A Highly Efficient and Simple Construction Strategy for Producing Recombinant Baculovirus Bombyx mori Nucleopolyhedrovirus

The silkworm baculovirus expression system is widely used to produce recombinant proteins. Several strategies for constructing recombinant viruses that contain foreign genes have been reported. Here, we developed a novel defective-rescue BmNPV Bacmid (reBmBac) expression system. A CopyControl origin of replication was introduced into the viral genome to facilitate its genetic manipulation in Escherichia coli and to ensure the preparation of large amounts of high quality reBmBac DNA as well as high quality recombinant baculoviruses. The ORF1629, cathepsin and chitinase genes were partially deleted or rendered defective to improve the efficiency of recombinant baculovirus generation and the expression of foreign genes. The system was validated by the successful expression of luciferase reporter gene and porcine interferon γ. This system can be used to produce batches of recombinant baculoviruses and target proteins rapidly and efficiently in silkworms.

Baculoviruses are a group of large viruses with circular double-stranded DNA genomes of 88-153 kb [8], which makes it laborious to manipulate and generate recombinant baculoviruses. Therefore, several construction strategies have been developed to increase the efficiency of recombinant baculovirus generation. To generate the recombinant AcMNPV, Patel et al. have developed a recombinant baculovirus shuttle strategy in yeast with yeast Vector (containing CopyControl origin) and E. coli TransforMax EPI300 were obtained from Epicentre (Madison, WI, USA). DH10Bac/pMON7124 (carrying a tetracycline resistance gene), transfer vector pVL1393 and Lipofectin were obtained from Invitrogen. Low-melting agarose was obtained from Sigma-Aldrich (St. Louis, MO, USA). Anti-PoIFN-γ antibody (Catalogue Number: AB10624) was obtained from Millipore (Boston, MA, USA), and goat anti rabbit pAb-HRP (Code No. 458) was obtained from MBL (Japan). Standard recombinant porcine IFN-γ (Catalogue Number: 985-PI-050) was obtained from R&D SYSTEMS (Minnesota, MN, USA).

Construction of the gene-targeting vector
The pCC-chi-cat vector (GenBank accession number: KU749549) was used to construct BmBac via homologous recombination with BmNPV. Homologous targeting arms were amplified (the primers for fusion PCR, namely Bm-chi-F, Bm-chi-R, Bm-cat-F, and Bm-cat-R, are listed in S1 Table) to remove the cathepsin (GeneID: 1724490) and chitinase (GeneID: 1724489) genes and were inserted into pUC19 via EcoRI/HindIII digestion. A fragment that contained the CopyControl E. coli origin of replication and a chl R gene that was purified from the pCC1BAC vector using SalI digestion was located between the arms.
The pPolh-1629 vector (GenBank accession number: KU749550) was used to construct the defective BmBac lacking a portion of the essential ORF1629 gene. The homologous arms were amplified (the primers for fusion PCR, namely Bm-1629-F, Bm-1629-R, Bm-polh-F and Bmpolh-R are listed in S1 Table) from the BmNPV genome and inserted into the pMD18-T simple vector. The tetracycline resistance (tet R ) gene, which served as an antibiotic selectable marker, was amplified (tet-F, tet-R) from the pMON7124 plasmid and inserted between the arms by XhoI digestion.
The transfer plasmid pVL1393-luc was used to deliver the luc+ gene into reBmBac, and it achieved rescue with a complete ORF1629 gene via homologous recombination in vivo during cotransfection. The luc+ gene was removed from the pGL3-Basic vector by BglII/XbaI digestion and was then transferred to a BamHI/XbaI-digested pVL1393 vector. The transfer plasmid pVL1393-PoIFN-γ was used to deliver the PoIFN-γ gene (NCBI Reference Sequence: NM_213948.1) [35] into reBmBac and to rescue the defective viral DNA. The porcine IFN-γ gene was amplified (primers for PCR, IFN-F and IFN-R are listed in S1 Table) and inserted into the pVL1393 vector by BamHI/EcoRI digestion.

