CN110387385B - Baculovirus expression vector - Google Patents
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Abstract
The invention discloses a novel baculovirus expression vector, and relates to the technical field of biology. The novel baculovirus expression vector is obtained by knocking out four continuous non-essential genes Ac84, Ac85, Ac86 and Ac87 on the baculovirus expression vector at the same time. The novel baculovirus expression vector of the invention improves the expression level of foreign protein by 80%. Meanwhile, the propagation property of the virus is not influenced. The improvement of the expression yield can obviously reduce the production cost of enterprises. The baculovirus expression vector can be used in the field of biological product industry, in particular in the field of subunit vaccine industry.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a baculovirus expression vector.
Background
Baculovirus is double-stranded DNA virus capable of specifically infecting arthropod, and Autographa californica polyribonucleotis virus
(Autographa californica nucleolyticus, AcMNPV) is a model species of baculovirus. Since Smith GE, etc. expressed human interferon-beta gene in insect cells with baculovirus for the first time in 1983 (Mol Cell biol. 1983; 3: 2156-65), baculovirus expression vector systems have been widely used in research and production, especially in the fields of biological products and vaccine industry, due to their advantages of low cost, high yield, and various post-translational modification systems. Disclosure of Invention
However, compared to the prokaryotic expression systems (E.coli, B.subtilis) and yeasts which are commonly used industrially
The production of the expression system, the baculovirus expression vector system, is not yet satisfactory, which results in a high production cost of the baculovirus expression system.
Therefore, specific technical means to improve the yield of the above expression vector system need to be studied.
Disclosure of Invention
In view of this, the embodiment of the present invention provides a baculovirus expression vector, which mainly aims to increase the expression yield of foreign proteins.
In order to achieve the purpose, the invention mainly provides the following technical scheme:
in one aspect, the embodiment of the invention provides a baculovirus expression vector, which is obtained by knocking out four continuous non-essential genes Ac84, Ac85, Ac86 and Ac87 on the baculovirus AcMNPV expression vector at the same time.
Preferably, the Ac84 knockout means that the coding region of Ac84 is disrupted or that the promoter region of Ac84 and its coding region are disrupted simultaneously.
Preferably, the Ac85 knockout means that the coding region of Ac85 is disrupted or that the promoter region of Ac85 and its coding region are disrupted simultaneously.
Preferably, the Ac86 knockout means that the coding region of Ac86 is disrupted or the promoter region of Ac86 and its coding region are disrupted at the same time.
Preferably, the Ac87 knockout means that the coding region of Ac87 is disrupted or the promoter region of Ac87 and its coding region are disrupted at the same time.
On the other hand, the invention provides a recombinant protein, wherein the baculovirus expression vector is used for constructing a recombinant virus, then insect host cells are infected, and the host cells are cultured, proliferated and expressed into the recombinant protein, so that the recombinant protein is obtained.
In yet another aspect, the present invention provides the use of the baculovirus expression vector in the vaccine industry.
Compared with the prior art, the invention has the beneficial effects that:
the invention discovers that a large number of non-essential genes exist in the baculovirus genome at the time of research, and some non-essential genes exist in clusters adjacent to each other, and the non-essential genes existing in clusters (containing unknown functional genes) comprise four genes which are continuous from Ac84 to Ac 87; after four continuous virus non-essential genes Ac84, Ac85, Ac86 and Ac87 are knocked out simultaneously, the protein expression level is found to be remarkably improved, and the proliferation level of the virus is not obviously changed, which shows that the baculovirus expression vector with excellent production characteristics and high yield characteristics can be provided by the knocking-out technology.
Drawings
FIG. 1 is a schematic diagram of the gene knockout strategy of the baculovirus vector of the invention;
FIG. 2 is a diagram of the baculovirus vector expressing GFP whole cell protein electrophoresis immunoblot hybridization;
FIG. 3 is a histogram of total fluorescence intensity of GFP expressed by the baculovirus vector of the present invention; FIG. 4 is a primary growth curve of the baculovirus vector of the present invention.
Detailed Description
To further illustrate the technical means and effects of the present invention adopted to achieve the predetermined objects, the following detailed description of the embodiments, technical solutions, features and effects according to the present invention will be given with preferred embodiments. The particular features, structures, or characteristics may be combined in any suitable manner in the embodiments or embodiments described below.
The following detailed description of the principles of the invention is provided in connection with the accompanying drawings.
Example 1
1. Ac84-87 Gene knockout
The ampicillin resistant gene fragment was amplified using the pTriEx1.1 plasmid as a template using primers (SEQ ID NO:1 and SEQ ID NO:2) having homology arms of 50bp upstream and downstream of the Ac84-87 DNA fragment to obtain a PCR product (SEQ ID NO:3) of 1023 bp.
