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Article Cite This: ACS Omega 2018, 3, 17341−17347
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Reversible Mannosylation as a Covalent Binding Adjuvant Enhances Immune Responses for Porcine Circovirus Type 2 Vaccine Yuanzi Wu,*,† Chao Yan,† Jian’an He,*,‡,§ Wenli Xiong,† Shuigen Wu,† Shutao Liu,† and Zhen Cai† †
College of Biological Science and Engineering, Fuzhou University, 2 Xueyuan Road, Fuzhou 350108, China Central Laboratory of Health Quarantine, International Travel Health Care Center, Shenzhen Entry-Exit Inspection and Quarantine Bureau, 2006 Shennan Road, Shenzhen 518045, China § Shenzhen Academy of Inspection and Quarantine, 2049 Heping Road, Shenzhen 518010, China
ACS Omega 2018.3:17341-17347. Downloaded from pubs.acs.org by 5.62.152.61 on 12/20/18. For personal use only.
‡
ABSTRACT: Capsid protein of porcine circovirus type 2 (PCV2) is an ideal subunit vaccine candidate for the postweaning multisystemic wasting syndrome. In this study, mannan-mediated targeting of PCV2ΔCap42‑233 protein to antigen presenting cells (APCs) was investigated for the development of PCV2 vaccine. Mannan was attached to PCV2ΔCap42‑233 protein via an acid sensitive Schiff base reaction. The mannosylated protein was endowed with the capacity to target the mannose receptor on APCs as well as the ability of controlled release of the antigen in the acidic condition of the lysosome. Finally, the immune response of mannosylated PCV2ΔCap42‑233 protein in mice was evaluated. The mannosylated protein exhibited the ability to stimulate humoral immune response and enhance the immunity. Thus, acid-sensitive and APCs-targeting mannosylated PCV2ΔCap42‑233 protein represents a promising candidate for the potential commercial application as an efficient vaccine against porcine circovirus.
1. INTRODUCTION Porcine circovirus type 2 (PCV2) is the primary causative agent of the postweaning multisystemic wasting syndrome, one of the most serious swine diseases worldwide.1−6 The open reading frame 2 of PCV2 genome encodes the major structural capsid protein, which is also the major antigen protein of PCV2.7,8 This capsid protein shows the ability to serve as an effective candidate immunogen for designing a new recombinant subunit vaccine against PCV2.9−11 Initiating the adaptive immune response, vaccine based on antigenic protein plays an important role in protecting against various infectious diseases. However, a crucial restriction for recent vaccine development is the inefficiency in the process of activating cytotoxic T lymphocytes by antigen presenting cells (APCs).12 To achieve this, one of the essential steps is to enhance the antigen capture and internalization by APCs. Mannose receptor (MR) belongs to the C-type lectin superfamily, a major component in the phagocytosis of pattern recognition receptors, which is highly presented on APCs.13−16 MR-mediated antigen uptake has shown simultaneous activation cellular and humoral immune responses to mannosylated antigens.17−21 Mannosylated antigens can significantly enhance the immune response to specific antigens, which shows the ability to favor the recruitment and activation of APCs. For the vaccine design, mannan can serve as a promising APCs targeting adjuvant to enhance the antigen uptake and increase the vaccine immunity.22−24 © 2018 American Chemical Society
Nanometer-sized particles have attracted growing interests as drug carriers for protein-based vaccines. Such nanoparticles are designed to degrade under mildly acidic conditions to allow for control release of the antigen in the acidic environment of the phagosomes of APCs.25−28 On the other hand, for the APCs targeting purpose, the adjuvant should be solidly and covalently bonded to the antigen properly. Thus, for efficient vaccine design, the ideal vaccine will be composed of entities that promote the antigen internalization by APCs, as well as facilitate the release of the antigen in the lysosomal environment and further cross-presentation of APCs. In this study, we focus on the acid-sensitive mannosylated antigen serving as a potential control release vaccine. The mannan after facile oxidization is capable of reacting with free amino groups in a protein antigen through a reversible Schiff base reaction, which is cleavable in the acidic environment.29,30 Truncated PCV2ΔCap42‑233 protein was used as an antigen for a subunit vaccine. Mannosylated PCV2ΔCap42‑233 (M-PCV2) protein nanoparticles were prepared by covalently attaching the oxidized mannan to the amine groups of PCV2ΔCap42‑233 (Scheme 1). We studied the degradation of the mannosylated antigen containing an acid-sensitive covalent bond with a Schiff base in the lysosomal environment of pH 4.5.31 Immunity of Received: September 4, 2018 Accepted: December 4, 2018 Published: December 14, 2018 17341