Manipulation of the baculovirus genome in E. coli
The homologous recombination mediated by phage λ red recombinase and the elimination of the FRT-flanked fragment by FLP recombinase were performed according to Wanner et al., as described briefly below [30]. For the recombination, the targeting element with homologous arms was isolated by digestion or PCR from the targeting vector and purified using gel electrophoresis. A 1 μg sample of the targeting element was transferred into electrocompetent cells (E. coli BW25113 with phage λ Red recombinase) using an ECM 630 Electro Cell Manipulator with a voltage of 15 kV/CM according to the manufacturer's instructions (BTX Instrument Division of Genetronics, Inc.). Positive recombinant clones were selected after culture with the appropriate antibiotic. The positive clones were further screened by PCR with primers that spanned the viral genome sequence and targeted element sequence. The BmBac vector with the rpsL-neo gene was transferred into electrocompetent cells (E. coli DH10B with FLP recombinase) to eliminate the FRT-flanked fragment (the rpsL-neo counter-selection gene). The counter-selection gene was removed with FLP recombinase. The positive clones were selected on str R antibiotic plates after culture. The positive clones were also further screened by PCR.
A large amount of high-quality reBmBac DNA was induced by L-arabinose [36] and prepared according to the CopyControl BAC cloning kit instructions (Epicentre) in E. coli DH10B. Purified DNA was resuspended in 100 μL of TE buffer, and the concentration was measured with a UV-Vis Spectrophotometer (NanoDrop 2000, Thermo).

Cell culture and virus amplification in cells
The Bombyx mori-derived cell line, Bm5, was cultured in TC100 insect cell culture medium (Applichem, Darmstadt, Germany) with 10% fetal bovine serum (FBS, Gibco, USA) at 27°C according to published procedures [37]. For the manipulation of transfection or cotransfection, Bm5 cells were cultured in 6-well plates at a constant cell density of 1x10 6 cells per well for 12 h using TC100 medium with FBS. Then cells were washed twice in TC100 medium without FBS and added with the mixture of transfection or cotransfection. After incubation for 4-6 h, the FBS were added into the medium for culture.
For virus amplification or expression in cells, the cells were infected at an MOI of 0.1 (multiplicity of infection of 0.1 p.f.u. per cell) for 1-2 h.

Cotransfection of the transfer vector and reBmBac for recombinant virus with foreign genes
The Bm5 cells were cultured as description. For transfection, 0.5 μg of reBmBac DNA and 1-2 μg of transfer vector DNA were mixed with 6 μL of Lipofectin in a total volume of 60 μL and incubated at room temperature for 15-30 min [38]. Cotransfection was performed with the mixture according to the Lipofectin transfection instruction manual (Invitrogen). The supernatants of cells transfected with recombinant virus was collected after culturing for 5 days at 25-27°C and stored at 4°C.

Expression of foreign genes in silkworm larvae and pupae
Fifth instar silkworm larvae or pupae were injected with recombinant viruses (approximately 10 5 PFU) between the abdominal knobs on the backside. Silkworm larvae and pupae were reared or incubated at 25-27°C and 65% humidity for 108-120 h. Larval haemolymph was collected by cutting the prolegs, and 1-phenyl-2-thiourea was added at final concentration of 0.1 μM to prevent melanization. Larval haemolymph and pupae were stored at -20°C for subsequent assays.
A plaque assay was performed to test the recombination efficiency using conventional methods [37]. The expression level of luciferase in 50 μg of protein lysate was assayed with a Luciferase Assay kit (Promega). The amount of protein in the lysate was measured by the Bradford method [39]. The expression of recombinant porcine IFN-γ was detected by western blotting according to the Protein Blotting Guide (Bio-Rad). The antiviral activity of recombinant porcine IFNs was assayed in a GFP-reduction assay with recombinant vesicular stomatitis virus (VSV-GFP) [40].