Transferring the obtained PCR product into an escherichia coli strain HS996 with RedET plasmid and Bacmid by an electrotransformation method, generating recombinase by arabinose induction, and screening the recombined escherichia coli by an ampicillin resistance plate to obtain positive clone. At this time, the Ac84-87 DNA fragment had been replaced by an ampicillin resistance gene fragment. Bacmid is extracted from escherichia coli, and is named as Bacmid delta Ac84-87 after sequencing and identification (the knockout strategy is shown in figure 1).
Bacmid delta Ac84-87 is used for subsequent experiments after being linearized by plasmid miniplasmid and Bsu36I restriction enzyme.
2. Protein production of fluorescent protein GFP
Linearized Bacmid Δ Ac84-87 was co-transfected with pTriEx-GFP plasmid (GFP gene fragment cloned between pTriEx1.1 NcoI/XhoI sites) into Sf9 insect cells, and P0 recombinant viruses were collected 5 days after transfection. The P0 generation virus was amplified to the P1 generation virus, Sf9 cells were infected at 3 MOI, and cells were harvested on the fourth day after infection. After cell lysis, polyacrylamide gel electrophoresis, membrane transfer, and hybridization with anti-GFP antibody were performed (FIG. 2). The results show that the vector yield after modification is significantly higher than that of the control.
3. Activity to express Green fluorescent protein GFP
Sf9 cells were infected at 3 MOI with the recombinant virus carrying the GFP gene described above, and the cells were harvested on the fourth day after infection. The green fluorescence intensity of the cells was measured by flow cytometry and the geometric mean was calculated (FIG. 3). The yield of GFP, a green fluorescent protein, was found to be 83% higher than that of the wild type after comparison.
4. Virus propagation properties
Sf9 cells were infected at 0.1 MOI using the recombinant virus with green fluorescent protein, and then samples were taken every 24 hours, and the virus titer was determined by limiting dilution to plot a primary virus growth curve (FIG. 4). Compared with wild-type virus, the Ac84-87 knockout virus has similar virus propagation characteristics as the control.
The invention has the innovative ideas that: the invention discovers that a large number of non-essential genes exist in the baculovirus genome at the time of research, and some non-essential genes exist adjacent to each other in clusters, and the clustered non-essential genes (containing unknown functional genes) comprise four genes which are continuous from Ac84 to Ac 87.
Ac84 and Ac85 have not been reported in any study so far, and are presumed to be non-essential genes.
Ac86 encodes a protein with RNA ligase, 5 'polynucleotide-kinase and 3' polynucleotide-phosphatase activities, and is likely involved in RNA repair (J Biol chem.2004; 279: 18220-31.). It has been considered by some studies to be a non-essential gene expressed early in viral infection (J Gen Virol. 1998; 79: 629-37.).
Ac87 was not reported in AcMNPV. The homologous gene Bm70 in silkworm polyhedrosis Virus probably encodes a 15kDa nucleocapsid protein (Gene. 1997; 185:69-75.), but knocking out Bm70 does not affect the replication of the Virus (Virus Res.2012; 165: 197-.
Overall, Ac84, Ac85, Ac86, and Ac87 are non-essential or suspected non-essential genes of baculoviruses, and at least when replicated in insect cell lines, there is no published information showing that these four genes have important functions. The present inventors tried to knock out the above four unnecessary genes and found that the protein expression level of the novel expression vector was significantly improved and the virus proliferation level was not significantly changed by examining the yield and proliferation characteristics of the novel expression vector, thereby demonstrating that a baculovirus expression vector having an excellent production shape and high-yield characteristics can be obtained by the method of example 1 above.
The embodiments of the present invention are not exhaustive, and those skilled in the art can select them from the prior art.
The above disclosure is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and shall be covered by the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the above claims.
Sequence listing
<110> northwest agriculture and forestry science and technology university
<120> a novel baculovirus expression vector
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<170> SIPOSequenceListing 1.0
<210> 1
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tcgtaaagcg agttgaattt tgattacaaa tattttgttt atgatagcaa aacgcgcgga 60
catgag 66
<210> 2
<211> 66
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
gtttacataa cattctactt taatgtaata atattcttca atttcttggg ttaccaatgc 60
ttaatc 66
<210> 3
<211> 1023
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
tcgtaaagcg agttgaattt tgattacaaa tattttgttt atgatagcaa aacgcgcgga 60
catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat 120
tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc 180
tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg 240
ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg 300
ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga 360
cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta 420
ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc 480
tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc 540
gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg 600
ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc 660
aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca 720
acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct 780
tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat 840
cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg 900
gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgtt 960
tacataacat tctactttaa tgtaataata ttcttcaatt tcttgggtta ccaatgctta 1020
atc 1023
Claims (6)
1. A baculovirus expression vector is characterized in that four continuous non-essential genes Ac84, Ac85, Ac86 and Ac87 on a baculovirus AcMNPV expression vector Bacmid are knocked out simultaneously to obtain the baculovirus expression vector.