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Figure 1. Characterization of the PCV2ΔCap42‑233. (A) PCV2ΔCap42‑233 protein was characterized by SDS-PAGE. Protein marker, purified PCV2ΔCap42‑233 protein, flow-through from Ni-chelating affinity chromatography, bacterial lysates from isopropyl β-D-thiogalactopyranoside (IPTG)-induced recombinant bacteria, and lysates from recombinant bacteria without IPTG, for lanes 1−5, respectively. WB: Western blotting of the IPTG-induced recombinant strain lysate. (B) Indirect ELISA curves for the determination of PCV2ΔCap42‑233. The values for each point corresponding to the average of three replicates. (C) The transmission electron microscopy (TEM) image of PCV2ΔCap42‑233 protein nanoparticles. (D) Dynamic light scattering (DLS) analysis of PCV2ΔCap42‑233 protein nanoparticles.
this mannosylated antigen vaccine was evaluated by comparing the PCV2-specific immune response in mice with two established adjuvants.
Scheme 1. Representation of the Synthesis of Mannosylated PCV2 Nanoparticles, APCs Targeting Delivery, and AcidSensitive Degradation in the Lysosome
2. RESULTS AND DISCUSSION 2.1. Generation of PCV2ΔCap42‑233 Nanoparticle. Because the arginine-rich nuclear localization signal (NLS) sequence at the N-terminus of the PCV2 capsid protein is difficult to produce in the standard Escherichia coli expression system, because the corresponding aminoacyl arginine-tRNA is rarely expressed in this bacterium. 32 In this study, PCV2ΔCap42‑233 protein, a truncated variant of the capsid protein that lacks the NLS sequence, was expressed in E. coli. The PCV2ΔCap42‑233 protein was purified by Ni2+ affinity chromatography and further examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The final protein was obtained with an apparent molecular weight of 25 kDa with a purity of electrophoresis level (Figure 1A). After removing urea by gradient dialysis, the PCV2ΔCap42‑233 protein was identified using a rabbit anti-PCV2 polyclonal antibody via Western blotting (WB). The reactivity of the PCV2ΔCap42‑233 was evaluated by indirect enzyme-linked immunosorbent assay (ELISA). The purified protein showed strong a immunoreaction with the antibody against PCV2 with a detection limit of about 100 ng/mL. These results confirmed that the PCV2ΔCap42‑233 protein was highly expressed and well purified, with the retention of the immunoreactivity. Transmission electron microscopy (TEM) and dynamic light scattering (DLS) was used to determine the physical characteristics of the PCV2ΔCap42‑233 protein after renaturation (Figure 1B,C). Data showed that the protein self-assembled and formed homogeneous nanoparticles with an average dry
diameter of approximately 28 nm, which is of comparable size with the PCV2 virus (about 17 nm). However, a monomodal distribution with a hydrodynamic diameter of approximately 217 nm (dispersity of 0.144) was detected by DLS, which is much larger than the size detected by TEM. The differences between the hydrodynamic diameter and the dry diameter might be because of the swelling of the protein nanoparticle 17342
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Figure 2. Identification of mannan oxidization and mannosylated PCV2ΔCap42‑233 protein nanoparticles. (A) UV−vis spectra of mannan and oxidized mannan. (B) IR spectra of mannan and oxidized mannan by Fourier transform infrared spectroscopy. (C) Mannan conjugates with PCV2ΔCap42‑233 measured by SDS-PAGE. PCV2ΔCap42‑233 protein, PCV2ΔCap42‑233 conjugates with mannan with the protein/mannan molar ratio of 1:1, 1:2, and 1:4, respectively (lanes 1−4). Identity of mannosylated PCV2ΔCap42‑233 protein by fluorescein isothiocyanate (FITC)-lectin blotting. PCV2ΔCap42‑233 protein, PCV2ΔCap42‑233 conjugates with mannan with the protein/mannan molar ratio of 1:1, 1:2, and 1:4, respectively (lanes 5−8). (D) DLS analysis of the PCV2ΔCap42‑233 protein nanoparticles conjugated with mannan (sample 2 in (C)).