Constructing BmBac (BmNPV shuttle vector) by inserting of the CopyControl origin of replication
The CopyControl origin of replication in E. coli (which is formed by E. coli F factor-based partitioning, a single-copy origin of replication [41] and an inducible oriV high-copy origin of replication [42]) was chosen to construct the BmNPV shuttle vector (BmBac) which can be manipulated in E. coli. The pCC-chi-cat vector was constructed to insert this origin into the BmNPV genome and replace the cat and chi genes. Homologous arms of the cat and chi genes were present in the vector. The CopyControl origin of replication and a chloramphenicol resistance (chl R ) selectable marker gene were placed between the arms. Fig 1A shows the structure of the pCC-chi-cat vector.
The CopyControl origin was inserted into BmNPV genomic DNA to inactivate the cat and chi genes. A 7.9 kb fragment (chl R -ori) was obtained from the pCC-chi-cat vector by EcoRI/ HindIII digestion and was then purified using gel electrophoresis. A mixture of the purified fragment (1 μg) and BmNPV DNA (2 μg) was transformed by electroporation into E. coli containing Red λ recombinase. The chl R transformants contained a recombinant BmNPV in which the chi and cat genes were deleted and replaced by the origin fragment. Positive clones were identified and screened by a PCR assay. A fragment spanning the viral genome sequence and targeting vector sequence was amplified with the primers, dchi-F, dchi-R, dcat-F and dcat-R, which are listed in S1 Table. Positive clones were further confirmed by DNA sequencing (Fig  2A). The recombinant BmNPV shuttle vector was named BmBac.
BmBac DNA was extracted by the alkaline lysis method and transfected into Bm5 cells to confirm that the shuttle vector could replicate in both E. coli and insect cells. The extracted BmBac DNA was then transformed back into E. coli for the following study. The results showed that the CopyControl origin was successfully inserted into baculovirus DNA and that BmBac could reproduce in Bm cells.
The effectiveness of the CopyControl origin induction was examined to test the BmBac vector. Selected BmBac DNA was transformed into E. coli TransforMax EPI300, which carries an inducible trfA gene. L-arabinose (0.01%) was added to the E. coli host culture medium to induce DNA replication [36]. Both induced and uninduced BmBac DNA was purified and

Construction of an ORF1629-defective BmBac
The ORF1629 gene, which is located downstream of the polyhedrin gene, was partly deleted in our system on the basis of BacPAK and flashBAC strategies. Baculoviruses that lack a portion of the essential ORF1629 gene cannot replicate in insect cells [12]. Therefore, the defective ORF1629 gene can be rescued only during homologous recombination with an intact ORF1629, which allows the recombinant baculovirus to replicate in insect cells, when the foreign gene is inserted downstream of the polyhedrin gene promoter. This feature allows for more efficient acquisition of recombinant baculoviruses [12]. The pPolh-1629 vector was constructed to knock out the function of this gene. This vector contained two homologous arms of the polyhedrin and ORF1629 genes, with a tetracycline resistance gene inserted between the arms as a selectable marker (Fig 1B).
A portion of the essential ORF1629 gene was deleted from the BmBac DNA. A 2.3 kb tet R element fragment that was flanked by homologous arms of the partial polyhedrin gene and ORF1629 was obtained from pPolh-1629 via PstI/EcoRI digestion and purification. DNA (1 μg) of the fragment was used to electroporate BW25113 electrocompetent cells that contained the BmBac and phage λ Red recombinase. The tet R and chl R transformants were selected on antibiotic-containing plates. The partial polyhedrin gene and ORF1629 gene were deleted and replaced with the tet R gene via homologous recombination. The defective BmBac was also identified and screened by a PCR assay (primers d1629-F, d1629-R, dpolh-F and dpolh-R in S1 Table) and further confirmed by DNA sequencing (Fig 2B).
BmBac DNA without an essential gene replicates in E. coli cells but not insect cells. The cotransfection of reBmBac alone was performed in Bm5 cells and showed no sign of infection. However, cotransfection of the mixture containing the above-mentioned BmBac DNA and the pVL1393 transfer vector was successful. The PCR and cotransfection results confirmed that the essential ORF1629 gene-defective BmBac had been constructed successfully.

