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VP23 is a herpes simplex virus type I (HSV-I) capsid protein that plays critical roles in virion assembly and maturation, DNA packaging, and infection. The assembly process, and specifically the VP23 capsid protein, has been regarded as the potential target of new drugs that can block the viral infection. However, there is a lack of tools and commercial antibodies for studying VP23. To further investigate VP23 using proteomics approaches and to facilitate targeted drug screen, this study was undertaken to express recombined VP23 in a bacterial expression system and generate polyclonal antibodies against VP23. The UL18 gene encoding VP23 was cloned into the prokaryotic expression vector pET-28a(+) in frame with a 6Ã-His tag and expressed as soluble protein in E.coli BL21 (DE3). After purification by nickel affinity chromatography and gel filtration, the recombinant protein was injected as antigen into rabbits to produce polyclonal antibodies. Western blot analysis demonstrated that VP23 was specifically recognized by the polyclonal antibodies. This report presents a protocol to obtain an over-expressed and high-purity recombinant VP23 as well as high titer of rabbit polyclonal antibodies specifically against VP23. The polyclonal antibodies generated in this study provide a useful and effective tool for structural and functional characterization of HSV-I VP23 and targeted drug screen of agents disrupting HSV-I assembly.
Keywords: herpes simplex virus; VP23; triplex; prokaryotic expression; purification; polyclonal antibody
HSV-I, herpes simplex virus type I; PVDF, polyvinylidene difluoride; HRP, horseradish peroxidase; ELISA, enzyme-linked immunosorbent assay; TMB, tetramethyl benzidine; IMAC, immobilized metal-chelate affinity chromatography.
The herpes simplex virus type 1 (HSV-I) is a common human pathogen that can cause the infection of mucocutaneous membranes and lead to the development of several disorders including encephalitis, retinitis, hepatitis, pneumonia, and esophagitis when reactivated from latency. Immunocompromised patients, such as those undergoing chemotherapy or receiving organ transplants, are at particular risk of developing life-threatening complications due to reactivation of latent herpesvirus infections.
The HSV-I virion is bounded by a membrane envelope in which a proteinaceous layer called the tegument surrounds the icosahedral capsid, and the capsid contains the double-stranded linear DNA viral genome inside. The capsid plays critical roles in virion assembly and maturation, DNA packaging, and delivery of the genome into a host nucleus. Herpesvirus capsids are assembled in the infected cell nucleus where a closed DNA-free procapsid is first formed and later filled with DNA . Capsid can also protect nucleic acid from environmental damage, and act as a vector to mediate it into cells. Capsid is a major component of antigen, so it's the main target of vaccine studied for preventing virus.
The mature herpes simplex virus type 1 (HSV-I) capsid is an icosahedral shell that is 125 nm in diameter and 15 nm thick [2-4]. Its major structural features are 162 capsomers (150 hexons and 12 pentons) that lie on a T=16 lattice. The major capsid protein, VP5, is the structural subunit of both the hexons and the pentons; hexons are VP5 hexamers, while pentons are VP5 pentamers. Hexons are found on the faces and edges of the icosahedral capsid, while one penton is found at each of the 12 capsid vertices [5-7].
In addition to the capsomers, the capsid shell contains a total of 320 trivalent structures called triplexes. The triplexes lie above the capsid floor connecting capsomers in groups of three. Triplexes may vary in composition, but on average they are heterotrimers, composed of one copy of VP19C and two copies of VP23 per triplex. Six copies of VP26 are located on the outer rim of each hexon where they appear as horn-shaped protrusions [7-11].
Although the triplex proteins make up a relatively small percentage of the total capsid protein, the triplexes appear to provide essential support for the capsid shell as it is formed, capsid assembly will not occur in the absence of the triplex proteins [12-15].
