CN111378687A - High-yield baculovirus expression vector - Google Patents

Abstract

The invention discloses a gene knockout high-yield baculovirus expression vector. The vector increases the expression level of the foreign protein by at least 60 percent by simultaneously knocking out two adjacent baculovirus non-essential genes Ac15 and Ac 16. At the same time, the proliferation properties of the virus are also significantly improved. 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

High-yield baculovirus expression vector

Technical Field

The invention belongs to the technical field of recombinant protein expression, and particularly relates to a gene knockout high-yield baculovirus expression vector.

Background

Baculovirus is a double-stranded DNA virus which specifically infects arthropods, and Autographa californica polynuclear polyhedrosis virus (AcMNPV) is a model species of baculovirus since Smith GE, etc. express β -interferon gene in insect cells for the first time with baculovirus in 1983 (Mol CellBiol, 1983; 3: 2156-65.), the baculovirus expression vector system is widely applied in scientific research and production due to its low cost, high yield and advantages of various post-translational modification systems, etc.

However, the yield of baculovirus expression vector systems is less than satisfactory than the prokaryotic expression systems (E.coli, B.subtilis) and yeast expression systems which are commonly used in industry. For this purpose, various strategies have been adopted to increase the yield of the expression system, including:

1. the promoter and surrounding elements are modified. For example, GFP production can be increased 4.4-fold by concatenating the p6.9 and p10 promoters (PLoS ONE 2014; 9(5): e 96562.); a repeat of the Burst sequence downstream of the polh promoter increased the GUS enzyme activity by 1.5-fold (Biotechnol Bioeng. 2010; 107: 909-16.).

2. Constructing an anti-apoptotic vector or cell line. One subject group in Taiwan expresses Sf-caspase-1 dsRNA in insect cells Sf9 by using an RNA interference vector, successfully silences the Sf-caspase-1 in the cells, and obviously improves the yield of foreign proteins (Biotechnol Appl biochem, 2007; 48: 11-19.). Zhang Xiaoyue et al cloned the double-stranded small RNA coding sequence targeting Sf-caspase-1 directly into the baculovirus genome, increasing the expressed luciferase activity by 10-fold (BMCBiotechnol. 2018; 18: 24.).

3. Knocking out non-essential genes. For example, the knock-out of chitinase and cathepsin (ChiA/Cath) contributes to the increased expression of secreted proteins (J Virol methods, 2004; 122: 113-118.); on the basis, three continuous non-essential genes p26, p10 and p74 are knocked out, so that the yield of the EGFP can be increased by 2.6 times (Cell Biol Toxicol. 2010; 26: 57-68.).

The mechanism of knocking out non-essential genes to improve the yield of foreign genes is diverse. The degradation of the protein can be reduced by knocking out a cathepsin (Cath) gene; knock-out of p10 may reduce the release of intracellular transcriptional resources late in infection. In addition, knockout of non-essential genes can also reduce the size of the baculovirus genome, facilitating the accommodation of larger foreign DNA fragments.

We found by searching the literature that a large number of non-essential genes are present in the baculovirus genome and that some of the non-essential genes are present adjacent to each other in clusters, these clusters including Ac15 and Ac 16.

Ac15 encodes ecdysteroid UDP-glucosyltransferase (EGT). EGT functions to prevent moulting and pupation of infected larvae by catalyzing the transfer of glucose from UDP-glucose to ecdysteroids. The presence of EGT can prolong the feeding stage of infected larvae, thereby enabling the virus to replicate in larger larvae for a longer period of time, resulting in higher virus production. AcMNPV and BmNPV (Bombyx mori nucleolylhydrovirus) mutants with inactivated EGT genes were found to be infectable and replicable (J. Virol. 1990; 64: 1321. sup. 1328.) but the survival times of insects were significantly shortened (Bio/technology. 1991; 9: 1086. sup. 1089.). Spontaneous deletion mutants of the EGT gene can often be isolated using cell-line-enhanced baculovirus (Virus Res. 1987; 7: 335. 349.).

Ac16 can interact with fp25 (Ac 61) and form a complex with cellular actin (virology, 1998;240: 64-75.). In BmNPV, the homologous gene to Ac16, Bm8, directly related to IE-1 (J. Gen Virol. 2005; 86: 3031-8.). Similar observations were made in AcMNPV and Ac16 was also found to work with IE-1/IE-0 (virology. 2009;385: 484-95.). In addition, the AcMNPV Ac16 insertionally inactivated mutant has replication activity and is not significantly different from wild-type in insect cells or larvae (J Gen Virol. 1990; 71: 1029-37.). Initial attempts to isolate BmNPV (Bm 8) null mutants were unsuccessful, but the C-terminal deletion mutant was replication-active (Arch Virol. 2004; 149: 1581-94.) but the BV titer of the mutant was lower than that of the wild type. One study in AcMNPV found that deletion of Ac16 resulted in a delay in DNA synthesis and BV production (virology 2010; 404: 168-79.), but in another study had a lesser effect (virology 2009;385: 484-95.).

Overall, Ac15 and Ac16 are non-essential genes of baculoviruses, which do not exhibit important functions, at least when replicated in insect cell lines. The influence of knocking out these two genes on the expression level of a foreign gene has not been noticed.

