Recombinant baculovirus expression vector and cell

Abstract

A recombinant baculovirus expression vector or cell comprising an engineered baculovirus fp25k gene with one to three modified or mutated spots, the modified spots comprise the two 7-adenine mononucleotide repeats (MNR) and the 10.sup.th TTAA site. The invention also provides the method of making the vector and baculovirus.

Claims

1. A recombinant baculovirus expression vector, comprising an engineered baculovirus fp25k gene, which comprises a nucleic acid of SEQ ID NO. 2.

2. The vector of claim 1, wherein the sequence set forth by SEQ ID NO: 2 is modified at nucleotide sequence 181-187 to AAGAAAA and nucleotide sequence 421-430 to CTGAAGAAGA.

3. The vector of claim 1, wherein the baculovirus is an Autographica californica multiple nucleopolyhedrovirus (AcMNPV) with a nucleic acid of SEQ ID NO. 1.

4. A recombinant baculovirus cell comprising the engineered baculovirus fp25k gene of claim 1.

5. A method of preparing a baculovirus vector suitable for production of a gene product in a host cell, the baculovirus vector being an engineered baculovirus fp25k gene of a nucleic acid of SEQ ID NO. 2, comprising the steps of: providing a plasmid that contains the wild-type fp25k gene modifying nucleotide sequence 181-187 of the wild-type fp25k gene to AAGAAAA to produce a first modified plasmid of SEQ ID NO. 7; and using the first modified plasmid of SEQ ID NO. 7 as a template to modify nucleotide sequence 421-430 to CTGAAGAAGA with one or more primers to generate the engineered baculovirus fp25k gene of SEQ ID NO. 2.

6. A method of preparing a recombinant baculovirus with the engineered baculovirus fp25k gene of claim 1, comprising the steps of providing one or more baculovirus cells, and co-transfecting the baculovirus cells with the engineered baculovirus fp25k gene and a viral DNA with a gfp expression cassette at the fp25k locus.

Description

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

(1) FIG. 1A is a schematic drawing of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) fp25k (a nucleic acid of SEQ ID NO. 1) mutational hot spots (A7-1, A7-2 and 10.sup.th TTAA) that are mutagenized into the recombinant baculovirus with a nucleic acid of SEQ ID NO. 2 (also referred to as AcP3SDM). Small circles above the open reading frame (ORF) represent TTAA sites, while the black small circle represents the 10.sup.th TTAA site. Mutated bases underlined corresponding to that in the wild type AcMNPV (AcP3) with a nucleic acid of SEQ ID NO. 1 are shown in brackets for the engineered fp25k gene of AcP3SDM.

(2) FIG. 1B depicts PCR detection of fp25k gene mutations using DNA templates extracted from the passaged budded viruses (BV) (P1 to P3, P5, and P10) showing increased PCR products of 1.5 kbp and decreased products of 1.2 kbp from the wild-type (wt) AcMNPV E2 plaque 3 (AcP3) strain (a nucleic acid of SEQ ID NO. 1). On the other hand, no change of the 1.2 kbp products for the recombinant baculovirus AcP3SDM (a nucleic acid of SEQ ID NO. 2) is detected. P0 is non-passaged virus stock; P4 and 6 to 9 are not shown.

(3) FIG. 1C has two partial histograms of DNA sequencing reactions: one is 1.2 kbp PCR product of AcP3 P0 (no passage) (a nucleic acid of SEQ ID NO. 1), showing the wt 10.sup.th TTAA site; the other is 1.5 kbp PCR product of P5 (passaged 5 times) of AcP3, a nucleic acid of SEQ ID NO. 10, showing the 10.sup.th TTAA site with a host DNA insertion (*). The putative transposition site is underlined.

(4) FIG. 2A refers to photographs illustrating polyhedra production between the cells infected with AcP3 (a nucleic acid of SEQ ID NO. 1) and the cells infected with AcP3SDM (a nucleic acid of SEQ ID NO. 2) during Hi5 cell passage. Arrow heads point to cells with poor polyhedra product. Arrows point to cells with good polyhedra production.

