Introduction

The silkworm, Bombyx mori is an important economic insect and lepidopteran model insect. At present, over 30 million farmer households are involved in sericultural production in China across ten provinces. Unfortunately, the silkworm, B. mori is particularly susceptible to virus diseases, especially due to B. mori nucleopolyhedrovirus (BmNPV), which results in great loss in sericulture [1]. BmNPV is a member of the Baculoviridae family, which has an enveloped, circular, and double-stranded DNA ranging from approximately 80–180 kbp. They infect only invertebrates, and the majority of baculoviruses described are from insects in the order Lepidoptera [2].

The envelope glycoprotein, GP64, of BmNPV is a type I integral membrane protein that is present on the infected cell surface and on the virion. It is a highly conserved gene among the group I alphabaculoviruses [3, 4]. GP64 was also a viral fusion protein mediating pH-triggered membrane fusion during virus entry by endocytosis. Evidences suggested that GP64 was necessary and sufficient for pH-dependent membrane fusion during viral entry. In addition, GP64 is also necessary for efficient budding and production of infectious virions [5, 6]. Recently, a region important for receptor-binding was mapped onto the N-terminal portion of the GP64 ectodomain [7].

Bacillus subtilis is a Gram-positive bacterium, which has been widely used as a model organism for laboratory studies, especially of sporulation. In addition, this organism is not pathogenic for both human and animal, and has been adopted as a probiotic added in the consumption. Previous findings indicated that B. subtilis spore was a powerful vehicle for delivery of heterologous antigens or some bioactive molecule [8, 9]. In this article, an integrative B. subtilis strain, which exhibited GP64 on the B. subtilis spore surface, has been successfully constructed, and the antiviral effect of B. mori orally immunized with B. subtilis spores displaying GP64 examined.

Materials and Methods

Bacterial Strains and Transformation

B. subtilis strain 168 (trp−) was obtained from Bacillus Genetic Stock Center, Department of Biochemistry, The Ohio State University. Preparation and transformation of B. subtilis strain 168 (trp−) competent cells was performed as previously described [10]. Plasmid amplification for nucleotide sequencing, subcloning experiments, and transformation of E. coli competent cells were carried out in the E. coli strain DH5α. Expression of target protein GP64 was carried out in E. coli strain DE3 cells. Bacterial strains were transformed as previously described [11].

Expression of GP64 in E. coli and Preparation of GP64-Specific Antibody

Two primers GP64-F1: 5′-ATGAATTCAATCAGTCATACCAAGGCTTCGA-3′ (EcoRI site was underlined) and GP64-R1: 5′-GCAAGCTTCCAAGTGGGTGGCCG -3′ (HindIII site was underlined) were designed to amplify a 904-bp fragment of BmNPV GP64. The PCR product was cloned first into pMD18-T (TaKaRa) and then into the expression vector pET28a (Promega), which generated plasmid pET28a-GP64 with 6×His-tag sequence at the N-terminus. E. coli DE3 cells containing pET28a-GP64 were grown to an optical density at about 0.6 of OD600 and induced by addition of 0.5 mM isopropyl-β-d-thiogalactopyranoside (IPTG). After incubation for 10 h at 37°C, cells were harvested by centrifugation at 7,000 g for 15 min at 4°C. The fusion protein present in the pellet was separated in 15% SDS-polyacrylamide gels and stained with Coomassie brilliant blue. The induced GP64 band was excised directly, and the antiserum was raised in rats according to the method of Sambrook et al. using Freund’s adjuvant [11].

Construction of Plasmid pJS700-GP64 and Site-Specific Gene Integration in B. subtilis amyE Locus

To obtain an integration of fusion gene cotC-GP64 in B. subtilis amyE locus, a recombinant plasmid for double cross-over with B. subtilis chromosome was constructed. The authors first amplify a 1489-bp fragment from BmNPV genomic DNA with primer pair GP64-F2: 5′-CCGGTACCTGCAACGCGCAAATGA-3′ (KpnI site was underlined) and GP64-R2: 5′-CGGAATTCTTAATATTGTCTACTATTACGGTTTC-3′ (EcoRI site was underlined). The PCR product was digested with KpnI and EcoRI and then was cloned into vector pJS700 (Ning et al. published) to generate the recombinant plasmid named pJS700-GP64, in which the upstream or downstream flanking region of Erythromycin (Em)-cotC-GP64 was homologous to B. subtilis amyE, and it was subsequently verified by restriction analysis and complete sequencing.