Capsid assembly involves interaction of the major capsid protein with a scaffolding protein. The triplex proteins, VP19C and VP23, are unable to associate with major capsid-scaffold protein complexes independently of one another, suggesting that VP19C and VP23 must interact with each other prior to binding to major capsid-scaffold protein subunits for capsid assembly . Capsid assembly is necessary for replication of HSV-I, and it would not produce infective progeny virus if this process lost. The assembly process of capsid proteins has been regarded as the target of screening new drugs, which can be compound or extracts from traditional Chinese herbs, effectively aim at capsid proteins.
The capsid protein VP23 that we studied is a 34.3 kDa protein composed of 318 amino acids, encoded by UL18 gene (cDNA sequence has 957 bps). It's an important structural protein of HSV-I capsid and is closely connected with the package of progeny virion. However, there still is no an effective tool for studying capsid protein VP23. To further investigate capsid protein VP23 and its structural and functional characterization in targeting drug screening, this study was undertaken to construct a prokaryotic expression vector which can largely express VP23, and it was used to produce polyclonal antibodies.
In the present study, the UL18 gene was cloned into pET-28a(+) to yield pET-28a-UL18. The His-tagged VP23 was then expressed in E.coli BL21 (DE3) cells and purified by a nickel-nitrilotriacetic acid (Ni2+-NTA) affinity resin. Subsequently, the polyclonal antibodies specifically against the purified His-tagged VP23 were raised in rabbits. Finally, the reactivity and specificity of the polyclonal antibody were characterized by Western blot and immunofluorescent assays.
Materials and Methods
Chemicals and Reagents
rTaq DNA Polymerase, restriction enzymes NdeI and XhoI, and T4 DNA ligase were obtained from TaKaRa Company (Japan). KOD DNA Polymerase was obtained from TOYOBO Company (Japan). Cycle-Pure kit, Gel extraction kit and plasmid mini kit were purchased from the Omega Company (USA). Goat anti-Rabbit and Goat anti-Mouse IgG were purchased from Millipore (USA). His-tag Mouse mAb was purchased from Abmart (China). IPTG was purchased from Promega Company (USA). Freund's complete adjuvant (FCA) and Freund's incomplete adjuvant (FIA) were purchased from Sigma (USA). Sepharose G25 column were purchased from GE Healthcare (England). Ni-NTA agarose was obtained from Invitrogen (USA). All other chemicals were of analytical grade.
Cells, Plasmids and animals
Herpes Simplex Virus type 1 strain F (HSV-I F; ATCC VR-733) was supplied by University of Hong Kong. Vero cell line (ATCC CCL81) was obtained from ATCC Company (USA). Escherichia coli DH5Î± was obtained from Promega Company (USA). Escherichia coli BL21(DE3) and plasmid pET-28a(+) were obtained from Novagen (Germany). New Zealand rabbits were purchased from Guangdong Provincial Center of Medical Laboratory Animals (China).
Construction of the prokaryotic expression plasmid pET-28a(+)-UL18
The vero cells were cultured in DMEM medium with 2% fetal bovine serum for 24 h, and then HSV-I virus was added to infect vero cells. When 80% of all vero cells pathologically changed, total RNAs of vero cells were extracted by TRIzol reagent (Invitrogen, USA) and reverse transcribed using the PrimeScript RT reagent Kit (Takara, Japan). The UL18 cDNA strand was amplified by PCR (Polymerase Chain Reaction). The primers used for PCR amplification were: UL18 sense primer (5'-TAACCATGGGCATGCTGGCGGACGGCTTT-3'), and UL18 antisense primer (5'-GCGCTCGAGGGGATAGCGTATAACGGGGGC-3'), where the underlined nucleotides were digestion sites for the restriction enzymes Nco I and Xho I, respectively. The reaction was carried out under following conditions: initial denaturation at 94 °C for 2 min followed by 30 cycles of denaturation at 94 °C for 30 sec, annealing at 58 °C for 40 sec and extension at 68 °C for 60 sec. The PCR product was digested with Nco I and Xho I, and then ligated into pET-28a(+) between the Nco I and Xho I restriction sites. The ligated products were amplified in E. coli DH5Î±. The positive recombinant clones were identified by PCR, restriction enzyme analysis and finally confirmed by DNA sequencing.