high-molecular-weight smear with a wide distribution. With the increase in the mole ratio of oxidized mannan to protein, the conjugate band was found to appear at a much higher molecular weight, and the band corresponding to the free PCV2ΔCap42‑233 disappeared. The results revealed that there was only partial conjugation with the molar ratio of 1:1, while reach to reaction completely at the molar ratio of 1:4 (protein/ mannan). Furthermore, the affinity of the modified mannan to the mannose receptor was investigated. The immunoblotting of mannan was stained by incubating with fluorescein isothiocyanate (FITC) labeled lectin, which is commonly used to recognize and bind mannosyl peptides in glycoproteomics workflows.15,37 The fluorescence pattern was consistent with the protein distribution shown in SDS-PAGE, confirming that the mannan conjugated to PCV2ΔCap42‑233 retained free mannose residue for lectin binding. The size distribution of the synthetic mannosylated PCV2ΔCap42‑233 protein nanoparticles was analyzed by using DLS. The hydrodynamic diameter increased as expected to a broader range of values from 200 to 800 nm (Figure 2D), which suggests that part of mannosylated protein nanoparticles aggregated slightly. The moderate aggregation is advantageous for vaccines here because decent size of protein nanoparticles at 500 nm scale or larger could promote the class I antigen presentation of protein antigens.38 Taken together, these results indicated that the oxidized mannan was covalently conjugated to PCV2ΔCap42‑233 to form a mannose receptor targeting complexes. 2.3. Acid Degradable Mannosylated Protein at Lysosomal pH in Vitro. For the PCV2 vaccine design, mannan has been demonstrated as an adjuvant that targets APCs through the mannose receptor and facilitates antigen endocytosis. Moreover, mannan also can be regarded as a shell protecting antigen from degradation. Schiff base bond between protein and mannan is chemically stable in the neutral
with a loose conjunction in the solution, and a possible slight aggregation of particles in the suspension. The controllable protein particles with a hydrodynamic radius of hundreds of nanometers could serve as the self-adjuvant by promoting internalization by APCs through phagocytosis, and enhancing the major histocompatibility complex class I antigen presentation of antigens.33,34 Moreover, compared with the nanocarrier based on biomacromolecular networks, the selfassembled protein nanoparticle could disassemble much easier intracellularly and might process and present into the cytoplasmic compartment before lysosomal trafficking. 2.2. Mannosylation of PCV2 Nanoparticles. Aldehyde groups were introduced to mannan by mild oxidation with sodium periodate.35 The functional aldehyde groups were identified by UV−vis spectra and Fourier transform infrared (FTIR) spectra, respectively. Oxidized mannan shows an absorption peak at approximately 230 nm in the UV−vis spectrum, which represents the aldehyde group in aqueous solution (Figure 2A). Figure 2B shows the IR spectra of mannan before and after oxidation, respectively. Oxidized mannan showed an absorption peak at 1735 cm −1 , representing a stretching vibration of the carbonyl group of the aldehyde moiety.36 However, the characteristic C−H vibration peak of aldehyde group was concealed by the stretching vibration band of C−H of alkane at 2930 cm−1. The other characteristic IR peaks of mannan and oxidized mannan were almost identical, indicating that most of the mannose structures have not been destroyed under mild oxidation conditions. Mannosylation was carried out by simply mixing the oxidized mannan with PCV2ΔCap42‑233 for 24 h at room temperature. The coupling of mannan and the antigens was verified by the denaturing SDS-PAGE (Figure 2C). The PCV2ΔCap42‑233 yielded a single band at 25 kDa (lane 1), whereas the mannosylated protein appeared on the gel as a 17343
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Figure 3. Identification of mannosylated protein release at the lysosomal pH. (A) Mannosylated protein release identified by SDS-PAGE. Native PCV2, mannosylated PCV2 at pH 7.4, and pH 4.5 for lanes 1−3, respectively. (B) UV−vis spectra of mannosylated protein conjugated with rhodamine 123, dialyzed at pH 7.4 and 4.5, respectively. (C) The dynamic intensity changes of A450nm with dialysis time at pH 7.4 and 4.5, respectively.