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.

According to the invention, the Ac15 and Ac16 are knocked out simultaneously, so that the baculovirus expression vector with high foreign protein yield is obtained.

The invention is realized by amplifying an ampicillin resistance gene by using primers (SEQ ID NO:1 and SEQ ID NO: 2) with an upstream and downstream 50bp homologous arm of Ac15-16, transforming an obtained PCR product (SEQ ID NO: 3) into bacillus coli containing Bacmid, realizing homologous recombination in the bacillus coli by inducing and expressing RedET recombinase, and obtaining the Bacmid (named Bacmid delta Ac 15-16) knocked out by Ac15-16 through ampicillin resistance screening.

In Bacmid delta Ac15-16, the whole coding region of Ac15, the promoter region of Ac16 and the 528bp partial coding region at the 5' end are knocked out, so that Ac15-16 is completely inactivated.

Bacmid delta Ac15-16 and a contrast Bacmid are used as expression vectors, and luciferase genes FluC and GFP genes are respectively inserted into a polyhedron locus through recombination. Through determination, the yield of the luciferase protein is improved by 60 percent, and the fluorescence intensity of GFP is improved by nearly one time.

By testing the primary growth curve of the virus, we find that the proliferation property of the Ac15-16 knockout virus is better than that of the control virus.

After two adjacent virus nonessential genes are knocked out simultaneously, the protein expression level is obviously improved, the virus multiplication level is obviously improved, and the baculovirus expression vector with excellent production characteristics and high yield characteristics can be provided.

Another object of the present invention is to produce a protein preparation using the knock-out baculovirus vector described above.

Another object of the present invention is to prepare a vaccine using the knock-out baculovirus vector described above.

Drawings

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

FIG. 2 luciferase FluC expressed using the baculovirus vector provided by the present invention. The result of Coomassie brilliant blue staining after whole-cell protein electrophoresis shows that the yield of the baculovirus vector provided by the invention is obviously higher than that of a control, and the yield is improved by 60.3 percent through density scanning quantitative comparison.

FIG. 3 shows green fluorescent protein GFP expressed using the baculovirus vector provided in the present invention. Cells 4 days after virus infection were analyzed by flow cytometry, and the total fluorescence intensity was calculated from the geometric mean. The results show that the yield of GFP expressed by the baculovirus vector provided by the invention is improved by 94%.

FIG. 4 is a primary growth curve of the baculovirus vector provided by the invention. After Ac15-16 is knocked out, the multiplication of the virus is not influenced, but the multiplication capacity of the virus is obviously improved.

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.

1. Knock-out of Ac15-16 gene.

A1023 bp PCR product (SEQ ID NO: 3) was obtained by amplifying the 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 15-16.

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 Ac15-16 DNA fragment had been replaced with the ampicillin resistance gene fragment. Bacmid is extracted from escherichia coli, and is named as Bacmid delta Ac15-16 after sequencing identification (the knockout strategy is shown in figure 1).

After Bacmid delta Ac15-16 is subjected to plasmid miniextraction and Bsu36I restriction enzyme digestion linearization, the plasmid is used for subsequent experiments.

2. Expressing luciferase Fluc.

The linearized Bacmid Δ Ac15-16 was co-transfected with pTriEx-FluC plasmid (FluC gene fragment cloned between pTriEx1.1NcoI/XhoI sites) to Sf9 insect cells, and 5 days after transfection, recombinant viruses of P0 generation 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 that luciferase Fluc production was increased by 60.3% relative to wild type.

3. The green fluorescent protein GFP was expressed.

Linearized Bacmid Δ Ac15-16 was co-transfected with pTriEx-GFP plasmid (GFP gene fragment cloned between pTriEx1.1NcoI/XhoI sites) into Sf9 insect cells, and recombinant viruses were collected 5 days after transfection for P0 generations. 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). After comparison, the yield of GFP, a green fluorescent protein, was increased by 94% relative 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 Ac15-16 knockout virus has better virus propagation characteristics.

The above-mentioned expression of luciferase FluC and green fluorescent protein GFP are only examples of the present invention, and are not intended to limit the present invention. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

<110> Shanxi bacon Biotech Co., Ltd

<120> a high-yield baculovirus expression vector

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<213> Artificial sequence (Autographa californica multiple nucleolyhydrovirus)

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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

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tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat 840

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gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat 960

taagcattgg taactgctga cggcggcgtt tggttcgcga gtcatagttt atgaaaatag 1020

tcg 1023

Claims (5)

1. A gene knockout high-yield baculovirus expression vector is characterized in that two adjacent non-essential genes Ac15 and Ac16 on the baculovirus expression vector are simultaneously knocked out.

2. The Ac15 knockout of claim 1, wherein the promoter region of Ac15 is disrupted, or the coding region is disrupted, or both the promoter region and the coding region are disrupted.

3. The Ac16 knockout of claim 1, wherein the promoter region of Ac16 is disrupted, or the coding region is disrupted, or both the promoter region and the coding region are disrupted.

4. Use of the baculovirus expression vector of claims 1 to 3 for protein expression.

5. Use of the baculovirus expression vector of claims 1 to 3 in the vaccine industry.

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