Plasmids pJS700-GP64 were digested with enzyme BglII and BamHI, and the resulting linear fragment containing Em-cotC-GP64 gene cassette and amyE flanking region was gel purified, resuspended in distilled water to a final concentration of 200 ng/μl. 20 ng of target fragment was used to transform the competent cells of B. subtilis strain 168 (trp−). The transformed cells were incubated at 37°C, 80 rpm for 30 min in 1 ml LB medium, then 100 μl was spread onto LB medium containing Em 0.4 μg/ml. Plates were incubated at 37°C overnight, and colonies resistant to Em were selected, and target colony with cotC-GP64 integrated in B. subtilis amyE locus was analyzed by amylase activity and PCR confirmation.

Erythromycin-resistant (Emr) clones were the result of a double cross-over recombination, resulting in the interruption of the non-essential amyE in the B. subtilis chromosome (Fig. 2a). Several Emr colonies were selected and were grown on LB plates containing 1% starch overnight, and then the plates were stained with iodine to examine the amylase activity. A blue color was produced by starch in the presence of free iodine when fusion gene cotC-GP64 was integrated in the B. subtilis chromosome. However, no blue color was observed when iodine was added in the starch plates, owing to the expression of amyE from B. subtilis 168 (trp−) chromosome hydrolyzing the starch in the plates.

Interruption of amyE from its locus in B. subtilis chromosome and correct insertion of GP64 and cotC partner at the amyE locus were confirmed by PCR. The relative positions of the primer pairs are shown in Fig. 2b. Primers amyE-F and amyE-R will amplify a 1098-bp wild-type fragment and an 4.5-kb integrated fragment. Primer pair GP64-F2 and GP64-R2 were used to detect the correct insertion of the GP64 gene. Primer pairs amyE-F/GP64-R2 and GP64-F2/amyE-R were used to examine the junction between the upstream or downstream flanking region and GP64.

Preparation of B. subtilis Spores

Sporulation of wild type B. subtilis 168 (trp−) and recombinant strain DRJS711 (cotC-GP64) was induced in DSM (Difco-sporulation media) using the exhaustion method as previously described [12]. Cultures were harvested 48 h after the initiation of sporulation. Spores were collected, washed several times, and purified by lysosome treatment as described by Nicholson and Setlow [12] to break any residual sporangial cells. PMSF was added to inhibit proteolysis. After the final suspension of spores in water treated at 68°C for 1 h to kill any residual cells, the number of purified spores harvested was measured by direct counting using hemocytometer under an optical microscope.

Immunodetection of GP64 in the Extraction from B. subtilis Spore Coat Proteins

Spore coat proteins were extracted from spores suspensions of strain DRJS711 or 168 (trp−) at high density (1 × 1010 spores/ml), using an SDS–DTT extraction buffer as previously described [12]. Extracted proteins were assessed for concentration using the Bio-Rad DC Protein Assay kit.

To confirm that GP64 was expressed on the B. subtilis spore surface of strain DRJS711, culture was harvested after 48 h of the initiation of sporulation. Then, the spore coat proteins were extracted from strain DRJS711 or 168 (trp−), mixed with 1× SDS-PAGE loading buffer and analyzed by Western blot. Gp64 antibodies and pre-serum were used at a dilution of 1:1000. Immunoreactive proteins were visualized using goat anti-rat IgG and horseradish peroxidase. Antibodies against the GP64 protein were used to perform western blot analysis. Western blot filters were visualised by the enhanced chemiluminescence (Amersham Pharmacia Biotech) according to the manufacturer’s instruction.