2. The baculovirus expression vector of claim 1, wherein said Ac84 knockout means that the coding region for Ac84 is disrupted or that the promoter region for Ac84 and its coding region are disrupted simultaneously.
3. The baculovirus expression vector of claim 1, wherein said Ac85 knockout means that the coding region for Ac85 is disrupted or that the promoter region for Ac85 and its coding region are disrupted at the same time.
4. The baculovirus expression vector of claim 1, wherein said Ac86 knockout means that the coding region for Ac86 is disrupted or that the promoter region for Ac86 and its coding region are disrupted simultaneously.
5. The baculovirus expression vector of claim 1, wherein said Ac87 knockout means that the coding region for Ac87 is disrupted or that the promoter region for Ac87 and its coding region are disrupted simultaneously.
6. Use of the baculovirus expression vector of any one of claims 1-5 to express a foreign protein in insect cells.
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Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114317608B (en) * | 2020-12-28 | 2023-08-22 | 陕西杆粒生物科技有限公司 | Gene knockout type baculovirus expression vector |
| CN114292877B (en) * | 2021-01-11 | 2023-08-22 | 陕西杆粒生物科技有限公司 | Baculovirus expression vector for Ac63-64 gene knockout |
| CN114292878B (en) * | 2021-04-13 | 2023-08-22 | 陕西杆粒生物科技有限公司 | Baculovirus expression vector with Ac68-72 knocked out |
| CN113106125B (en) * | 2021-04-16 | 2023-08-22 | 陕西杆粒生物科技有限公司 | Ac129-131 deleted baculovirus vector |
| CN113416751B (en) * | 2021-07-01 | 2023-08-22 | 陕西杆粒生物科技有限公司 | Ac96 inactivated baculovirus vector |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4879236A (en) * | 1984-05-16 | 1989-11-07 | The Texas A&M University System | Method for producing a recombinant baculovirus expression vector |
| CN101285072A (en) * | 2008-06-05 | 2008-10-15 | 复旦大学 | Process for enhancing gene level of expression of baculoviral expression carrier |
| CN104017826A (en) * | 2014-05-30 | 2014-09-03 | 江苏大学 | Engineered bombyx mori baculovirus vector and method of increasing the NS1 expression amount by utilization of the vector |
| CN106636207A (en) * | 2016-09-22 | 2017-05-10 | 西北农林科技大学 | Recombinant baculovirus vector resistant to host apoptosis |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2954050A2 (en) * | 2013-02-08 | 2015-12-16 | European Molecular Biology Laboratory | Improved baculoviral expression system and methods of producing the same |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4879236A (en) * | 1984-05-16 | 1989-11-07 | The Texas A&M University System | Method for producing a recombinant baculovirus expression vector |
| CN101285072A (en) * | 2008-06-05 | 2008-10-15 | 复旦大学 | Process for enhancing gene level of expression of baculoviral expression carrier |
| CN104017826A (en) * | 2014-05-30 | 2014-09-03 | 江苏大学 | Engineered bombyx mori baculovirus vector and method of increasing the NS1 expression amount by utilization of the vector |
| CN106636207A (en) * | 2016-09-22 | 2017-05-10 | 西北农林科技大学 | Recombinant baculovirus vector resistant to host apoptosis |
Non-Patent Citations (5)
| Title |
|---|
| "Complex genetic interactions among non-essential genes of BmNPV revealed by multiple gene knockout analysis";Hitomi Taka et al.;《Journal of Insect Biotechnology and Sericology》;20131231;第82卷;第25-32页 * |
| "HearNPV ORF83和ORF86的多抗制备与基因敲除重组Bacmid构建";李坚;《中国优秀博硕士学位论文全文数据库(硕士) 农业科技辑》;20101115(第11期);第D046-104页 * |
| "ORF85 of HearNPV encodes the per os infectivity factor 4 (PIF4) and is essential for the formation of the PIF complex";Huachao Huang et al.;《Virology》;20120302;第427卷;第217-223页 * |
| "Reduced expression of Autographa californica nucleopolyhedrovirus ORF34,an essential gene, enhances heterologous gene expression";Tamer Z. Salem et al.;《Virology》;20121103;第435卷;第225-238页 * |
| "敲除Ac148-150对苜蓿银纹夜蛾核型多角体病毒复制和外源蛋白表达的影响";赵凯霞 等;《西北农林科技大学学报（自然科学版）》;20191104;第48卷(第5期);第42-48页 * |
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