(5) FIG. 2B is a diagram comparing polyhedrin protein yields between the cells infected with AcP3 (a nucleic acid of SEQ ID NO. 1) and the cells infected with AcP3SDM (a nucleic acid of SEQ ID NO. 2) during Hi5 cell passage. Error bars denote standard deviation (SD); n=3. Treatments with different number of asterisk indict significant differences at P0.05. Bars, 10 m.

(6) FIG. 3A refers to photographs illustrating polyhedra production between the cells infected with AcP3 (a nucleic acid of SEQ ID NO. 1) and the cells infected with AcP3SDM (a nucleic acid of SEQ ID NO. 2) during Sf9 cell passage. Arrow heads point to cells with poor polyhedra product. Arrows point to cells with good polyhedra production.

(7) FIG. 3B is a diagram comparing polyhedrin protein yields between the cells infected with AcP3 (a nucleic acid of SEQ ID NO. 1) and the cells infected with AcP3SDM (a nucleic acid of SEQ ID NO. 2) during Sf9 cell passage. Error bars denote standard deviation (SD); n=3. Treatments with different number of asterisk indict significant differences at P0.05. Bars, 10 m.

(8) In describing the preferred embodiment of the invention which is illustrated in the drawings, specific terminology will be used for the sake of clarity. However, it is not intended that the invention be limited to the specific term so selected and it is to be understood that each specific term includes all technical equivalents which operate in a similar manner to accomplish a similar purpose. For example, the words connected or terms similar thereto are often used. They are not limited to direct connection, but include connection through other elements where such connection is recognized as being equivalent by those skilled in the art.

DETAILED DESCRIPTION OF THE INVENTION

(9) Broadly, the present invention provides for an improved baculovirus expression vector or a recombinant baculovirus comprising an engineered baculovirus fp25k gene that is more mutation resistant. The expression vector of the present invention is more resistant to mutation during the process of producing a desired protein, virus, protein hybrid, or virus hybrid. The engineered baculovirus fp25k gene has one to three modified or mutagenized (or mutated) spots, including two 7-adenine mononucleotide repeats (A7 MNR) and the 10.sup.th TTAA site. The first A7 MNR can be referred to as A7-1 or A7-1 MNR. Preferably, the A7-1 MNR is modified or mutagenized into AAGAAAA; the second A7 MNR (A7-2 or A7-2 MNR) and the 10.sup.th TTAA site are collectively mutagenized into CTGAAGAAGA. These are silent mutations. Silent mutations are DNA mutations that do not result in a change to the amino acid sequence of a protein. Other suitable sequences can also be used for the present invention so long as they are silent mutations.

(10) As used herein, the terms modified and mutated refer to a mutation and/or deletion and/or insertion into the gene and can include genetic events that occur naturally, and/or in a laboratory setting and by design e.g., through cell passaging, and/or through one or more recombinant genetic methods.

(11) Preferably, the A7-1 MNR is mutated by converting AAAAAAA to AAGAAAA to generate an engineered plasmid (a nucleic acid of SEQ ID NO. 7) using a pair of suitable mutagenesis primers. The preferred primers are pA7-1F, 5-CGA CAG CAG GCT GAA TAA TAA GAA AAT TAG AAA C-3 (a primer of SEQ ID NO. 3), and pA7-1R 5-CTA ATT TTC TTA TTA TTC AGC CTG CTG TCG TGA ATA CCG-3 (a primer of SEQ ID NO. 4). Other suitable primers can also be used. Suitable primers are the primers that can provide the silent mutations mentioned above.

