Expression of human papillomavirus 6b L1 protein in silkworm larvae and enhanced green fluorescent protein displaying on its virus-like particles
© Palaniyandi et al.; licensee Springer. 2012
Received: 19 July 2012
Accepted: 2 October 2012
Published: 4 October 2012
Human papillomavirus (HPV) 6b L1 capsid protein was expressed using the Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid expression system in silkworm larvae. Two constructs, full-length L1 (500 a.a) and C-terminal-deleted short L1 (479 a.a), and three PCR-manipulated antigenic loops at amino acids 55–56, 174–175, and 348–349 regions were incorporated with whole enhanced green fluorescent protein (EGFP). Expressed in full, short L1 proteins and variants were purified in heparin affinity column chromatography and confirmed by SDS-PAGE and western blot. The presence of self-assembled virus-like particles (VLPs) and EGFP incorporation on the surface of VLPs were confirmed by the observation of transmission electron and immunoelectron microscopies, respectively. HPV 6b L1 major capsid protein was successfully expressed in silkworm, and effective manipulation on the antigenic regions showed the path to versatile vaccine development based on HPV L1-VLPs.
Virus-like particles (VLPs) are empty virus particles, which lack virus-derived genome DNA or RNA, and composed of virus-capsid or matrix proteins. A non-enveloped virus, VLPs are mainly composed of virus-capsid proteins, which can be self-assembled both in vitro and in vivo. VLPs have been utilized as a vaccine because of its high immunogenicity and capability of inducing cellular and humoral immune responses ([Grgacic and Anderson 2006]). Alternatively, VLPs can be used as functional nanoparticles for drug delivery system, cell and tissue imaging by modification of the surface of VLPs and incorporation of functional materials into VLPs (Georgens et al. ).
To modify the surface of VLPs, chemical and genetic modifications have been adopted (Ma et al. ; [Pokorski and Steinmetz 2011]). An enhanced green fluorescent protein (EGFP) variant and outer surface protein C (OspC) from Borrelia burgdorferi were inserted into the immunodominant c/e1 epitope region of hepatitis B virus capsid protein without the disruption of VLP’s shape (Kratz et al. ; Nassal et al. ). In general, it is difficult to display a whole full-length protein on the surface of VLPs by genetic modification.
In this study, human papillomavirus (HPV) 6b L1 protein was expressed in silkworm larvae, and HPV 6b L1-VLPs were purified from the fat body of silkworms. HPV is a non-enveloped virus that has approximately 50–60 nm in diameter and ~8 kbp of double-stranded circular DNA genome. HPV L1 protein can be self-assembled as a VLP when this protein is expressed in various expression systems (Trus et al. ). To display this whole protein on the surface of HPV 6b L1-VLPs, EGFP, as a model of a whole full-length protein, was inserted into BC, EF, and HI loop domains (Bishop et al. ; Chen et al. ) of HPV 6b L1 protein. EGFP-incorporated HPV-VLPs were analyzed and discussed targeting for using alternative vaccine candidate.
Results and discussion
Expression of HPV 6b L1 protein and its short variant in silkworm larvae
Expression of L1 protein inserted with EGFP at its loop domain
Three L1-EGFP fusion proteins did not have green fluorescence, but EGFP was displayed on the surface of its capsomeres. To date, a genetic whole protein display system on the VLP has not been established, except for hepatitis B virus nucleocapsid protein VLP (Kratz et al. ; Nassal et al. ). These results suggest that this whole protein display system using HPV L1 protein can be applied to develop new vaccines against infectious diseases, and HPV L1 protein can serve as a potential vaccine template.
Construction of recombinant BmNPV bacmids
Primers for amplification of the HPV L1 gene and chimeric variants
5’ – 3’
HPV 6b L1-F
HPV 6b L1-R
HPV 6b L1 short-R
Eco-HPV 6b L1-F
HPV 6b L1-55R-LINKER
HPV 6b L1 LINKER-56F
Kpn-HPV 6b L1-R
HPV 6b L1-174R-LINKER
HPV 6b L1 LINKER-175F
HPV 6b L1-348R-LINKER
HPV 6b L1 LINKER-349F
Each L1-EGFP fusion gene was obtained by two-step PCR. To obtain L1-EGFP55, two truncated L1 genes coding a.a.1–55 and 56–500 were amplified by PCR using primers Eco-HPV 6b L1-F and HPV 6b L1-55R-LINKER and HPV 6b L1 LINKER-56F and Kpn-HPV 6b L1-R (Table 1), respectively. In addition, an EGFP gene was amplified by PCR using primers LINKER-EGFP-F and EGFP-R-LINKER (Table 1). A second PCR was performed using the amplified EGFP gene as a template and the two amplified truncated L1 gene as primers to obtain L1-EGFP55 fusion gene. The EGFP gene was inserted between 55 and 56 amino acids in an L1-coding gene. Moreover, linker-region coding sequences (GGGGSGGGGS) were also added between L1 and EGFP genes. By this two-step PCR, L1-EGFP174 and L1-EGFP348 were obtained by PCR using primers (Table 1). Each amplified fusion gene was inserted at Eco RI-Kpn I site in pFastBac1. The recombinant BmNPV CP- bacmid containing each L1-EGFP fusion gene was constructed according to the protocol described above.
