CN114317608A

CN114317608A - Gene knockout type baculovirus expression vector

Info

Publication number: CN114317608A
Application number: CN202011573495.9A
Authority: CN
Inventors: 不公告发明人
Original assignee: Shaanxi Bacmid Biological Technology Co ltd
Current assignee: Shaanxi Bacmid Biological Technology Co ltd
Priority date: 2020-12-28
Filing date: 2020-12-28
Publication date: 2022-04-12
Anticipated expiration: 2040-12-28
Also published as: CN114317608B

Abstract

The invention discloses a gene knockout type baculovirus expression vector, and relates to the technical field of biology. The gene knockout baculovirus expression vector is obtained by simultaneously knocking out five continuous non-essential genes Ac29, Ac30, Ac31, Ac32 and Ac33 on a baculovirus expression vector. The gene knockout type rhabdovirus expression vector of the invention can improve the expression level of foreign protein by 80 percent. While the proliferative properties of the virus are not affected. 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

Gene knockout type baculovirus expression vector

Technical Field

The invention relates to the technical field of biology, in particular to a gene knockout type baculovirus expression vector.

Background

Baculovirus is a double-stranded DNA virus that specifically infects arthropods, and Autographa californica polynuclear polyhedrosis virus (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 due to their low cost, high yield, and various post-translational modification systems.

However, the production of baculovirus expression vector systems is not satisfactory compared to the prokaryotic expression systems (E.coli, B.subtilis) and yeast expression systems commonly used in industry, which leads to high production costs of baculovirus expression systems.

Therefore, it is necessary to develop specific technical means for improving the yield of the above expression vector system.

Disclosure of Invention

The invention aims to provide a gene knockout high-yield recombinant baculovirus vector, aiming at solving the problem of low expression quantity in the background technology.

In order to achieve the purpose, the invention mainly provides the following technical scheme:

in one aspect, the embodiment of the invention provides a gene knockout baculovirus expression vector, which is obtained by simultaneously knocking out five continuous non-essential genes Ac29, Ac30, Ac31, Ac32 and Ac33 on a baculovirus AcMNPV expression vector.

Preferably, the Ac29 knockout means that the promoter region of Ac29 is disrupted or the coding region of Ac29 is disrupted or the promoter region of Ac29 and its coding region are disrupted simultaneously.

Preferably, the Ac30 knockout means that the promoter region of Ac30 is disrupted or the coding region of Ac30 is disrupted or the promoter region of Ac30 and its coding region are disrupted simultaneously.

Preferably, the Ac31 knockout means that the promoter region of Ac31 is disrupted or the coding region of Ac31 is disrupted or the promoter region of Ac31 and its coding region are disrupted simultaneously.

Preferably, the Ac32 knockout means that the promoter region of Ac32 is disrupted or the coding region of Ac32 is disrupted or the promoter region of Ac32 and its coding region are disrupted simultaneously.

Preferably, the Ac33 knockout means that the promoter region of Ac33 is disrupted or the coding region of Ac33 is disrupted or the promoter region of Ac33 and its coding region are disrupted simultaneously.

In another aspect, the invention provides a recombinant protein, wherein the recombinant protein is obtained by constructing a recombinant virus with the novel baculovirus expression vector, then infecting insect host cells, culturing, proliferating and expressing the host cells into the recombinant protein.

In yet another aspect, the present invention provides the use of the novel 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 clustered non-essential genes (containing unknown functional genes) comprise five genes which are continuous from Ac29 to Ac 33; after five continuous virus non-essential genes Ac29, Ac30, Ac31, Ac32 and Ac33 are knocked out simultaneously, the protein expression level is found to be remarkably improved, and the virus multiplication level is not obviously changed, which shows that the invention can provide a baculovirus expression vector with excellent production traits and high yield characteristics through the knocking-out technology.

Drawings

FIG. 1 is a schematic diagram of a gene knockout strategy for a baculovirus vector provided by the invention;

FIG. 2 is an electrophoretogram of luciferase FluC whole cell protein expressed using the baculovirus vector provided by the present invention;

FIG. 3 is a histogram of total fluorescence intensity of GFP expressed using the baculovirus vector provided in the present invention;

FIG. 4 is a primary growth curve of the baculovirus vector provided by the invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

The following detailed description of the principles of the invention is provided in connection with the accompanying drawings.

Example 1.

1. Ac29-33 gene knockout.

A1023 bp PCR product (SEQ ID NO: 3) was obtained by amplifying an ampicillin resistant gene fragment using pTriEx1.1 plasmid as a template with primers (SEQ ID NO:1 and SEQ ID NO: 2) having an upstream and downstream 50bp homology arm of Ac 29-33.

Transferring the obtained PCR product into an escherichia coli strain HS996 with RedET plasmid and Bacmid by an electrotransformation method, inducing the escherichia coli strain to generate recombinase by arabinose, screening the recombined escherichia coli by an ampicillin resistance plate, and carrying out colony PCR verification to obtain positive clone. At this time, the Ac29-33 DNA fragment had been replaced with the ampicillin resistance gene fragment. Bacmid is extracted from Escherichia coli HS996, and is named as Bacmid delta Ac29-33 after sequencing and identification (the knockout strategy is shown in figure 1).

After Bacmid delta Ac29-33 is linearized by Bsu36I restriction enzyme, the linearized product is used for subsequent experiments.