(12) This plasmid (a nucleic acid of SEQ ID NO. 7) is then used as a template to mutate the A7-2 MNR and the overlapping 10th TTAA site collectively (also called the combined A7-2 MNR and 10th TTAA site) by mutagenizing CTTAAAAAAA to CTGAAGAAGA using suitable primers. After the mutation, the collective site can be referred to as the modified A7-2 MNR/10th TTAA site. The preferred primers are pTTAA-F, 5-GTT ACT GAA GAA GAC TCG CGA CGC TCT GTT GC-3 (a primer of SEQ ID NO. 5), and pTTAA-R 5-CGT CGC GAG TCT TCT TCA GTA ACA GCT TTT G-3 (a primer of SEQ ID NO. 6). Other suitable primers can also be used so long as they can provide the silent mutations mentioned above. The resulting engineered plasmid (also called the engineered fp25k gene vector) includes the fp25k gene with three mutated spots (a nucleic acid of SEQ ID NO. 2). In other words, the resulting plasmid vector lost the three mutational hot spots of the fp25k gene. In some embodiments of the present invention, the engineered plasmid includes the fp25k gene with one or two mutated spots.

(13) Mutations of AcMNPV fp25k are reported to be caused by slippage replication errors of the two 7-adenine mononucleotide repeats (MNR) and a 287 bp host DNA insertion at the 10.sup.th TTAA site in Sf21 cells (Cheng et al., 2013 J. Gen. Virol 94: 166-176; Gin et al., 2010 J. Gen. Virol. 91: 3053-3064). In Example 1 below, during the Hi5 cell infection, the 287 bp host cellular DNA was also found inserted at the 10.sup.th TTAA site at the wt fp25k locus. The other two A7 MNRs might also mutate in Hi5 cells, but were not specifically detected in Example 1.

(14) In some embodiments of the present invention, a recombinant virus with the engineered fp25k gene is generated by co-transfecting Sf9 cells with the engineered fp25k gene vector and viral DNA that contained a gfp expression cassette at the fp25k locus. The recombinant virus is more resistant to mutation. Suitable baculovirus cells can be Hi5 cell, Sf21 cell, Sf9 cell, or a combination thereof.

(15) The method of co-transfecting the virus/plasmid DNA to cell can be found in Cheng et al., 2013, J. Gen. Virol. 94: 166-176, Reduction of polyhedrin mRNA and protein expression levels in Sf9 and Hi5 cell lines, but not in Sf21 infected with Autographa californica multiple nucleopolyhedrovirus fp25k mutants, and in Ogay et al. 2006, Cytotechnology 51, 80-98, both of which are incorporated by reference herein. In the cell transfection method, a monolayer of Sf9 cells in a 30 mm diameter tissue culture dish were co-transfected with 1 g of engineered fp25k gene vector and 250 ng of viral DNA that contained a gfp expression cassette at the fp25k locus by using polyethylenimine (PEI) (Ogay et al, 2006, Cytotechnology 51, 80-98). Using this method, the resulting recombinant virus contained the engineered fp25k gene but not the gfp expression cassette.

EXAMPLE

(16) The present invention will be described below in more detail. The examples are exemplification only and do not limit the present invention.

Example 1

(17) Wild-type (wt) AcMNPV E2 plaque 3 (AcP3) strain with a nucleic acid of SEQ ID NO. 1 was used for the mutagenesis of the fp25k gene. To eliminate the three hyper-mutational hot spots, a plasmid transfer vector (pGEMT25k) that contained the wt fp25k gene with about 500 bp flanking sequences on each side of the fp25k gene was used as the template for site-directed mutagenesis using a QuikChange I Site-Directed Mutagenesis Kit (Agilent Technology). The titers of both AcP3 and AcP3SDM were determined by real time qPCR using a pair of AcMNPV pp34 primers (see Cheng et al., 2013 for the method).