Expression of VP23 in E.coli
To optimize the induction conditions, a single colony of the cells was cultured in 50 mL LB medium containing kanamycin (50 Î¼g/mL) at 37 °C and grown with constantly shaking at 180 rpm/min in a constant temperature oscillator (XHX-82, XinJia, China). Add IPTG for induction when the OD600 value of culture medium in each flask reached between 0.6 and 0.8. For IPTG optimization, the bacterial culture was induced with different concentrations of IPTG (0.2, 0.4, 0.6, 0.8, 1.0, 1 and 1.5 mM) and cultured at 37 °C for 4 h. For temperature optimization, the bacterial culture was induced with optimized concentration of IPTG and cultured at four different temperatures (15, 20, 25, 30 and 37 °C) for 8 h. For time optimization, the bacterial culture was induced with optimized concentration of IPTG and cultured at optimized temperature for 4, 6, 8, 10, 12, 16, 20 and 24 h respectively. The sample from each optimization experiment was collected and the absorbance at 600 nm was determined in order to monitor bacterial growth. The expression levels were analysed by 12% SDS-PAGE.
Purification of VP23
After induction under optimized condition, the cells were harvested by centrifugation with 8000 g for 10 min at 4 °C. Cell pellets were resuspended in 1:10 volume of PBS buffer (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.8) and lysed by sonication on ice at 600 W for 30 min (sonication for 4 sec and intermission for 4 sec). The soluble and the insoluble fractions were separated by centrifugation at 17000 g for 30 min at 4 °C. Both protein fractions of cell lysate were analyzed by 12% SDS-PAGE.
The purification of the recombinant VP23 was based on the His-tag protocol. The supernatant was loaded onto the Ni-NTA column, which was pre-equilibrated with five column volumes of PBS buffer. After incubation for 1 h at 4 °C, loosely bound contaminants were removed from the Ni-NTA columns with PBS buffer containing 40 mM imidazole until the 280 nm absorbance returned to the baseline. Subsequently, the target protein was eluted with PBS buffer containing 400 mM imidazole at a flow rate of 1 mL/min. The fraction containing target protein was further pooled and desalted by a Sephadex G-25 column. This was done to eliminate remaining salt and imidazole. Purified VP23 was condensed and stored at âˆ’20 °C. The fraction from each step was visualized by 12% SDS-PAGE.
Western blot analysis
Inductive bacterial was heated to lyse at 100 °C for 10 minutes. Proteins of Vero cells and HSV-I infected Vero cells were simultaneously extracted in RIPA buffer when 80% of Vero cells were pathological changes. Proteins was electrophoresed in a 12% SDS-PAGE, and then transferred onto a polyvinylidene difluoride (PVDF) membrane by a Bio-Rad apparatus at 200 mA for 1.5 h. The membrane was blocked with 5% skim milk in PBST buffer (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, 0.05% Tween20) for 2 h at room temperature. After probed with primary antibodies at 4 °C overnight, the membrane was then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature. Specific protein bands were visualized using the chemiluminescence method and imaged by autoradiography.
Production of polyclonal rabbit VP23 antibodies
Three New Zealand white rabbits were immunized with 3.0 mg of purified VP23 in Complete Freund's Adjuvant (CFA) via intradermal injection (total dose = 250 Î¼g/rabbit), and given other three injections of same amount of VP23 with Incomplete Freund's Adjuvant (IFA) at 2 weeks intervals. The rabbits were bled in 2 weeks after the last immunization. The titer of polyclonal antiserum raised against VP23 was analyzed by enzyme-linked immunosorbent assay (ELISA). Briefly, The 96-well plates were coated with 1 Î¼g/mL of purified VP23 and incubated overnight at 4 °C. After blocking with 5% BSA, the wells were incubated with polyclonal anti-VP23 antisera for 2 h at 37 °C. Following the thoroughly washing, HRP conjugated goat anti-rabbit IgG was incubated for 2 h at 37 °C. Finally, tetramethyl benzidine (TMB) was added to each well in the dark at 37 °C for 10 min. The color reaction was stopped with 2 M H2SO4 before measuring the absorbance at 450 nm in microplate reader (Bio-Rad 550).