Figure 4. Immune response after intradermal injection of ICR mice with different vaccine candidates. (WO: white oil adjuvant; Alu: aluminum salt adjuvant; CV: commercial vaccine.) PCV2-specific IgG antibody (A), IL-2 (B), IL-4 (C), and IFN-γ (D) 15 days after the second immunization. Data are shown as individual data points, and scale bars represent mean ± standard of five mice per group (* p < 0.05, ** p < 0.01, *** p < 0.001).
corresponding to rhodamine 123 was negligible, whereas at pH 7.4, the absorbance value reaches a certain value after thorough dialysis for over 6 h to get rid of the excess dye from the solution (Figure 3B,C). The results confirmed the acid sensitivity of the Schiff base bond in mannosylated PCV2 nanoparticles at the lysosome conditions, which indicated the capability of controlled release of the antigens in the APCs. 2.4. Immune Mice and Detection of Immunoglobulin G (IgG), Interleukin-2 (IL-2), Interleukin-4 (IL-4), and Interferon-γ (IFN-γ). To test the efficacy of the mannosylated PCV2ΔCap42‑233 complexes, diversified vaccine candidates were subcutaneously intraperitoneally injected into the model mice to stimulate an immune response in vivo. PCV2-specific IgG antibody, IL-2, IL-4, and IFN-γ levels of the immunized animals 15 days after the booster vaccination were assessed via enzyme-linked immunosorbent assay (ELISA). The adjuvantfree truncated PCV2ΔCap42‑233 showed a distinct PCV2 specific IgG antibody production. Moreover, levels of IgG antibody
condition but quickly decomposes under acidic condition, which makes the reaction an ideal tool for drug delivery system.38 We investigated the stability of mannosylated PCV2 protein nanoparticles at pH 4.5 and 7.4 in vitro to understand the antigen release properties in the acidic environments of lysosome. SDS-PAGE was used to confirm the controlled release of protein antigens in lysosomal environments at pH 4.5 (Figure 3A). After incubation at pH 4.5 overnight, the smear at higher molecular weight corresponding to mannosylated PCV2 faded, whereas the protein concentrated again at the pristine PCV2 position. Furthermore, excessive amounts of rhodamine 123 with an amino group were induced to couple with aldehyde residue of mannosylated PCV2 nanoparticles via the same Schiff base reaction. The complex was dialyzed against phosphate-buffered saline (PBS) at pH 4.5 and 7.4, respectively. Intensity changes in rhodamine served here as an internal marker for the breakage of the Schiff base bond. At pH 4.5, after 9 h, UV−vis absorbance peak at 450 nm 17344
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promise as acid-sensitive covalent adjuvant for a variety of protein vaccines with the capacity of APCs targeting, as well as controlled release.