Deletion of the excessive resistance gene and construction of reBmBac
We separately introduced the chl R and tet R genes in the ORF1629 gene-defective BmBac vector in combination with the CopyControl origin of replication and partially deleted ORF1629. The tet R gene would be replaced during cotransfection, and it was not present in the recombinant baculoviruses, but the chl R gene could not be replaced. One antibiotic resistance gene is sufficient for genetic manipulation in E. coli. Thus the chl R gene should also be deleted to ensure environmental biosafety. The strategy for deleting the chl R gene involved the use of the FLP/ FRT recombinase system combined with the rpsL-neo counter-selection marker. The rpsL-neo gene (GenBank: GU084141.1) flanked by FRT sites was synthesized and inserted into the pMD18 vector to construct the pRN-FRT vector (Fig 1C). The FRT sites used for this step have been optimized for high efficiency and stable knockout [43,44]. The remaining FRT site was inactivated by FLP recombinase after recombination (Fig 1D).
The FRT-rpsL-neo element fragment was flanked by chl R deletion homologous arms by using PCR (primers, Re-chl-F and Re-chl-R, in S1 Table) and was purified through gel electrophoresis. A 1 μg sample of the fragment was transformed into BW25113 electrocompetent cells that contained the ORF1629-defective BmBac and phage λ Red recombinase. The tet R and kan R (kanamycin resistance) transformants were selected on antibiotic-containing plates. The chl R gene was replaced with the FRT-rpsL-neo fragment by homologous recombination. The positive plasmid was transformed into electrocompetent DH10B cells that contained FLP recombinase. The rpsL-neo counter-resistance gene was removed through FLP recombination, and the streptomycin antibiotic resistance of the DH10B cells was restored. Positive clones that lacked the chl R and rpsL-neo genes were selected on tet R and str R (streptomycin resistance) antibiotic-containing plates. The ORF1629 gene-defective BmBac with only one antibiotic resistance gene (tet R ) was screened with a PCR assay (primers, dchl-F and dchl-R, in S1 Table) and further confirmed using DNA sequencing (Fig 2C). The deletion of chi and cat genes, introduction of CopyControl ori and the defective ORF1629 gene were verified again by using the PCR analyze and sequencing as described before. This construct was named reBmBac (GenBank accession number: KU749552).
A novel reBmBac expression system was developed though the steps described above. Fig 3  illustrates an overview of this system (with reference to Airenne et al. [45]).

Luciferase expression
The luciferase gene is one of the most sensitive reporter genes, and it is remarkably easy to detect. Therefore, this gene was used to test the reBmBac expression system. The luciferase gene was obtained from the pGL3-Basic vector by using BglII/XbaI digestion and was . In BmBac DNA, the chi and cat genes was replaced by the Copycontrol origin and a chl R gene. Then the essential ORF1629 gene was partially deleted by using tet R gene. The chl R gene was removed by using FLP/ FRT system. The finally acquired bacmid DNA was reBmBac.
doi:10.1371/journal.pone.0152140.g003 transferred into the BamHI/XbaI-digested pVL1393 vector to construct the pVL1393-luc vector. A mixture of pVL1393-luc and reBmBac DNA was cotransfected into Bm5 cells. The supernatant, which contained recombinant BmNPV (reBm-luc), was collected after 4-5 days of incubation, and it was used to infect silkworms. A 50 μg sample of protein from lysed cells or larval haemolymph was used in the luciferase assay. The luminescence of the cotransfected cells was approximately 4.86±0.47×10 6 RLU (relative light unit), and the luminescence of silkworm larval haemolymph was approximately 3.42±0.52×10 8 RLU. The background luminescence of the cell culture and larval haemolymph from luc-negative virus-infected samples was 150-300 RLU (S1 Fig). We continuously amplified the recombinant reBm-luc from cotransfected viral stocks in Bm5 cells for three rounds, and infected silkworms to verify the purity of the recombinant baculovirus. If the recombinant BmNPV is not pure, the expression levels of luciferase in cells and larvae will reduced to 10% or even less in the first two rounds of amplification. Here, the luciferase expression in silkworms was not significantly different from that of the original viral stocks. This result indicated that the stable deletion of ORF1629 and the use of CopyControl ori ensured the efficiency of recombinant BmNPV in cotransfection. It also showed that the viral stock acquired during cotransfection was pure to be directly used for viral amplification and protein expression. There was scarcely any wild baculovirus mixing in the target recombinant virus. S1A Fig shows the luminescence data from cells and larvae in the three rounds of amplification and expression. Therefore, the recombinant virus acquired in cotransfection was pure and with stably expression level of luciferase.
Twenty-four reBm-luc plaques were isolated and used to infect Bm5 cells cultured in 24-well plates to measure the recombination efficiency. Viral supernatants were collected from 24 dishes after 108-120 h of incubation. Groups of ten silkworm larvae were injected with the supernatant samples. Luciferase activity was detected in each of the 24 samples by a luminescence assay. The luminescence of 50 μg of protein from infected larval haemolymph samples that had been lysed ranged from 5×10 7 to 5×10 8 RLU (S1 These results demonstrated that the reBmBac expression system could be used to express foreign genes successfully. The recombinant BmNPV in the cotransfection viral stock could be used directly for expression.