The specificity of anti-VP23 antibodies was verified by western-blot analysis. We used diluted antiserum as the primary antibody and HRP-conjugated goat anti-rabbit IgG as the secondary antibody, respectively.
Cloning and expression of VP23
PCR amplification and double enzymes digestion were conducted to examine the recombinant plasmid. As shown in Fig. 1A, the size of target fragment from double digestion of pET-28a (+)-UL18 was about 975 bp (6Ã-His tags included), the same as the PCR product showed in Fig. 1B. DNA sequencing further confirmed that the recombinant plasmid was constructed correctly (data not shown). Recombinant plasmid was then transformed into expression host E. coli BL21 (DE3) for protein expression. Expression induction revealed a band which with an approximate molecular weight of 35 kDa (6Ã-His tags included) which was expected for VP23 protein (Fig. 1C).
Optimization of the expression conditions for VP23 in E. coli BL21 (DE3) was conducted by varying IPTG concentrations, culture temperatures and induction time. As shown in Fig. 1C, the expression level of VP23 did not obviously increased at 37 °C compared with 20 °C, so the increase of induction temperature did not result in obvious improvement of expression. Besides, the VP23 can be expressed even though IPTG was not used. But the amount of VP23 increased significantly as the induction time extended. Thus, human VP23 was successfully expressed in E. coli BL21 (DE3) and the optimized expression conditions were identified to be at 20 °C for 24 h without IPTG induction.
Purification of recombinant protein VP23
Before purification, the expression of the recombinant Vp23 protein was efficiently accomplished by induction with 1 mM IPTG at 20 °C for 24 h. Following cell disruption and centrifugation, most of the VP23 was found in soluble form (Fig. 2A, lane 1). Thus, the supernatant fraction was applied to the affinity chromatography in chelated nickel resin, as described in detail in the method section above. Large amounts of VP23 were efficiently bound to the Ni-NTA resin, while only a small amount was lost in the flow-through (Fig. 2A, lane 2). The 40 mM imidazole wash was successful in the removal of contaminating proteins (Fig. 2A, lane 3). Increasing the imidazole concentration to 400 mM, majority of VP23 was then eluted, only several very faint additional bands were observed (Fig. 2A, lane 4). Then the eluted VP23 was desalted and de-imidazole by crossing Sepharose G-25 column (Fig. 2A, lane 5). Thus, the purity of VP23 was 97% after a series of purification processes above. At last, VP23 was condensed to a concentration of 1.6 mg/mL by using 15 mL ultrafiltration centrifugal tube (Millipore, USA). Western blotting confirmed the identity of the VP23 protein (Fig. 2B).
Immunogenicity analysis of recombinant VP23
The immunogenicity of obtained VP23 was assayed by immunizing rabbits. They were immunized four times by the purified protein and the antisera were pooled after the last immunizations. The immunized rabbits produced a high titer of antiserum against the purified VP23 as determined by ELISA, where the titers reached up to 1:102,400 (Fig. 3A). However, the pre-immunized rabbit serum used as the negative control did not display signal by ELISA, indicating that purified VP23 displayed good immunogenicity. VP23 protein in whole lysis of inducted E. coli BL21 (DE3) and HSV-I infected Vero cells were then analyzed by Western blotting using our antibodies, and uninfected vero cell lyses were used as a negative control. A single band corresponding to the expected size of VP23 was detected in extracts of HSV-I infected vero cells and E. coli BL21 (DE3) lysates, while the same band was not present in uninfected Vero cells (Fig. 3B). Overall, these results indicate that purified VP23 preserved a high immunogenicity and specificity.