were increased for the mice in the mannosylated PCV2ΔCap42‑233 group (M-PCV2), which was significantly higher than that of the adjuvant-free PCV2 group, adjuvant with PCV2 group (aluminum salt (Alu) and white oil (WO)), and blank control group (PBS) (Figure 4A). The IgG antibody titer of M-PCV2 was a little lower than that of the commercial vaccine (CV) expressed by baculovirus vector. The results indicated that the mannosylation of antigen might stimulate the production of the IgG antibody against PCV2. IL-2 is involved in the secretion of IgG by B cells and the proliferation and differentiation of cytotoxic T cells. IL-4 promotes lymph B-cell activation, proliferation, differentiation, and secretion of IgG. Both IL-2 and IL-4 play major roles in regulating antibody-mediated humoral immune responses by the immune system. Levels of IL-2 and IL-4 were significantly higher for the mice in the M-PCV2 group, compared with that in other groups (Figure 4B,C). The results indicated that the mannosylation displayed superior immunity as compared with the PCV2 by stimulating an enhanced humoral immune response to the PCV2 antigens. The M-PCV2 group showed a higher level of IL-4 but a lower level of IL-2 when compared with the CV group. IFN-γ activates NK cells and enhances their capacity to kill target cells, which primarily regulate the cellular immune response. Changes in IFN-γ levels were not that noticeable among all the vaccine group except the PBS control, indicating that cell-mediated immune response activated by mannan was indistinctive (Figure 4D). Taken together, the results showed that M-PCV2 induced similar levels of humoral and cell-mediated immune responses compared with the commercial vaccines. For a systematic and comprehensive study, PCV2 antigens were formulated with two traditional adjuvants, aluminum salts (Alu), and white oil (WO), respectively. One of the most important capabilities of the two established adjuvants is to protect the antigens for long period release. However, for the mannan-free group, there was no significant difference for the two adjuvanted vaccines in the level of antibodies. We ascribe the reason to the special conformation of PCV2: compared with the antigen monomer, the PCV2 nanoparticle with a diameter over hundred nanometers may serve as the antigen reservoir in vivo and resist the clearance from the body itself. To note that the M-PCV2 was significantly higher than that in the two adjuvanted groups with or without mannosylation. We presume that (1) the mannosylation may play a role in protecting the antigen from clearance and (2) the embedding of the antigen nanoparticles in the adjuvants might limit the targeted delivery capacity of M-PCV2. As a whole, as a covalent bonding adjuvant, the mannosylated PCV2 showed a significantly stronger immunity compared with any other control groups.
4. EXPERIMENTAL SECTION 4.1. Preparation of Mannan Dialdehydes. Two hundred milligrams of mannan (Sigma) was dissolved in 10 mL of 0.05 mM aqueous solution of sodium periodates and stirred in the dark at 20 °C for 2 h. Then, the reaction was quenched by adding 1 mL glycerol. The solution then was stirred in the dark at 20 °C for 4 h. Low-molecular-weight compounds were removed from the reaction mixture by dialysis (NW10000, Spectrum) against distilled deionized water in the dark at room temperature for 48 h. The obtained oxidized mannan was lyophilized and stored at −20 °C. Fourier transform infrared (FTIR) spectroscopy was performed on a Nicolet 380 FTIR spectrometer. UV−vis spectra were conducted with a Varian Cary 500 Scan UV/vis system. 