2. Expressing luciferase Fluc.

The linearized Bacmid Δ Ac29-33 co-transfected Sf9 insect cells with pTriEx-FluC plasmid (FluC gene fragment cloned between pTriEx1.1 NcoI/XhoI sites), and 5 days after transfection, P0 recombinant viruses were collected. The P0 generation virus was amplified to P1 generation virus, High Five cells were infected at 3 MOI, and cells were harvested on the fourth day after infection. After cell lysis, polyacrylamide gel electrophoresis and Coomassie blue staining were performed (FIG. 2). The stained gel was quantified by density scanning and comparison of the calculated results revealed an 80% increase in luciferase Fluc production relative to wild type.

3. The green fluorescent protein GFP was expressed.

Linearized Bacmid Δ Ac29-33 co-transfected Sf9 insect cells with pTriEx-GFP plasmid (GFP gene fragment cloned between pTriEx1.1 NcoI/XhoI sites), and recombinant virus generation P0 was collected 5 days after transfection. The P0 generation virus was amplified to P1 generation virus, Sf9 cells were infected at 3 MOI, and 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 increased by 86% compared to the wild type.

4. Virus propagation properties.

Sf9 cells were infected with the recombinant virus carrying green fluorescent protein at 0.1 MOI, and then samples were taken every 24 hours, and virus titers were determined by limiting dilution to plot a primary virus growth curve (FIG. 4). Compared with wild viruses, the proliferation characteristics of the Ac29-33 knockout virus are not changed significantly.

The invention has the innovative ideas that: the present inventors have found at the time of their research that a large number of nonessential genes are present in the baculovirus genome and that some nonessential genes are present adjacent to each other in clusters, and these nonessential genes present in clusters (containing unknown functional genes) include five genes in succession from Ac29 to Ac 33.

The Ac29 gene has unknown function. The deletion of the homologous gene Bm20 in silkworm polyhedrosis Virus does not affect the replication of the Virus (Virus Res.2012; 165: 197-206.).

Ac30 may be a non-essential gene, and the knockout of the homologous gene Bm21 in Bombyx mori polyhedrosis virus does not affect the replication of the virus, but the survival time of the infected host is prolonged (J Gen Virol. 2008;89: 922-30.).

Ac31 encodes a protein similar to superoxide dismutase SOD. No experiments have demonstrated that the SOD encoded by Ac31 has enzymatic activity, and that Ac31 knock-out has no effect on virus proliferation, even in the presence of superoxide ion inducers (virology, 1991: 149-161.). The studies in silkworm polyhedrosis Virus are contradictory, with some studies showing that the Ac31 homolog Bm23 is essential for the Virus (Insect Biochem Mol biol 2008;38: 1080-6.), and another that the Bm23 knockout does not affect the replication of the Virus (Virus Res 2012;165: 197-.

The Ac32 gene encodes an epidermal growth factor, FGF, homolog protein that interacts with the insect epidermal growth factor receptor. The Ac32 knock-out did not affect viral replication (virology 2006;346: 258-65.), but death of the host was significantly delayed (virology 2007;365: 70-8.).

The Ac33 gene encodes a polynucleotide kinase, whose role in the viral life cycle is unknown. Ac33 has no homologous gene in silkworm polyhedrosis virus, and is presumed to be a non-essential gene.

Overall, Ac29, Ac30, Ac31, Ac32, and Ac33 are nonessential or suspected nonessential genes of baculoviruses, and at least when replicated in insect cell lines, there is no published information showing that these five genes have important functions. The present inventors tried to knock out the above five unnecessary genes and found that the protein expression level of the novel expression vector was significantly improved without significant change in the virus proliferation level 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.

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

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Claims

1. A gene knockout baculovirus expression vector is characterized in that five continuous non-essential genes Ac29, Ac30, Ac31, Ac32 and Ac33 on a baculovirus AcMNPV expression vector are knocked out simultaneously to obtain the gene knockout baculovirus expression vector.

2. The knock-out baculovirus expression vector of claim 1, wherein the Ac29 knock-out is disruption of the Ac29 promoter region or disruption of the Ac29 coding region or disruption of both the Ac29 promoter region and the Ac29 coding region.

3. The knock-out baculovirus expression vector of claim 1, wherein the Ac30 knock-out is disruption of the Ac30 promoter region or disruption of the Ac30 coding region or disruption of both the Ac30 promoter region and the Ac30 coding region.

4. The knock-out baculovirus expression vector of claim 1, wherein the Ac31 knock-out is disruption of the Ac31 promoter region or disruption of the Ac31 coding region or disruption of both the Ac31 promoter region and the Ac31 coding region.

5. The knock-out baculovirus expression vector of claim 1, wherein the Ac32 knock-out is disruption of the Ac32 promoter region or disruption of the Ac32 coding region or disruption of both the Ac32 promoter region and the Ac32 coding region.

6. The knock-out baculovirus expression vector of claim 1, wherein the Ac33 knock-out is disruption of the Ac33 promoter region or disruption of the Ac33 coding region or disruption of both the Ac33 promoter region and the Ac33 coding region.

7. A recombinant protein, which is obtained by constructing a recombinant virus using the knockout baculovirus expression vector of any one of claims 1 to 6 and then infecting insect host cells, which are cultured, proliferated and expressed as a recombinant protein.

8. Use of the knockout baculovirus expression vector of any one of claims 1-6 in the vaccine industry.