Expression of recombinant porcine interferon-γ
PoIFN-γ gene expression was tested to further examine the utility of the reBmBac expression system. First, the PoIFN-γ gene was cloned into the transfer vector pVL1393. Second, the gene was inserted into the viral genome to construct recombinant reBm-PoIFN-γ via homologous recombination. The recombinant reBm-PoIFN-γ virus was injected into larvae or pupae. The expression product of porcine IFN-γ was analysed by western blotting (Fig 4) with an anti-PoIFN-γ antibody (Millipore, USA). Fig 4 shows that an approximately 19 kDa protein band that reacted with the anti-PoIFN-γ antibody was detected in the samples. No corresponding immunoreactive protein was detected in the negative control sample from larval haemolymph infected with WT BmNPV. Therefore, these results indicate that porcine IFN-γ was successfully expressed in silkworms.
The antiviral activity assay indicated that the reBm-PoIFN-γ product exhibited antiviral activity that exceeded 1 x 10 6 IU/mL in haemolymph. Twenty-four plaques were also isolated and used to infect silkworms. The antiviral activity assay demonstrated that the activity of these plaque samples were approximately 6.2 ± 1.3×10 5 to 2.4 ± 0.7×10 6

Discussion
The above data demonstrated the successful development of the novel defective-rescue reBmBac expression system. Recombinant BmNPV viral stocks were obtained within 4-5 days with this system, and recombinant proteins expressed in silkworm larvae or pupae were harvested after an additional 4-5 days (Fig 5). This system is remarkably efficient. Many experiments have shown that the expression level of a heterologous protein in five silkworm larvae or pupae is approximately equal to the expression levels of fermentation products from 1 L of sf9-AcMNPV cells [7]. Therefore, this reBmBac-silkworm expression system is a rapid and highly efficient system for producing large amounts of foreign protein.
The BmNPV-silkworm expression system has been reported that it is a powerful and potential system for foreign gene expression. Scientists have long attempted to develop more convenient methods for generation of recombinant BmNPV and high-level expression of recombinant proteins in silkworm. The recombinant baculovirus generation strategies of BmNPV are similar to those corresponding ones of AcMNPV [46]. The Bac-to-Bac system and the BacPAK system are two typical and commonly used baculovirus expression system. A foreign gene can be inserted into BmNPV baculovirus DNA in E. coli by using the Bm-Bac-to-Bac system, which is a convenient and easy-to-used expression vector for recombinant baculovirus construction and expression [17]. There were studies shown that the recombinant Bacmid DNA or special E. coli containing Bacmid could be used to inject the silkworm directly for expression, but the expression efficiency and level were not ideal [17,47]. The Bac-to-Bac system is widely used in the BEVS expression fields, which benefit from the convenience and stabilization of its operation in E. coli. However, this system is also relatively inconvenient for the heterologous expression of many genes because each gene must be manipulated independently in E. coli. In the BmBacPAK system, there are several Bsu36I sites in baculovirus DNA [48]. The Bsu36I digested baculovirus DNA is linear and lacks part of ORF1629. Incomplete enzyme digestion dramatically reduces the ratio of recombinant virus. In comparison with these two BEVSs, the reBmBac expression system combines their advantages, i.e., convenient viral genome DNA preparation in E. coli and easy recombinant baculovirus production via cotransfection. The stable deletion of the essential ORF1629 gene in E. coli beforehand made the digestion step could be omitted and ensured the efficiency and purity of recombinant baculoviruses that were obtained by cotransfection. In addition, some disadvantages of these two BEVSs were avoided. It always takes at least two weeks to produce one recombinant baculovirus using Bac-to-Bac BEVS, whereas it takes only approximately five days when using reBmBac BEVS to produce various recombinant baculoviruses that contain different genes of interest at the same time. The batch production of different baculoviruses can be achieved conveniently via one-step cotransfection, and thus, a large amount of time is saved. It is much more convenient to prepare a large amount of high-quality parental viral genome DNA by using the reBmBac BEVS rather than the BmBacPAK system because of the CopyControl ori [33]. Besides, the ORF1629-defective Bacmid bBpGOZA expression system is another easy-to- use system [19]. Our reBmBac system has two major improvements relative to this system. First, the fragment replaced during homologous recombination in our reBmBac system is only a 1.2 kb tet R gene. In contrast, the fragment in bBpGOZA is more than 6 kb, and it contains a miniF replicon and a kan R gene. Second, the CopyControl origin in our system facilitates the large-scale preparation of high-quality reBmBac DNA. Our results over the course of many experiments indicated that the high quality of reBmBac DNA and transfer plasmid DNA are key factors for achieving good recombination results. We observed a greater than fivefold difference in the luciferase expression level and the expression levels of other proteins of interest depending on the quality of the DNA (S2 Table). The quality of well-preserved (fresh or store at -20°C) and badpreserved (freeze-thawed for five rounds) transfer vectors are different. And the quality of parent reBmBac viral DNA, extracted from E. coli by induction or non-induction methods, are different. Over many protein expression trials in this reBmBac system, we found that when we used the same amount but different quality of pVL1393-luc transfer vector DNA to mix with the same tube of reBmBac DNA and to cotransfect cells, the luminescence from the cells and larvae could reach a tenfold difference. When we use the same amount but different quality of reBmBac DNA to mix with the same tube of well-preserved pVL1393-luc vector and to carry out the cotransfection, the luminescence from cells and larvae were at least fivefold different.