4.2. Expression and Purification of PCV2ΔCap42233. To express the protein, the recombinant expression plasmid pET28a-PCV2ΔCap42233 was transformed into E. coli BL21 (DE3). Positive colonies containing the expression plasmid were grown at 37 °C in germ-free Luria−Bertani medium with 50 μg/mL kanamycin. When the OD600 reached 0.6−0.8, isopropyl β-D-thiogalactopyranoside (IPTG) (BBI Life Sciences, China) was supplemented to the pellet at a final concentration of 1 mM. After the bacteria were cultured at 37 °C for 10 h and shaken at 200 rpm/min, bacterial pellet was harvested by centrifuging at 10 000 rpm and 4 °C for 10 min. The cells were resuspended in disrupted buffer (50 mM sodium phosphate, 500 mM NaCl, 20 mM imidazole, 8 M solid urea, 1 mM phenylmethylsulfonyl fluoride, pH 7.4) and broken by sonication in a water−ice mixture. The homogenate was centrifuged at 12 000 rpm at 4 °C for 30 min. The supernatant containing PCV2ΔCap42‑233 protein was filtered with a 0.45 μm filter membrane (Jinteng, China), then the PCV2ΔCap42‑233 protein was purified by Ni Sepharose 6 Fast Flow (GE Healthcare). After flow-through of the sample, 10-column volumes of disrupted buffer were used to equilibrate the column. Then, the PCV2ΔCap42‑233 protein was eluted with elution buffer (50 mM sodium phosphate, 500 mM NaCl, 200 mM imidazole, 8 M solid urea, 1 mM phenylmethylsulfonyl fluoride; pH 7.4). The eluted protein solution was dialyzed gradually by buffer A (50 mM sodium phosphate, 200 mM NaCl, 20 mM imidazole, 1 mM phenylmethylsulfonyl fluoride; 1 mM ethylenediaminetetraacetic acid, 5 mM dithiothreitol, 30% (v/v) glycerol, pH 7.4). The purified protein was stored at −20 °C before further use. 4.3. Characterization of PCV2ΔCap42‑233 Protein. The quantity of PCV2ΔCap42‑233 protein was performed by the Bradford assay that used bovine serum albumin (BSA, BBI Life Sciences, China) as a standard. PCV2ΔCap42‑233 protein was analyzed by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then stained with Coomassie brilliant blue R-250 (BBI Life Sciences, China). For Western blotting, the PCV2ΔCap42‑233 protein is gel transferred onto PolyScreen poly(vinylidene difluoride) transfer membrane (Merck Millipore, Germany) using a DYCP-40C semidry blot electrophoresis cell (Beijing, China) in transfer buffer containing 39 mM glycine, 50 mM Tris, 1 mM SDS, 20% (v/v) methanol at 100 mA for 1 h. Then, the membrane was incubated in blocking buffer 1 (T-TBS (20 mM Tris−HCl (pH 8.0), 150
3. CONCLUSIONS In this report, PCV2ΔCap42‑233 protein was modified with oxidized mannan via Schiff base reaction to the development of PCV2 vaccine. The mannosylated PCV2ΔCap42‑233 formed nanoparticles with the size range of around 200−800 nm. It was demonstrated that the protein/mannan complex could couple with mannose receptor and might decompose to release the PCV2 under mildly acidic conditions. Furthermore, immunizing results confirmed that the mannosylated PCV2 induce humoral immunity response well and show the strongest immunity among the control groups. The mannosylation method developed in the report, therefore, shows 17345
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4.6. Immune Mice and Detection of Total IgG, IL-2, IL4, and IFN-γ. Commercial PCV2 vaccine were used as a positive control (Ingelvac CircoFLEX PCV2 baculovirus vector vaccine, Boehringer Ingelheim). Male mice (4 weeks old, 20−22 g) free of specific pathogen were obtained from Shanghai Laboratory Animal Center (Shanghai, China) and bred and maintained under specific-pathogen-free environment. All the animal experiments were approved by the Fujian Institute of Laboratory Animals Committee (permit number: SYXK2012-0001). All the mice were randomly split into eight groups, each with five mice. Every mouse was immunized via intradermal injection of 0.2 mL manufactured sample, which is PCV2ΔCap42‑233 protein, PCV2ΔCap42‑233 protein with an adjuvant of white oil or Alu, mannosylated PCV2ΔCap42‑233 protein, mannosylated PCV2ΔCap42‑233 protein with an adjuvant of white oil or Alu, two commercial vaccines, PBS for blank, and mannan for control. Then, the mice were boosted by injection on days 15 by the accordant formulation. Tail blood of mice was taken at 29 days after initial immunization. The antibody levels of mice serum were performed by ELISA and indicated using the highest dilution of serum. Briefly, the concentration of IL-2, IL-4, and IFN-γ in mice serum at 29 days of initial immunization was detected by mouse IL-2, IL-4, and IFN-γ ELISA Kit (Boshide, China) according to the manufacturer’s direction.