Expression of HPV 6b L1 full- and short-protein and variants in silkworm
A recombinant BmNPV CP- bacmid was prepared by alkaline extraction described in the Bac-to-Bac manual (Invitrogen). Five micrograms of extracted BmNPV CP- bacmid DNA, together with a helper plasmid, was mixed with 1/10 volume of DMRIE-C reagent (Invitrogen) and incubated at room temperature for an hour. This mixture was injected into silkworm larvae. The DNA-injected silkworm larvae were reared using Silkmate 2S (NOSAN Co. Yokohama, Japan) as a diet for 6–7 days and followed by the collection of fat body from silkworm larvae. Collected fat body was stored at −80°C before use.
Partial purification of HPV VLPs
The fat body was suspended in a homogenate buffer (50 mM Tris–HCl buffer [pH 7.5] containing 150 mM NaCl) and sonicated to extract expressed proteins. The homogenate was centrifuged at 30000 × g for 15 min. A supernatant was applied to the HiTrap heparin affinity column chromatography (GE Healthcare, Pittsburgh, PA, USA). After loading the sample, the column was washed with 20-column volumes of homogenate buffer, followed by an elution by NaCl concentration gradient to 2 M. Fractions containing L1 protein were collected and dialyzed against homogenate buffer overnight. The dialyzed sample was applied to Mono S 5/50GL column chromatography (GE Healthcare). The column was washed with 10-column volumes of homogenate buffer, and proteins were eluted by NaCl concentration gradient to 1 M.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis
The samples were subjected to SDS-PAGE on a 10 or 12% polyacrylamide gel with the Mini-protean II system (Bio-Rad, Hercules, CA, USA). The total number of proteins on the SDS-PAGE gel was detected with Coomassie Brilliant Blue (CBB) R-250. In the case of western blot, the proteins found in the gels were blotted onto a polyvinylidene fluoride (PVDF) membrane using the Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad). After being blocked in 5% skim milk in a Tris-buffered saline containing 0.1% Tween 20 (TBST), the membrane was incubated in a 1:10000 diluted mouse anti-HPV 16 L1 antibody (Novus Biologicals Inc. Littleton, CO, USA) for an hour. This antibody can also recognize HPV 6b L1 protein. The membrane was washed with TBST and then incubated in a 1:20000 diluted either anti-mouse or anti-rabbit labeled with horseradish peroxidase (HRP) (GE Healthcare) for an hour. Detection was performed using ECL Plus Western Blotting Reagent (GE Healthcare). Specific bands were detected using a Fluor-S/MAX Multi-Imager (Bio-Rad). Protein band intensity was analyzed by Quantity One software (Bio-Rad).
Sucrose density gradient centrifugation analysis
The homogenate of the fat body was laid on a 25–60% sucrose density gradient. This suspension was centrifuged at 96000 × g for 3 h at 4°C. Each 0.5 ml of the fraction was collected at the top of the tube. Each fraction was analyzed by SDS-PAGE and western blot.
Transmission electron and immunoelectron microscopic analyses
Purified VLPs were immobilized on the grid and blocked with 4% BSA in PBS (pH 7.4). This grid was soaked in either mouse anti-HPV 16 L1 antibody diluted by 30-fold with 1% BSA in PBS for 2 h. After washing with PBS, the grid was soaked in either 10 nm gold-conjugated goat polyclonal anti-mouse IgG+IgM (H+L) (British BioCell International, Cardiff, UK) diluted by 25-fold with 1% BSA in PBS for 1 h. After washing with PBS, the grid was stained with 2% phosphotungstic acid. VLPs were observed by JEM-2100F (JEOL Ltd., Japan) at 200 kV.
This work was funded by Promotion of Nanobio-Technology Research to support Aging and Welfare Society from the Ministry of Education, Culture, Sports, Science & Technology (MEXT), Japan (to EP). MP was supported by Japanese Government Scholarship fellow MEXT, Japan.
