CN113416751B - Ac96 inactivated baculovirus vector - Google Patents

Ac96 inactivated baculovirus vector Download PDF

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CN113416751B
CN113416751B CN202110742717.3A CN202110742717A CN113416751B CN 113416751 B CN113416751 B CN 113416751B CN 202110742717 A CN202110742717 A CN 202110742717A CN 113416751 B CN113416751 B CN 113416751B
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Shaanxi Bacmid Biological Technology Co ltd
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    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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Abstract

The invention discloses a gene inactivation type baculovirus expression vector. The carrier baculovirus nonessential gene Ac96 is inactivated, so that the expression level of the exogenous protein is obviously improved. At the same time, the proliferation properties of the virus were not significantly changed. The improvement of the expression yield can reduce the production cost of enterprises, and the inactivation of Ac96 can improve the biosafety of the recombinant baculovirus. The baculovirus vector can be used in the field of biological product industry.

Description

Ac96 inactivated baculovirus vector
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to a gene-inactivated baculovirus vector.
Background
Baculoviruses are double-stranded DNA viruses that specifically infect arthropods, and the noctiluca californica nuclear polyhedrosis virus (Autographa californica nucleopolyhedrovirus, acMNPV) is a model species of baculovirus. Since the first expression of human interferon-beta gene in insect cells by baculovirus such as Smith GE in 1983 (Mol Cell biol. 1983; 3:2156-65.), baculovirus expression vector systems have been widely used in scientific research and production due to their low cost, high yield, and various post-translational modification systems.
However, the yield of baculovirus expression vector systems is still unsatisfactory compared to prokaryotic expression systems (E.coli, B.subtilis) and yeast expression systems commonly used in industry. For this purpose, various strategies have been adopted to increase the yield of the expression system, including:
1. modifying the promoter and the peripheral elements. GFP production can be increased 4.4-fold, for example, by tandem p6.9 and p10 promoters (PLoS ONE.2014; 9 (5): e 96562.); repeating the Burst sequence once downstream of the polh promoter increases GUS enzyme activity 1.5-fold (Biotechnol bioeng. 2010; 107:909-16.).
2. An anti-apoptotic vector or cell line was constructed. The dsRNA of Sf-caspase-1 is expressed in insect cells Sf9 by using RNA interference vectors, so that the Sf-caspase-1 in the cells can be successfully silenced, and the yield of the exogenous protein is remarkably improved (Biotechnol Appl biochem.2007; 48:11-19.). Zhang Xiaoyue et al cloned the Sf-caspase-1 targeting double-stranded small RNA coding sequence directly onto the baculovirus genome, increasing the expressed luciferase activity 10-fold (BMC Biotechnol. 2018; 18:24.).
3. Knocking out non-essential genes. Such as knockdown chitinase and cathepsin (ChiA/V-Cath) to help increase expression of secreted proteins (J Virol methods 2004; 122:113-118.); on this basis, the knockout of three consecutive nonessential genes p26, p10, p74 increased EGFP production by a factor of 2.6 (Cell Biol Toxicol. 2010; 26:57-68.).
The mechanism by which knocking out the non-essential gene increases the yield of the foreign gene is diverse. Knocking out the cathepsin (V-Cath) gene can reduce the degradation of protein; knocking out p10 can release intracellular transcription resources in late infection.
In addition, knocking out non-essential genes can also reduce the size of baculovirus genome, facilitating the accommodation of larger foreign DNA fragments. Wild-type baculoviruses can insert up to about 40kb of exogenous DNA fragments, and if multiple proteins are expressed with one recombinant virus, or another viral vector is loaded with a baculovirus vector, the 40kb space is often left open. If more space can be released by knocking out genes, the application range of the baculovirus expression vector can be greatly expanded.
The inventors found, by searching the literature, that a large number of non-essential genes are present in the baculovirus genome, including Ac96.
Ac96 encodes a protein (PIF-4) of 173 amino acids with a molecular weight of 19.8 kDa, an oral infectious agent, ac96, and a fourth protein identified in the AcMNPV genome, which is present in all baculovirus genomes. Baculoviruses from which the gene was knocked out were able to replicate in cell lines but not in insects (J virol 2009; 83:12569-78.).
Downstream of Ac96 there is a potential gene Ac97 which may encode a protein of 56 amino acids having a molecular weight of 6.5 kDa. This potential gene appears to be present only in AcMNPV, and the lack of a homologous gene in BmNPV suggests that the gene is not a functional gene. Even in AcMNPV, some whole genome sequencing results were not labeled as potential genes, such as KM667940, further indicating that it is not a functional gene.
Overall, ac96 (including Ac 97) is a non-essential gene of baculovirus, and at least when replicated in insect cell lines, both genes do not exhibit important functions. At the same time, the influence of deletion of these genes on the expression level of exogenous genes has not been paid attention to the proliferation of viruses. In addition, deletion of Ac96 can prevent the virus from infecting insects, and can increase the biological safety of recombinant baculovirus.
Disclosure of Invention
The invention aims to provide a gene deletion type baculovirus vector, which aims to solve the problems of low expression quantity and low capacity of exogenous DNA fragments in the background technology.
According to the invention, the Ac96 gene is inactivated, so that a baculovirus vector with improved yield of foreign proteins is obtained.
According to the invention, the Ac96-97 gene is inactivated simultaneously, so that a baculovirus vector with improved exogenous protein yield is obtained.
The invention is realized by knocking out Ac96 fragments from baculovirus vectors by homologous recombination or gene editing and other technologies, thus obtaining the Ac96 inactivated baculovirus vectors.
The invention is realized by knocking out Ac96 fragments and Ac97 and Ac96 overlapping sequences from baculovirus vectors by homologous recombination or gene editing and other technologies, thus obtaining the Ac96-97 inactivated baculovirus vector.
Or when synthesizing the baculovirus vector, the Ac96 gene is not contained, namely the Ac96 and Ac96-97 inactivated baculovirus vector is obtained.
Or the promoter region and/or the coding region and/or the 3' -non-coding region of the Ac96 and Ac97 genes are subjected to base deletion, insertion and substitution, so that the genes lose the original activity, and the Ac96 or Ac96-97 inactivated baculovirus vector is obtained.
The invention also aims at providing a recombinant baculovirus, which is obtained by recombining the Ac96 or Ac96-97 inactivated baculovirus vector with a DNA fragment with an exogenous sequence.
Another object of the present invention is to provide a recombinant protein obtained by constructing a recombinant virus using the gene-inactivated type baculovirus vector, and then infecting insect host cells and expressing as the recombinant protein.
