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.
KeywordsVirus-like particles Human papillomavirus silkworm expression system BmNPV bacmid
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.
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