Larval Bioassays

In order to examine whether B. subtilis spore displaying GP64 has any effect on inhibiting the BmNPV attack in B. mori, bioassays were performed as described following. In brief, spores were harvested from DSM culture, purified by centrifugation, and resuspended in double-distilled water. To determine the antiviral effect of recombinant spores, 300 μl samples containing 0 (control), 1 × 103, 1 × 104, and 1 × 105 B. subtilis spores separately, were applied onto three mulberry leaves. Newly molted second-instar larvae of B. mori were reared at 25°C in canteen and were fed on leaves sprayed with above concentrations of 168 (trp−) wt or DRJS711 recombinant spores. After 12 h, fresh mulberry leaves contaminated with 1 × 106 BmNPV PIBs were added into the canteens when the remaining diet sprayed with spores was cleared out. Fresh diet was added into the canteens when all the virus-contaminated diet was consumed. After 72 h, the number of B. mori with typical symptom of BmNPV infection was recorded. Thirty larvae per dosage were used in the experiment, and each dose was repeated in triplicate. Statistic analysis was performed using the Student’s t test. P value of <0.05 was considered statistically significant.

Results

Construction of Recombinant Plasmid pJS700-GP64

A 1489-bp fragment was amplified from BmNPV genomic DNA by specific primer pair GP64-F2 and GP64-R2 (Fig. 1b Lane 1). The PCR products obtained were cloned into pJS700 to generate the recombinant plasmid pJS700-GP64 (Fig. 1a), in which GP64 and cotC are co-expressed under the control of the cotC promoter. The resulting pJS700-GP64 was subjected to the analysis of restriction–digestion (Fig. 1b Lane 2) and subsequent sequencing. The size of obtained sequences matched with the expected fragments of BmNPV GP64.

Fig. 1
figure 1

Schematic diagram showing the construction of integrative recombinant plasmid pJS700-GP64 and identification of pJS700-GP64 by PCR and enzyme digestion. a The flow chart of plasmid pJS700-GP64 construction. amyE 5′-terminal and amyE 3′-terminal are integrative fragments from amylase gene of B. subtilis 168 (trp−), Em r gene resistant to erythromycin; cotC B. subtilis spore coat. b Lane M DNA marker; Lane 1 1489-bp fragment product amplified from BmNPV genomic DNA; Lane 2 restriction analysis of pJS700-GP64 with KpnI and EcoRI

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Identification of GP64 Integrated in B. subtilis DRJS711 Chromosome

To exhibit GP64 on the spores surface of B. subtilis, a recombinant B. subtilis DRJS711 was generated, in which fusion gene cotC-GP64 was integrated into the B. subtilis chromosome at the amyE locus by double cross-over recombination events (Fig. 2a).

Positive colonies with cotC-GP64 integrated in B. subtilis amyE locus were screened primarily by analysis of amylase activity. Several B. subtilis colonies with integrative chromosome were identified by blue color, but control with no integration in B. subtilis chromosome were identified by no blue color (Fig. 2c).

Fig. 2
figure 2

Strategy for construction of fusion gene cotC-GP64 integrated in B. subtilis amyE locus and by analysis of amylase activity and PCR confirmation. a Schematic diagram showing the structure of amyE locus in wild type and integrative chromosome and the integration of fusion gene cotC-GP64. b Positions of primer pairs used in the confirmation of the GP64 gene insertion. c Identification of recombinant strains by analysis of amylase activity. 1 B.subtilis 168 (trp−) as a control; 2, 3, and 4 recombinant strain with amyE disruption. d PCR analysis of different primer pairs. M DNA marker with sizes indicated. The virus templates are shown above each lane, and the primer pairs used are shown below

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The disruption of amyE was further confirmed by PCR with several primer pairs (Fig. 2d). Primer pair amyE-F/amyE-R produced a 4.5 kb product from the DRJS711 chromosome, in comparison with a 1098-bp product from the 168 (trp−) chromosome. Primer pair GP64-F2/GP64-R2 produced 1489-bp product in DRJS711 chromosome, but no PCR product in 168 (trp−) chromosome. Primer pair amyE-F/GP64-R2 produced 3.8 kb product in DRJS711 chromosome, but no PCR product in 168 (trp−) chromosome. Primer pair GP64-F2/amyE-R produced 2.3 kb product in DRJS711 chromosome, but no PCR product in 168 (trp−) chromosome.