The expression tests of luciferase and PoIFN-γ revealed that the purity of the recombinant viruses acquired by cotransfection was sufficient to directly infect silkworms for expression. The amount of recombinant virus from one cotransfection was sufficient to infect at least 5,000 silkworm larvae or pupae. The expression was sufficiently high to perform functional research on the expressed protein. Furthermore, the three continuous rounds of viral reproduction demonstrated that the efficiency and purity of recombinant BmNPV were sufficient for virus amplification and protein expression. The plaque assays of reBm-luc and reBm-PoIFN-γ showed that the reBmBac system allows 100% recombinant BmNPV formation. Combining the three rounds of amplification test, the direct expression in silkworms obviates the need for further virus plaque purification assay. Therefore, the time required from expression plasmid production to harvesting a large amount recombinant protein is only 10 days, in theory. Obtaining recombinant virus requires only a simple cotransfection, which could be performed in batch mode and would be suitable for the expression of different foreign proteins after batches. In general, we performed 8-12 cotransfections in two 6-well cell culture plates (6 x 35 mm) for one batch and used the luciferase reporter to evaluate the recombination and expression efficiency in silkworms. We were confident that a group of recombinations and gene expression trial in the silkworms was successful when the luminescence of the cell lysate from the cotransfection reached 2 x 10 6 RLU or greater, and the larval haemolymph achieved 5 x 10 7 RLU or greater. These features are beneficial for the rapid and efficient expression of foreign proteins.
The three rounds of amplification and infection of reBm-luc in cells and silkworms showed that the luciferase expression level was stable by using this reBmBac system. We have used the uninduced reBmBac DNA to generate the luciferase gene recombinant BmNPV. The luminescence reduced dozens-fold in the first round of amplification and expression in larvae, which indicated that the recombinant BmNPV acquired in cotransfection is not pure and not suitable for directly expressing in larvae. The same phenomenon sometimes appears in the using of BacPAK6 system. Here, the stable expression data in the three rounds indicated that the reBmluc acquired in cotransfection was pure and could be used for expression directly. In the plaque assay of reBm-luc, all of the screened virus stocks drive positive expression of luciferase. But the expression levels of them were steadily different. This is because of the reBm-luc acquired in cotransfection is a mixture of various types of recombinant viruses, while the screened virus stock is single clone recombinant virus in theory. Therefore, the expression level of luciferase of 24 plaque stocks were higher or lower than that of contransfection virus stock. And the plaque assay of reBm-PoIFN-γ also verified this result. Even so, the several virus stocks that drive higher expression levels could be selected for further researches or large-scale expression of target genes. Generally, the expression levels of these optimized recombinant virus stocks could be at least twofold higher than that of cotransfection stock. This optimization of expression level is recommended step for further large-scale producing.
The introduction of the CopyControl replicon facilitates the generation of large-plasmid DNA in E. coli. Wild [33] and the CopyControl BAC cloning kit instructions indicate that the copy number of the vectors, containing CopyControl origin, depends on the vectors' size. The copy number for Bacmids could increase by 10-to 20-fold upon induction. The reBmBac DNA yield was similar in our system. The oriV origin of replication should be silent during genetic manipulations in baculovirus genome. The single-copy replicon allows for the autonomous replication and stable segregation of plasmids at a low number, which facilitates positive clone selection. The high-copy oriV origin of replication is induced when it is necessary to prepare Bacmid DNA for cotransfection. High-quality reBmBac DNA, which can be prepared easily on a large scale, is one of the key factors for successful homologous recombination. The above mentioned results showed that the quantity of reBmBac DNA extracted from 3 mL of overnight culture medium was sufficient for performing at least 10 cotransfections.
Recombinant BmNPV that contained foreign genes contained no antibiotic resistance gene in our system, which provides improved environmental biosafety. The tet R gene was replaced with a foreign gene during cotransfection. The chl R gene was deleted with the FLP/FRT system and the rpsL-neo counter-resistance selection system. The deletion of the chl R gene was also an example of the genetic manipulation of viral genes or the introduction of foreign genes into the other baculoviral genome sites in E. coli. This makes the multigene expression can also be achieved by using our reBmBac expression system as the using of other published system in silkworms [49]. The FRT site used here is a mutant site [43,44]. The remaining FRT sequence is inactivated after recombination, which is favourable for ensuring the stability of the viral genome.