The proliferation curve of the recombinant baculovirus constructed by the gene-inactivated baculovirus vector is not significantly different from that of the wild type vector. The expression quantity of the exogenous recombinant protein of the gene inactivated baculovirus is obviously higher than that of a control vector.
After the Ac96 gene is inactivated, the protein expression level is obviously improved, and the proliferation level of the virus is not affected. It is demonstrated that the present invention can provide a baculovirus expression vector having excellent production traits and high yield characteristics.
In addition, baculovirus from which the gene was knocked out was unable to replicate in insects, increasing the biosafety of baculovirus expression vectors.
Another object of the present invention is to provide the use of said gene inactivated baculovirus expression vector in the biological industry.
Drawings
FIG. 1 is a schematic diagram of a gene knockout strategy for baculovirus vectors provided by the invention.
FIG. 2 shows the primary growth curve of Bacmid ΔAc96 baculovirus vector provided by the invention. The Ac96 knockout did not significantly affect viral proliferation compared to the wild-type vector.
FIG. 3 shows green fluorescent protein GFP expressed by Bacmid delta Ac96 baculovirus vectors provided by the invention. Results of SDS-polyacrylamide gel electrophoresis of cells 4 days after virus infection, coomassie brilliant blue staining.
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
The principle of application of the invention is described in detail below with reference to the accompanying drawings.
1. Knockout of the Ac96 gene.
The knocking-out strategy is shown in figure 1. The rpsL-AMP expression cassette with the Ac96 homology arm was amplified by PCR technique using primers with 54bp homology arm upstream and downstream of the Ac96 gene as template and prpsL-AMP plasmid (university of North agriculture and forestry science, report-Nature science edition, month 2015, volume 43, pages 181-190). The primer sequences used in this step are as follows:
Ac96U: ttggctatcgtgtttgtacttttcgtgttaatttatttaataatttcgatcaaagatggcctggtgatgg
Ac96D: aaacattatggattacgaacaatattttattaaataaaaatataaactctattcttaccaatgcttaatc。
the PCR product obtained (about 1.5kb in size) was electrotransformed into E.coli HS996 containing Bacmid Bac10: KO1629 (Nucleic Acids Research, 2003, vol.31, no. 2 e 6), pSC 101-BAD-gbaplasmid was induced by arabinose to express RedET recombinase, homologous recombination was achieved in E.coli, and positive clones were obtained by ampicillin resistance screening (see for methods see Counter-Selection BAC Modification Kit specification of GENE BRIDGES). After PCR identification and sequencing identification, the Bacmid with the Ac96 fragment replaced by rpsL-AMP is obtained and named Bacmid-Ac 96:rpsL-AMP.
In order to remove the selection marker rpsL-AMP expression cassette, a further counter-selection is required.
A108 bp reverse-screen substitution sequence was amplified in a PCR instrument with the following primers:
Ac96f: ttggctatcgtgtttgtacttttcgtgttaatttatttaataatttcgatcaaagaatagagtttatattttta
Ac96r: aaacattatggattacgaacaatattttattaaataaaaatataaactctattc。
the obtained reverse screening substitution sequence amplification product is electrically transformed into escherichia coli HS996 containing Bacmid-Ac96, rpsL-AMP, the expression of RedET recombinase is induced by arabinose, homologous recombination is realized in escherichia coli, and a resistant colony on a background lawn is selected through streptomycin resistance screening, so that a positive clone is obtained (see the specification of GENE BRIDGES company Counter-Selection BAC Modification Kit). And (3) carrying out PCR identification and sequencing identification to obtain the Bacmid with the knockout Ac96 fragment, which is named Bacmid delta Ac96. This is the baculovirus expression vector for Ac96 knockout.
E.coli HS996 containing Bacmid DeltaAc 96 was grown in an expanded culture, bacmid DeltaAc 96 was extracted with a "Bacmid miniprep kit" (product of Biyundian Co.) and then linearized by restriction with Bsu 36I. The linearized Bacmid delta Ac96 is extracted by phenol-chloroform, and after removing proteins, the extract is dissolved in sterile water for standby.
2. Constructing recombinant viruses.
Linearized wild-type Bacmid (Bac 10: KO 1629) and Ac96 knockout Bacmid were co-transfected with pTriEx-GFP plasmid (GFP gene fragment cloned between pTriEx1.1 NcoI/XhoI sites) into Sf9 insect cells and P0 generation recombinant virus was collected 5 days after transfection. Sf9 cells were infected with the appropriate amount of P0 generation virus and P1 generation virus was collected 4 days after transfection.
3. Virus proliferation curve.
Sf9 cells were infected with control recombinant virus and Ac96 knockout recombinant virus at 0.1 MOI, after which samples were taken every 24 hours until 144 hours post infection. The virus titer was determined by quantitative PCR and a virus primary growth curve was drawn (FIG. 2). The Ac96 knockdown virus has virus proliferation characteristics similar to those of the control virus, compared to the wild-type virus. In other words, knockout Ac96 did not significantly affect viral replication.
4. Expressing the recombinant protein.
Sf9 cells and High Five cells were infected with the recombinant virus at 3 MOI and the cells were harvested the fourth day after infection. After cell lysis, SDS-polyacrylamide gel electrophoresis and Coomassie brilliant blue staining were performed (FIG. 3). Compared with the wild type virus vector, the expression level of the exogenous recombinant protein GFP is remarkably improved in both Sf9 cells and High Five cells. Since the knock-out virus test signal is too strong to evaluate the yield exactly by density scanning, the GFP expression levels in both Sf9 cells and High Five cells were increased several times visually.
GFP can generate both an observable fluorescent signal and is not cytotoxic and therefore a common reporter protein in the field of protein expression. The increase in GFP expression in this example demonstrates that the Ac96 knockout recombinant virus has good recombinant protein expression properties. By utilizing the high-yield characteristic, any one of protein products and subunit vaccines can be expressed with high efficiency.
From the above experiments, it can be seen that the knockout of Ac96 does not affect virus proliferation, but can greatly improve the yield of exogenous proteins. Furthermore, the Ac96 knockout also reduces the viral genome by 0.6kb, increasing the capacity of the viral vector. And as an oral infectious agent, knockout of Ac96 also increases the biosafety of baculovirus expression vectors. Compared with the prior art, the method has outstanding substantive characteristics and remarkable progress.
The expression of green fluorescent protein GFP is only an example of the present invention and is not intended to limit the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
It is additionally noted that Ac97 is not a functional gene from the current perspective, and its open reading frame overlaps the carboxy terminus of the coding region of the Ac96 gene. This example knocks out Ac96 and also breaks the Ac97 open reading frame, which can be considered as Ac96-97 being knocked out simultaneously.

