 58 is one of the most common high2risk HPVs in Chinese cervical cancer patients. However, there is no effective vaccine to prevent HPV 58 infection so far. The HPV 58 L1 wt gene was modified by several strategies. The modified gene, named as HPV 58 ml1, was expressed by Baculovirus Expression System, and the recombinant protein was purified by CsCl ultracentrifugation. The transmission electron microscopy analysis showed that the recombinant HPV 58 ml1 protein could self2assemble into regular VLP with a diameter of about 55 nm. New Zealand white rabbit and guinea pigs were subcutaneously immunized with HPV 58 ml1 VLP. VLP ELISA showed that HPV 58 VLP could induce high titers of HPV 58 ml1 VLP specific antibodies. Dot blot assay indicated that the sera antibodies were against the surface epitopes of VLP. Taken together, HPV 58 ml1 VLP was produced by Baculovirus Expression : ; : (863,No. 2007AA02Z475) (No ) 3 Tel : ; E2mail : sina. com Received : February 15,2009 ; Accepted : April 3,2009 Supported by National High Technology Research and Development Program of China ( 863 Program, No. 2007AA02Z475) and National Natural Science Foundation of China (No ) 3 Corresponding author Tel : ; E2mail : sina. com

2 System and antisera were obtained from two different species, which were important for further developing effective HPV 58 VLP based prophylactic vaccines. Key words human papillomavirus type 58 ;virus2like particle ;gene modification ;antisera (human papillomavirus, HPV) DNA,,, 118 [1 ], HPV 15, HPV [2 ],, [1 ]., 50, 27., 416, 216 [3 ]., HPV [4 ], HPV., HPV L1 L1 (L2) (virus2like particle, VLP),VLP,, HPV [5 ]. HPV L1 VLP, HPV ( HPV 18 45,HPV ), HPV L1 VLP [1 ].,, HPV. 25 ( ) 3607, 3 HPV 16 ( 5315 %), 18 (1712 %) 45 (617 %), HPV58 7 (212 %) [1 ], 5954, HPV %, HPV 16 (5214 %) 18 (1415 %) [6 ], ,HPV % [3 ],, HPV 58. L1 VLP HPV, Gardasil ( HPV 16Π18Π6Π11 L1 VLPs) Cervarix( HPV 16Π18 L1 VLPs) HPV, HPV 58 L1 VLP. VLP HPV. HPV 58 ml1, 2 HPV 58 ml1 VLP, HPV 58 ml1 VLP, HPV 58 L1 VLP pfastbac1 E. coli DH10Bac Sf9 Invitrogen, HPV 16 ml1 ( HPV 16 L1, L1 C 31 ) SF2900II SFM Cellfectin Reagent Invitrogen ;CamVir21 IgG IgG IgG ;CsCl Amerasco ; R250 ; 300 ; BCA Pierce ; Corning ; ECL Transgen ,,6 8, 212 kg, ; 2,,6 8, 300 g,. 114 HPV 58 L1 HPV 58 L1 (D90400), C 25, : Sf9 ; mrna,,, HPV 58 L1, HPV 58 ml1. HPV 58 ml1. Kozak, HPV 58 ml1 pfastbac HPV 58 ml1

3 7 :HPV 58 L1 VLP 679 Bac2to2Bac 2, pfastbac1πhpv 58 ml1 E. coli DH10Bac, PCR, HPV 58 ml1 Bacmid (rbacmid). rbacmid, Cellfectin Reagent, Sf9. 3 d, 2, Sf9., 10 % SDS2PAGE, Western HPV 58 ml1, Sf9, HPV 16 ml1, CamVir21 ( ),ECL. 116 HPV 58 ml1,4,800 rπmin 6 min,,,4, CsCl VLP [7 ]. Western 10 mmolπl HEPES(pH 712),BCA. 5 g VLP 10 % SDS2PAGE, VLP. 20 l VLP 300,1 min, 20 l 1 %, J EM21010, VLP g HPV 58 ml 1 VLP 500 l,,, ; 20 g HPV 58 ml1 VLP 200 l,,, ,,. 1 1,, ELISA,,, IgG ELISA 100 ng HPV 58 ml1 VLPΠ 96,,,, IgG IgG, , OPD,37 5 min, 2 molπl H 2 SO 4, 490 nm. A HPV 58 ml1 VLP 014 g VLP, VLP 95 5 min, 5 % TBST 1 h, HPV 58 ml1 VLP HPV 58 ml1 VLP, ,4, TBST 3, IgG IgG, , 1 h,tbst 3,ECL HPV 58 ml1 Western Western, HPV 58 ml1 Sf9 53 kd, HPV 58 ml1, HPV 16 ml1 Sf9, Sf9 ( Fig. 1), HPV 58 ml1. Fig. 1 Western blot assay of HPV 58 ml1 protein expression in Sf9 cells Lysate from Sf9 cells (lane 1), Sf9 cells infected with rbaculovirus HPV 16 ml1 (lane 2) or HPV 58 ml1 (lane 3) were separated by 10 % SDS2PAGE. Proteins were transferred onto PVDF membrane and probed with CamVir21 mab at a dilution of The membrane was incubated with goat anti2mouse peroxidase2conjugated secondary antibody at a dilution of and processed by the enhanced chemiluminescence detection method. Protein markers are shown on the left. The position of the L1 protein (53 kd) is indicated by the arrow on the right 2. 2 VLP,5 g L1, SDS2PAGE, 53 kd, (Fig. 2),, 90 % , HPV 58