In general, bacterial expression system has been one of the most universal expression systems and commonly applied due to a variety of advantages such as relative inexpensive cost, ease of manipulation and rapid growth rate .
In the present study, IMAC purification was applied to establish an efficient approach to purify the VP23 protein. His tag, a small purification partner, has extremely small size and high binding capacity, mild and flexible elution conditions, as well as its ability to function well under denaturing conditions. So the use of His tag decrease the time and cost of protein production process without affecting protein well folding and bioactivity [18, 19]. Thus, a 6Ã-His tag was added to the C-terminus of VP23.
By exploring the induction conditions, it was found that recombinant VP23 expression rarely depended on IPTG concentration and induction temperature on a small scale of fermentation, but mainly depended on induction duration. It is not obvious to improve the expression level when using IPTG and increasing its concentration and elevating the induction temperature higher than 20 °C. IPTG is also toxic to cells to some extent, and the protein tends to be expressed as soluble form and structurally more stable at low temperature. Overall, after several attempts to determine the optimal conditions, the highest amount of VP23 was produced at 20 °C for 24 h without IPTG induction. Thus a large number of over-expressed VP23 was obtained for purifying.
After optimized expression, protein purification was achieved using nickel affinity chromatography and desalted by Sephadex G-25 gel filtration chromatography. To ensure a reasonable yield and high purity of VP23, we adjusted the imidazole concentrations in the purification buffers. It was found that the purification was the most efficient when using 40 mM and 400 mM of imidazole in the washing and elution buffers, respectively. What's more, it is still worthwhile to be noticed that condensed VP23 can easily crystallized when stored at -4 °C for a week, maybe it is caused by its structural instability. A better solution might be that to lyophilize VP23 after it had been purified.
Further, high titer of rabbit polyclonal antibodies was detected by ELISA assay with purified VP23, demonstrating the good immunogenicity of the protein and specificity of the antibodies. Western blot demonstrated that the self-made antibodies could detect VP23 protein expressed in HSV-I infected cells (Vero), but it has a high background. In the western blot result of the whole lysates of recombinant bacteria after induction, we also found another band above the VP23 band. It may be recognized for the reason of some remainder in the purified VP23 protein and simultaneously be injected into rabbits so that its antibodies were also produced.
Since VP23 was identified as an essential and irreplaceable structural protein in capsid which plays crucial roles in replication, assembly, maturation, release and infection of virus. Another research that we undertook showed that siRNA specific silencing UL18 can bring a very high inhibition rate of HSV-I plaque form (data was unpublished). What's more, capsid can be recognized as antigen recognition site and then be cleared out by specific proteic vaccin. The assembly process of capsid proteins has been regarded as the target of screening new drugs which effectively aim at capsid proteins. The assembly steps that occur in the nucleus and the proteins involved are highly conserved among all HSV family members, which suggests that antiviral agents that block these steps might be effective against many different herpesviruses and their associated diseases .
However, there is a lack of tools and commercial antibodies for studying capsid protein VP23. In the present study, we establish a cheap, rapid and highly reproducible protocol to largely express and purify proteins, and this is the first report on the production of polyclonal antibodies specifically against HSV-I capsid protein VP23. Finally, we got high-purity recombinant VP23 and high titer of rabbit polyclonal antibodies. The specific polyclonal antibodies provide a useful and effective tool for further studying HSV-I capsid protein VP23 and its structural and functional characterization in proteomics and targeting drug screening. We believe this protocol also can be used to produce more useful proteins and its polyclonal antibodies.
This study was supported by the National Natural Science Foundation of China (81274170), Key Projects in the National Science & Technology Pillar Program during the Twelfth Five-year Plan Period (SQ2011SF12B02099), Project of Innovative Technology on the Integration of Industry, Education and Research of Guangdong Province (2010b091000013), National High Technology Research and Development Program of China (863 Program) (2012AA02A405).