Surface Display of GP64 on the Recombinant Spores

To confirm that GP64 is expressed on the spore surface, antibodies against the GP64 protein were used to perform western blot analysis. The result showed that a 69 kDa band was detected in the extracts from recombinant spores, while no similar band in the extracts from wild-type spores was detected, indicating the presence of the cotC–GP64 fusion protein in the spore coat (Fig. 3).

Fig. 3
figure 3

Western blot analysis of proteins extracted from purified spores of strains 168 (trp−) or DRJS711. Extracted spores coat used for Western blot are indicated above the lanes. The protein standards are indicated on the left

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B. mori Larvae Following Oral Administration of Recombinant Spores Challenged with BmNPV Attack

To test for induction of local and systemic immunity, newly molted second-instard B. mori larvae were immunized orally with either a suspension of wild type B. subtilis 168 (trp−) spores or recombinant spores of strain DRJS711 respectively. The B. mori defense against BmNPV attack results showed that there was significant difference (P < 0.05) between recombinant spores group (percentage of infection, 53.3%) and wild spores or control group (percentage of infection, 73.3%) (Fig. 4).

Fig. 4
figure 4

Groups of B. mori were challenged with BmNPV after oral vaccination with B. subtilis spores. Dose of B. subtilis spores were indicated below. Data were presented as arithmetic means ± standard deviations. Each sample was performed in triplicate

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Discussion

B. subtilis display system is based on the construction of heterologous DNA fused to spore coat gene such as cotB, cotC, and cotG expressed on the surface of spores, which has been proven to be a novel and potentially powerful system to display heterologous antigens. Heterologous proteins and peptides being displayed on the surface of B. subtilis is becoming increasingly important, which has been used as a tool for fundamental and applied research in microbiology, molecular biology, vaccinology, and biotechnology.

Previous findings indicated that many antigens had been successfully exhibited on the surface of B. subtilis spores, including tetanus toxin fragment C (TTFC) of Clostridium tetani, the B subunit of the heat-labile toxin of E. coli (LTB) and alpha toxin of Clostridium perfringens [13–16]. In this work, GP64 from BmNPV was fused to the cotC, a major component of the B. subtilis spore coat, as a fusion partner for the expression of heterologous protein, and integrated into the B. subtilis chromosome at the amyE locus by double cross-over recombination. Western blot analysis against extracted spore coat proteins indicated that GP64 was successfully displayed on the B. subtilis spores.

Although it is arguable as to whether acquired immunity exists in invertebrates, some evidences indicated that invertebrates have a true adaptive immune system. For example, the shrimp vaccinated with VP 26 or VP 28 possessed resistance to White Spot Syndrome and reduced significantly the cumulative mortality of shrimp [17, 18]. Challenging successive mosquito generations with a densonucleosis virus yields progressive survival improvement, indicating that a specific, adaptive manner may be employed to reduce the incidence and severity of disease in arthropods [19].

Heterologous proteins and peptides displayed on the surface of B. subtilis spores was used for oral dosing and shown to generate specific systemic and mucosal immune responses. Based on the above opinion, we consider that B. mori may also employ a currently unknown specific adaptive manner in vivo. Therefore, GP64 was designed to display on the surface of B. subtilis spores using cotC as anchoring motifs, and we investigated whether B. mori vaccinated with the recombinant spores could escape from the attack of BmNPV. The results indicated that, after challenging with BmNPV, B. mori larvae induced with recombinant spores (104 spores) showed a significant lower cumulative infections compared to B. mori larvae induced with wild spores or blank control. It suggested that engineered spores may effectively deliver GP64 antigens to the host cell and induce an unknown adaptive resistance to BmNPV. In addition, BmNPV GP64 is a viral-encoded major envelope glycoprotein involved in host receptor-binding and fusion with the host cell membrane during viral entry. Therefore, it is possible that the competitive binding with the receptor of B. mori columnar epithelial cell between the displayed GP64 on recombinant spores surface and GP64 from BmNPV, which could block or diminish the attack on B. mori from BmNPV.

In conclusion, this study was designed to support the recent development of B. subtilis spores for heterologous antigen presentation and spore-based vaccine. Our results showed that B. subtilis spores have the potential for heterologous antigen presentation and ultimately for use as a vaccine system.