4 Fig. 2 Purified HPV 58 ml1 analyzed by 10 % SDS2PAGE with Coomassie blue staining Protein marker (lane 1) and purified HPV 58 ml1 ( lane 2) were separated by 10 % SDS2PAGE and stained with Coomassie blue. The 53 kd HPV 58 ml1 band is indicated by the arrow ml1 55 nm, (Fig. 3), HPV, HPV 58 ml1 VLP. Fig. 4 ELISA of antisera from New Zealand white rabbit and guinea pigs immunized with HPV 58 ml1 VLPs New Zealand white rabbit or guinea pigs were immunized three times with 50 g HPV 58 ml1 VLP or four times with 20 g the VLP, respectively. The post2immune sera were detected by VLP ELISA. ( A) The anti2hpv 58 ml1 VLP antibody titer of antiserum from New Zealand white rabbit. (B) The anti2hpv 58 ml1 VLP antibody titers of antisera from guinea pigs Fig. 3 Electron micrograph of purified HPV 58 ml1 VLPs Grids were stained with 1 % uranyl acetate and examined with a J EM 1010 electron microscope at magnification (bar = 100 nm) 2. 4 IgG HPV 58 ml1 VLP 96,ELISA HPV 58 ml1 VLP IgG. Fig. 4A 4B HPV 58 ml1 VLP IgG , HPV 58 VLP HPV 58 ml1 VLP, , HPV 58 ml1 VLP,, HPV 58 ml1 VLP, VLP (Fig. 5), VLP. 3 HPV 72 L1 12 L2 T = 7, 60 nm. HPV,, [8 ].,L1,, L1, T = 1 T = 4 VLP( L1 ), T = 7 VLP [9 11 ]. L1

5 7 :HPV 58 L1 VLP 681 Fig. 5 Dot blot assay Purified intact (1 and 3) and denatured HPV 58 ml1 VLPs (2 and 4) were directly loaded onto the nitrocellulose membrane. (A) Proteins were probed with the antiserum from New Zealand white rabbit, followed by goat anti2rabbit peroxidase2conjugated secondary antibody. (B) Proteins were probed with the mixture of antisera from guinea pigs, followed by rabbit anti2guinea pig peroxidase2conjugated secondary antibody. Both of membranes were detected by the enhanced chemiluminescence detection method, L1 L1 VLP [12 ]. VLP, T = 4 [10 ], VLP, T = 7 [11 ],., L1 VLP. HPV 58 L1,. 2, HPV 58 ml1, HPV 58 ml1 VLP.,L1 C 30,, DNA, [1 ], C, BPV L1 C 24 [13 ], HPV 16 L1 C 30 [9 ] VLP, L1 [13 ].,, mrna., HPV 58 L1,, Sf9 mrna Kozak HPV 58 L1 C 25, HPV 58 ml1, 2. CamVir21 HPV 16 L1 (L1 a. a GFGAMDF) [14 ], HPV, HPV 58 L1 1, HPV 58 L1 VLP, CamVir21 Western HPV 58 ml1, HPV 58 L1 [15 ]. HPV 58 L1 VLP, HPV 58 L1 VLP, HPV 58 L1 VLP. L1 VLP HPV 58, Cervarix HPV % ( 9719 % CI, % 2417 %) [16 ], Gardasil HPV 16 HPV % (95 % CI, 8 % 52 %) [17 ], HPV HPV 58. VLP,,. 75 g HPV 58 L1, 0 1 5, [15 ], 50 g 20 g HPV 58 ml1 VLP, VLP IgG , HPV 58 ml1 VLP, VLP, VLP. HPV 58 L1.,, T = 7 VLP [18 ], T = 1 VLP [9 ], L1 [12 ],, L1

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