(18) The mutagenesis work was accomplished in a two-step process. First, the A7-1 MNR was mutated by converting AAAAAAA to AAGAAAA to generate a plasmid pUCA7-1 (a nucleic acid of SEQ ID NO. 7) using a pair of mutagenesis primers (pA7-1F, 5-CGA CAG CAG GCT GAA TAA TAA GAA AAT TAG AAA C-3, a primer of SEQ ID NO. 3, and pA7-1R 5-CTA ATT TTC TTA TTA TTC AGC CTG CTG TCG TGA ATA CCG-3, a primer of SEQ ID NO. 4) (FIG. 1A). Secondly, the plasmid pUCA7-1 (a nucleic acid of SEQ ID NO. 7) was used as a template to mutate the A7-2 MNR and the overlapping 10th TTAA site by mutagenizing CTTAAAAAAA to CTGAAGAAGA using a pair of primers (pTTAA-F, 5-GTT ACT GAA GAA GAC TCG CGA CGC TCT GTT GC-3, a primer of SEQ ID NO. 5, and pTTAA-R 5-CGT CGC GAG TCT TCT TCA GTA ACA GCT TTT G-3, a primer of SEQ ID NO. 6) to generate the pFPSDM plasmid DNA (a nucleic acid of SEQ ID NO. 2) that lost all the three mutational hot spots of the wt fp25k gene (FIG. 1A). The pFPSDM plasmid DNA is also referred to as the transfer vector pFPSDM plasmid DNA or pFPSDM plasmid.

(19) To generate a virus with the engineered fp25k gene, a nucleic acid of SEQ ID NO. 2, the transfer vector pFPSDM plasmid DNA and the AcP325GFP viral DNA that contained a gfp expression cassette at the fp25k locus were used to co-transfect Sf9 cells to generate recombinant viruses that lacked gfp but contained the engineered fp25k gene. In the cell transfection method, a monolayer of Sf9 cells in a 30 mm diameter tissue culture dish were co-transfected with 1 g of engineered fp25k gene vector and 250 ng of viral DNA that contained a gfp expression cassette at the fp25k locus by using polyethylenimine (PEI). The method of co-transfecting the cell with the AcP325GFP viral DNA and pFPSDM plasmid DNA can be found in Cheng et al., 2013, J. Gen. Virol. 94: 166-176, Reduction of polyhedrin mRNA and protein expression levels in Sf9 and Hi5 cell lines, but not in Sf21 infected with Autographa californica multiple nucleopolyhedrovirus fp25k mutants; and also see Ogay et al, 2006, Cytotechnology 51, 80-98. The recombinant virus AcP3SDM was plaque-purified against the background of gfp expression parental viral plaques to be used in the comparison of fp25k stability in Hi5 cell passage between the parent AcP3 and the recombinant AcP3SDM.

(20) The titers of both AcP3 (a nucleic acid of SEQ ID NO. 1) and AcP3SDM (a nucleic acid of SEQ ID NO. 1) were determined by real time qPCR using a pair of AcMNPV pp34 primers (primers of SEQ ID NO. 11 and SEQ ID NO. 12) (see Cheng et al., 2013 for the method). The engineered viruses AcP3SDM and AcP3 were serially passaged in Hi5 cells according to the method stated in de Rezende et al. except for the modification stated below (de Rezende et al., 2009, J. Invertebr. Pathol. 100: 153-159): Before any passage and at each passage, the inoculum was tittered using real time qPCR. Hi5 cells at the density of 5105 cells/dish in six well plates were infected with a multiplicity of infection (MOI) of 10 plaque forming units (p.f.u)/cell using BV (budded virus) produced from a previous cell infection. Polyhedra production through the virus infected cells was monitored by phase contrast microscopy. At day four post infection, the majority of Hi5 cells were infected. The BVs in the media were tittered and harvested for infecting Hi5 cells in the next passage.

(21) A total of 10 passages were conducted for PCR analysis of the fp25k stability (see FIG. 1B). For the PCR analysis, an equal volume of the virus-containing medium (300 l) from each passage was used for viral DNA extraction, and the extracted DNA was dissolved in 10 l of water. In the PCR analysis of fp25k mutations, a pair of FP primers (FP-F, 5-ATGCATAGCAATGTCTTC-3, a primer of SEQ ID NO. 8, and FP-R, 5-TTTACGCACCATATACGC-3, a primer of SEQ ID NO. 9) and the high fidelity pfu DNA polymerase together with 1 l of each viral DNA from passaged viruses (either from AcP3 or AcP3SDM) were used. The amplified PCR products were analyzed by gel electrophoresis (0.7% agarose running at 90 V for 50 min in TAE buffer), and the resolved DNA products were recovered and directly sequenced. The PCR amplification of the fp25k locus showed in FIG. 1B that at the wt fp25k locus, in addition to the expected 1.2 kbp product, a 1.5 kbp product was found, suggesting the host DNA insertion had occurred.