mM NaCl, 0.05% (v/v) Tween) with 5% nonfat milk) at 1 h for 37 °C and shaken on a shaking table at 70 rpm. The membrane was first incubated with rabbit anti-PCV2 polyclone antibody (1:1000, TransGen, China) at 37 °C for 2.5 h, then followed with goat antirabbit IgG (H + L) horseradish peroxidase (HRP) conjugate (1:2000, TransGen, China). Finally, the immunoreactive signal was developed with Diaminobenzidine Horseradish Peroxidase Color Development Kit (Beyotime, China). Structure of the PCV2ΔCap42‑233 protein nanoparticles was determined by transmission electron microscopy (H7650, Hitachi). A copper grid was soaked in PCV2ΔCap42‑233 protein solution for 2 min. The superfluous liquid was isolated by a filter paper gently. Then, 2% phosphotungstic acid was used for negative staining. The dried copper grid was viewed by TEM at an 80 kV acceleration voltage. For ELISA, the 96-well plate was first coated with PCV2ΔCap42‑233 protein and incubated for 12 h at 4 °C. The plate was then blocked with 5% nonfat milk in PBS and washed three times with PBST (0.2% (v/v) Tween). The plate was further incubated with rabbit anti-PCV2 polyclone antibody (1:2000, TransGen, China) at 25 °C for 4 h and then with goat antirabbit IgG (H + L) HRP conjugate (1:3000, TransGen, China). Finally, the signal was developed with 3,3′,5,5′-tetramethylbenzidine ELISA substrate (TransGen, China). Readout was performed at a wavelength of 450 nm. Each point was tested in triplicate. 4.4. Generation and Determination of Mannan Conjugates with PCV2ΔCap42-233. The oxidized mannan was dissolved in the buffer A containing PCV2ΔCap42‑233 nanoparticles (400 μg/mL), followed by keeping in dark at room temperature and stirring at 200 rpm for 24 h. Then, the reaction compound was dialyzed against PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4). To determine the Schiff base reaction, the mannan-conjugated PCV2ΔCap42‑233 was identified by SDS-PAGE (12.5%) at 100 V for 1.5 h. The gel was then dyed by Coomassie brilliant blue R250. For immunofluorescence, the gel was transferred onto the nitrocellulose membrane (Merck Millipore, Germany) and then blocked in blocking buffer 2 (T-TBS (20 mM Tris−HCl (pH 8.0), 150 mM NaCl, 0.05% Tween 20 (v/v)) with 1% BSA) for 37 °C at 1 h, followed by washing with T-TBS for five times. Then, the membrane was immersed in T-TBS containing 100 μg/mL FITC-labeled lectin from Pisum sativum (Sigma). Then, the membrane was washed with PBS for three times. Finally, the membrane was photographed by Alpha imager HP (ProteinSimple) with the excitation 16 wavelength of 365 nm. The PCV2 nanoparticles were determined by dynamic light scattering at 37 °C (Zetasizer Nano ZS, Malvern, U.K.). The size distribution of intensity was analyzed by Zetasizer Nano ZS software. 4.5. Acid-Degradable Mannosylated Protein at Lysosomal pH in Vitro. Rhodamine 123 (Aladdin) (8 × 10−7 M) was added into prepared 400 μg/mL mannosylated PCV2 and stirred at 200 rpm for 24 h at room temperature in the dark. Then, the samples were divided into two aliquots. One was adjusted to pH 4.5 by HCl and the another maintain 7.4, respectively. Then, the two samples of different pH were dialyzed with PBS (pH 4.5) or PBS (pH 7.4) in the dark to remove the unbounded dye. Aliquots were fetched at different indicated time points for UV−vis analysis (Varian Cary 500). The samples treated with different pH were also loaded and verified by SDS-PAGE (12.5%) at 100 V for 1.5 h.
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected],
[email protected] (Y.W.). *E-mail:
[email protected] (J.H.). ORCID
Yuanzi Wu: 0000-0003-3523-8505 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank the National Natural Science Foundation of China (21504105 and 81703271), Initial Fund for Talents of Fuzhou University (XRC-1542), and Fuzhou University Testing Fund of precious apparatus for their financial support.
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REFERENCES
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DOI: 10.1021/acsomega.8b02264 ACS Omega 2018, 3, 17341−17347