Conclusions
A novel defective-rescue reBmBac expression system was successfully developed. The examples of luciferase and PoIFN-γ expression showed that foreign genes can be successfully expressed with this system. These results show that the novel reBmBac silkworm expression system is excellent and has the following advantages: (i) This system allows for simple and rapid generation of recombinant BmNPVs carrying foreign genes. Because the defective ORF1629 and the introduction of the CopyControl origin of replication, the purity of the recombinant virus harvested during cotransfection approaches 100%. The luciferase expression level could be used as the quality control for the cotransfection and expression efficiency of foreign genes in larvae. The amount and quality of recombinant viral stock is sufficient for various research applications. Plaque purification can be used to further improve the expression level but is not a necessary step. (ii) This system facilitates the batch generation of recombinant BmNPV with various foreign genes, thus allowing for the mass production of proteins of interest. (iii) No antibiotic resistance gene is present in recombinant BmNPV, and thus the biosafety of this system is ensured. The product of reBm-PoIFN-γ (cotransfection viral stock) exhibited antiviral activity that exceeded 1 x 10 6 IU/ mL haemolymph ("co-t" bar). 24 plaque viral stocks were screened using plaque assay and used to infect silkworms. The antiviral activity of the best sample was 2.4 ± 0.7x10 6 IU/mL, which exhibited a twofold improvement in antiviral activity compared with the cotransfection sample. The commercial positive control ("c-1" bar) sample was the standard recombinant porcine IFN-γ (R&D Systems, USA). The standard sample was reconstituted at 50 μg/mL and its antiviral activity was about 4.5 x 10 6 IU/mL. The negative control ("c-2" bar) sample was the non-interferon recombinant BmNPV infected larvae and it shown no antiviral activity. The activity of PoIFN-γ in one milliliter haemolymph of the best sample (in our system) was equal to that of 271.4 ± 81.5 ug standard control sample. (TIF) S1