(22) At passage 1, about 50% of progeny viruses had the host DNA insertion (1.5 kbp PCR product) in the fp25k gene, which suggests that the host DNA insertion occurred at a high frequency. After five passages of the viruses, almost all of the progeny viruses had the host DNA insertions (FIG. 1B). The intensity of the 1.5 kbp PCR product also continued to increase from passage 1 until passage 5 and then leveled off, reflecting the increased budding of viral progeny into the media. The increased budding of viral progeny is one hallmark of the fp25k mutation phenotype (Harrison et al., 1995 J. Gen. Virol. 76(Pt 6): 1451-1459; Kelly et al., 2008 Virus. Res. 133: 157-166; Wu et al., 2005 J. Gen. Virol. 86: 2439-2444). When the 1.5 kbp PCR product of wt AcP3 passage 5 was sequenced (a nucleic acid of SEQ ID NO. 10), the only mutation found was the host DNA insertion at the 10th TTAA site, and no other mutation in AcMNPV fp25k was found (please see FIG. 1C).

(23) In the engineered fp25k gene of the AcP3SDM with a nucleic acid of SEQ ID NO. 2 after ten passages, no host DNA insertion was found and no increase in progeny virus budding, indicating that there was no other fp25k mutations (FIG. 1B). Sequencing the PCR products from passage 5 of AcP3SDM confirmed the identity of the engineered fp25k gene. There is also no difference in the yield of the polyhedrin in Hi5 cell infection among passages 1 to 10 of AcP3SDM. These data suggest that the engineered fp25k gene of AcMNPV maintained a higher stability, leading to higher polh promoter activities and higher protein expression yields than that of the AcMNPV with a wt fp25 gene during Hi5 cell infection.

(24) This example demonstrates the improved stability of the engineered AcMNPV fp25k gene (a nucleic acid of SEQ ID NO. 2) over that of the wt fp25k gene (a nucleic acid of SEQ ID NO. 1) during the Hi5 cell passaging. The fp25k gene of other baculoviruses, such as the commercial Bacmid (Invitrogen) and flashBAC (BioNovus Life Sciences) can also be similarly engineered to increase protein stability during cell infection.

(25) While not wishing to be bound by theory, it is presently believed that insertion of the 287 bp Hi5 host DNA fragment at the 10.sup.th TTAA site of the fp25k gene is likely carried out by the Hi5 transposase via a transposition reaction. It also appears that Hi5 transposase was highly active so that about 50% of the BV from passage 1 carried the 278 bpHiS DNA insert (FIG. 1B). Even though high MOI infection was used in this example, the high transposase activity may suggest that even at low MOIs, such as 0.1 or 0.01, the Hi5 transposase may also insert the 287 bp Hi5 host DNA into the fp25k gene to inactivate the 25K protein and thus reduce the polh promoter power, which results in the reduction of protein expression yield. Therefore, blocking the function of the Hi5 transposase may reduce the possibility of the insertion of the host DNA into the fp25k gene, leading to an increased yield of protein expression.

Example 2

(26) This study evaluated whether the engineered fp25k gene of AcMNPV can lead to a higher protein expression yield than that of the AcMNPV with a wt fp25 gene during Hi5 cell infection.

(27) Wild-type (wt) AcMNPV E2 plaque 3 (AcP3) strain (SEQ ID NO.1) was used. The engineered fp25k gene of AcMNPV, a nucleic acid of SEQ ID NO.2, was produced according to the procedures listed in Example 1.