Claims (3)

1. The gene knockout type baculovirus expression vector is characterized in that a gene Ac96 on a baculovirus AcMNPV expression vector Bacmid is knocked out, and an open reading frame of the gene Ac97 is broken, so that the gene knockout type baculovirus expression vector is obtained.
2. A recombinant virus comprising the gene knockout baculovirus expression vector as defined in claim 1.
3. Use of the knock-out baculovirus expression vector of claim 1 for expression of a foreign protein in insect cells.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110387385A (en) * 2019-07-18 2019-10-29 西北农林科技大学 A kind of novel rhabdovirus expression vector
WO2020058118A1 (en) * 2018-09-17 2020-03-26 Universität Für Bodenkultur Wien Dual vector system for improved production of proteins in animal cells
CN111378687A (en) * 2018-12-27 2020-07-07 陕西杆粒生物科技有限公司 High-yield baculovirus expression vector
WO2020264139A1 (en) * 2019-06-26 2020-12-30 Virovek, Inc. Baculovirus expression system

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020058118A1 (en) * 2018-09-17 2020-03-26 Universität Für Bodenkultur Wien Dual vector system for improved production of proteins in animal cells
CN111378687A (en) * 2018-12-27 2020-07-07 陕西杆粒生物科技有限公司 High-yield baculovirus expression vector
WO2020264139A1 (en) * 2019-06-26 2020-12-30 Virovek, Inc. Baculovirus expression system
CN110387385A (en) * 2019-07-18 2019-10-29 西北农林科技大学 A kind of novel rhabdovirus expression vector

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Rohrmann GF.《Baculovirus Molecular Biology》.National Center for Biotechnology Information,2019,(第4版),第205-244页. *

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