- Bian T, Wang Y, Lu Z, Ye Z, Zhao L, Ren J, Zhang H, Ruan L, Tian H: Human papillomavirus type 16 L1E7 chimeric capsomeres have prophylactic and therapeutic efficacy against papillomavirus in mice. Mol Cancer Ther 2008, 7: 1329-1335. 10.1158/1535-7163.MCT-07-2015View ArticleGoogle Scholar
- Bishop B, Dasgupta J, Klein M, Garcea RL, Christensen ND, Zhao R, Chen XS: Crystal structures of four types of human papillomavirus L1 capsid proteins: understanding the specificity of neutralizing monoclonal antibodies. J Biol Chem 2007, 282: 31803-31811. 10.1074/jbc.M706380200View ArticleGoogle Scholar
- Chen XS, Garcea RL, Goldberg I, Casini G, Harrison SC: Structure of small virus-like particles assembled from the L1 protein of human papillomavirus 16. Mol Cell 2000, 5: 557-567. 10.1016/S1097-2765(00)80449-9View ArticleGoogle Scholar
- Georgens C, Weyermann J, Zimmer A: Recombinant virus like particles as drug delivery system. Curr Pharm Biotechnol 2005,6(1):49-55.Google Scholar
- Grgacic EV, Anderson DA: Virus-like particles: passport to immune recognition. Methods 2006, 40: 60-65. 10.1016/j.ymeth.2006.07.018View ArticleGoogle Scholar
- Hiyoshi M, Kageshima A, Kato T, Park EY: Construction of a cysteine protease deficient Bombyx mori multiple nucleopolyhedrovirus bacmid and its application to improve expression of a fusion protein. J Virol Methods 2007, 144: 91-97. 10.1016/j.jviromet.2007.04.005View ArticleGoogle Scholar
- Kratz PA, Bottcher B, Nassal M: Native display of complete foreign protein domains on the surface of hepatitis B virus capsids. Proc Natl Acad Sci USA 1999, 96: 1915-1920. 10.1073/pnas.96.5.1915View ArticleGoogle Scholar
- Li M, Cripe TP, Estes PA, Lyon MK, Rose RC, Garcea RL: Expression of the human papillomavirus type 11 L1 capsid protein in Escherichia coli: characterization of protein domains involved in DNA binding and capsid assembly. J Virol 1997, 71: 2988-2995.Google Scholar
- Ma Y, Nolte RJ, Cornelissen JJ: Virus-based nanocarriers for drug delivery. Adv Drug Deliv Rev 2012, 64: 811-825. 10.1016/j.addr.2012.01.005View ArticleGoogle Scholar
- Murata Y, Lightfoote PM, Rose RC, Walsh EE: Antigenic presentation of heterologous epitopes engineered into the outer surface-exposed helix 4 loop region of human papillomavirus L1 capsomeres. Virol J 2009, 6: 81. 10.1186/1743-422X-6-81View ArticleGoogle Scholar
- Nassal M, Skamel C, Vogel M, Kratz PA, Stehle T, Wallich R, Simon MM: Development of hepatitis B virus capsids into a whole-chain protein antigen display platform: new particulate Lyme disease vaccines. Int J Med Microbiol 2008, 298: 135-142. 10.1016/j.ijmm.2007.08.002View ArticleGoogle Scholar
- Pejawar-Gaddy S, Rajawat Y, Hilioti Z, Xue J, Gaddy DF, Finn OJ, Viscidi RP, Bossis I: Generation of a tumor vaccine candidate based on conjugation of a MUC1 peptide to polyionic papillomavirus virus-like particles. Cancer Immunol Immunother 2010, 59: 1685-1696. 10.1007/s00262-010-0895-0View ArticleGoogle Scholar
- Pokorski JK, Steinmetz NF: The art of engineering viral nanoparticles. Mol Pharm 2011, 8: 29-43. 10.1021/mp100225yView ArticleGoogle Scholar
- Sadeyen JR, Tourne S, Shkreli M, Sizaret PY, Coursaget P: Insertion of a foreign sequence on capsid surface loops of human papillomavirus type 16 virus-like particles reduces their capacity to induce neutralizing antibodies and delineates a conformational neutralizing epitope. Virology 2003, 309: 32-40. 10.1016/S0042-6822(02)00134-4View ArticleGoogle Scholar
- Schellenbacher C, Roden R, Kirnbauer R: Chimeric L1-L2 virus-like particles as potential broad-spectrum human papillomavirus vaccines. J Virol 2009, 83: 10085-10095. 10.1128/JVI.01088-09View ArticleGoogle Scholar
- Touze A, Mahe D, El Mehdaoui S, Dupuy C, Combita-Rojas AL, Bousarghin L, Sizaret PY, Coursaget P: The nine C-terminal amino acids of the major capsid protein of the human papillomavirus type 16 are essential for DNA binding and gene transfer capacity. FEMS Microbiol Lett 2000, 189: 121-127.View ArticleGoogle Scholar
- Trus BL, Roden RB, Greenstone HL, Vrhel M, Schiller JT, Booy FP: Novel structural features of bovine papillomavirus capsid revealed by a three-dimensional reconstruction to 9 A resolution. Nat Struct Biol 1997, 4: 413-420. 10.1038/nsb0597-413View ArticleGoogle Scholar
- Varsani A, Williamson AL, Jaffer MA, Rybicki EP: A deletion and point mutation study of the human papillomavirus type 16 major capsid gene. Virus Res 2006, 122: 154-163. 10.1016/j.virusres.2006.07.012View ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.