(28) Polyhedra Harvesting:

(29) Hi5 cells were seeded at 510.sup.5 cells/well in six-well plates. BVs of AcP3 passages 1, 5 and 10 as well as AcP3SDM passages 1, 5, and 10 were tittered by the end-point dilution method. In this end-point dilution tittering method, Sf9 cells in 60-well plates were infected with 10 serial dilutions (10 fold each) of the virus stocks. The numbers of wells with infected cells were counted to estimate the dilution of virus that produced infection in 50% of wells (O'Reilly, D. R., Miller, L. K. & Luckow, V. A. (1992). Baculovirus Expression Vectors: a Laboratory Manual. New York: W. H. Freeman & Co.). The Hi5 cells were then infected with the respective virus (AcP3 or AcP3SDM) at a multiplicity of infection (MOI) of 1 virus/cell in triplicate.

(30) At four days post infection, the media that contained BVs from the infected Hi5 cells were withdrawn. The cells were lysed with 1 ml of 0.5% Sodium Dodecyl Sulfate (SDS) for 30 minutes with rocking at 4 rocks/min at room temperature to release polyhedra from cells. Success of complete cell lyses was confirmed by microscopy. The released polyhedra were washed four times in 1 ml of 0.5% SDS by centrifugation at 16,100g for 1 min. The final polyhedra pellet was washed with sterile water to produce the purified polyhedra.

(31) Polyhedra Protein Measurement:

(32) The purified polyhedra were solubilized by treatment with 1 ml of 0.1 M Na.sub.2CO.sub.3 solution (pH 10.5) at 37 C. for 30 min or until no pellet was seen after centrifuging at 16,100 g for 1 min. Polyhedrin yields were estimated by the Bradford method using a Bio-Rad protein assay system. The purified bovine serum albumin (BSA) was used as a standard. The 1 dye reagent solution (2 ml) was added to a glass tube. Forty l of each diluted BSA in 0.1 M Na.sub.2CO.sub.3 solution (0.2, 0.4, 0.6 and 0.9 mg/ml) and unknown samples were added to individual glass tubes with dye and mixed. The mixed protein/dye solutions were incubated at room temperature for 5 minutes. The spectrophotometer was set to 595 nm. The absorbance of the standard and unknown samples was measured. A standard curve by plotting the 595 nm values (y-axis) versus their concentration of BSA in g/ml (x-axis) was constructed. The unknown sample concentrations were estimated using the standard curve. In this procedure, the unknown samples refer to the purified polyhedrin from AcP3 and AcP3SDM. The polyhedrin yields were then compared between that of AcP3 and that of AcP3SDM (FIGS. 2A and 2B).

(33) Results: The results (FIG. 2A and FIG. 2B) show that there was a progressive reduction of polyhedron during AcP3 passage whereas there was no detectable reduction of polyhedron of AcP3SDM in Hi5 cell passage.

Example 3

(34) This example evaluated the stability of the engineered fp25k gene of AcMNPV_in sf9 cell passage. The stability of the engineered fp25k gene is shown through the maintained or increased yield of polyhedrin protein. Wild-type (wt) AcMNPV E2 plaque 3 (AcP3) strain (a nucleic acid of SEQ ID NO. 1) was used. The engineered fp25k gene of AcMNPV (a nucleic acid of SEQ ID NO. 2) was produced according to the procedures listed in Example 1.

(35) Sf9 cells were seeded at 110.sup.6 cells/well in six-well plates. The procedures of polyhedrin harvesting and polyhedrin protein measurement were performed according to the procedures listed in Example 2. The results are shown in FIGS. 3A and 3B, and are similar to that of Example 2: There was a progressive reduction of polyhedrin during AcP3 passage whereas there was no detectable reduction of polyhedrin of AcP3SDM in Sf9 cell passage.

(36) This detailed description in connection with the drawings is intended principally as a description of the presently preferred embodiments of the invention, and is not intended to represent the only form in which the present invention may be constructed or utilized. The description sets forth the designs, functions, means, and methods of implementing the invention in connection with the illustrated embodiments. It is to be understood, however, that the same or equivalent functions and features may be accomplished by different embodiments that are also intended to be encompassed within the spirit and scope of the invention and that various modifications may be adopted without departing from the invention or scope of the following claims.