CN118291542A

CN118291542A - Multi-gene deleted high-yield baculovirus expression vector Bac5.0s and application thereof

Info

Publication number: CN118291542A
Application number: CN202410489149.4A
Authority: CN
Inventors: 刘琴; 张元兴; 俞越; 吴诗璟
Original assignee: Shanghai Bai'anzhen Biotechnology Co ltd; East China University of Science and Technology
Current assignee: Shanghai Bai'anzhen Biotechnology Co ltd; East China University of Science and Technology
Filing date: 2024-04-22
Publication date: 2024-07-05

Abstract

The invention provides a polygene-deleted high-yield baculovirus expression vector Bac5.0s and application thereof, which remarkably improves the stability of baculovirus genome and optimizes the expression of exogenous proteins by deleting about 19-22 genes on a large scale under the condition of keeping good replication capacity, and can be suitable for the production of various biological products and has application prospect.

Description

Multi-gene deleted high-yield baculovirus expression vector Bac5.0s and application thereof

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to a polygene-deleted high-yield baculovirus expression vector Bac5.0s and application thereof.

Background

The genome size of the noctuid nuclear polyhedrosis virus (Autographa californica nucleopolyhedrovirus, acMNPV) is about 134kb. BEVS as a eukaryotic expression system has its own limitations, mainly including: glycosylation problems, protein degradation problems, and genomic stability problems. Among them, the glycosylation problem has been solved to some extent by modification of the glycosylation pathway of insect cell lines.

Some researchers in the field put forward the concept of baculovirus genome minimization, on the basis of the proposal, the SynBac1.0 obtained after partial gene superposition deletion on the AcMNPV genome is remarkably improved, and the superiority brought by baculovirus genome shrinkage is presented. Guo et al have deleted the AcMNPV gene of about 17kb by biological synthesis method, and the obtained virus AcMNPV-Syn-mC1-1.1 has infectivity, but compared with wild type virus, the virus titer is obviously reduced, and the virus replication is obviously delayed.

Currently available baculovirus vectors are limited; in view of the mutual restriction and complex connection of genes or proteins, how to reduce genes to improve the genome stability of baculovirus and optimize the expression of exogenous proteins, the multi-version baculovirus expression vector is provided to adapt to the production of various biological products, and there are a plurality of difficulties for researchers, so that development of some new transformation strategies is needed.

Disclosure of Invention

The invention aims to provide a polygene-deleted high-yield baculovirus expression vector Bac5.0s and application thereof.

In a first aspect of the invention, there is provided a method of constructing a baculovirus vector comprising: the genome of the noctiluca californica nuclear polyhedrosis virus (AcMNPV) is taken as the basis for constructing a baculovirus vector, and polygene deletion (deletion) is carried out, wherein the polygene deletion comprises deletion of Ac15, ac16, ac20/21, ac22, ac42, ac43, ac44, ac55, ac56, ac57, ac58/59, ac60, ac61, ac69, ac70 and Ac72, and deletion of genes selected from the following groups: ac47, ac48 and Ac49 (Bac5.0-1, 14329bp reduced); ac84, ac85, ac86, ac87, ac88 and Ac91 (bac 5.0-2, reduced 17888 bp); or Ac148, ac149 and Ac150 (Bac5.0-3, 14558bp reduced).

In another preferred embodiment, the baculovirus vector further comprises elements required for expression of a foreign gene; preferably, the elements include (but are not limited to): promoters, replicons, transposition sites, foreign gene insertion sites (e.g., cleavage sites).

In another preferred embodiment, the element further comprises: resistance selection markers, reporter genes.

In another preferred embodiment, the promoter comprises (but is not limited to) a ph promoter, the replicon comprises (but is not limited to) a mini-F replicon, the transposition site comprises (but is not limited to) an attTn7 transposition site, the resistance selection marker comprises (but is not limited to) a kana (Kan) resistance selection marker, and the reporter gene comprises (but is not limited to) an eYFP fluorescent gene.

In another preferred embodiment, the alfalfa silver vein moth nuclear polyhedrosis virus (AcMNPV) genomic sequence is an artificial sequence from EmBacY bacmid.

In another preferred embodiment, the foreign gene insertion site is an enzyme cleavage site.

In another preferred example, the foreign gene insertion site may be one or more insertion sites.

In another preferred embodiment, the elements required for expression of the foreign gene and optionally the resistance selection marker and/or reporter gene in the baculovirus vector are operatively linked.

In another preferred example, the genomic sequence of the noctiluca californica nuclear polyhedrosis virus (AcMNPV) as a base sequence for constructing a baculovirus vector includes Ac1 to Ac154 genes (also referred to as Open Reading Frames (ORFs) 1 to 154).

In another preferred example, homologous recombination is performed using a λred recombinase, and deletion (deletion) of a gene is performed; preferably, deletion (deletion) of the gene is performed using the lambda Red recombinase/I-SceI enzyme traceless editing system.

In another aspect of the present invention, there is provided a recombinant baculovirus vector which is an engineered baculovirus vector based on the genomic sequence of the noctiluca californica nuclear polyhedrosis virus (AcMNPV) and which is subject to multiple gene deletion (deletion) comprising deletion of Ac15, ac16, ac20/21, ac22, ac42, ac43, ac44, ac55, ac56, ac57, ac58/59, ac60, ac61, ac69, ac70 and Ac72, and deletion of a gene selected from the group consisting of: ac47, ac48 and Ac49; ac84, ac85, ac86, ac87, ac88 and Ac91; or Ac148, ac149 and Ac150.

In another preferred embodiment, the recombinant baculovirus vector further comprises elements required for expression of a foreign gene; preferably, the elements include (but are not limited to): promoters, replicators, transposition sites, foreign gene insertion sites; optionally, the element further comprises: resistance selection markers, reporter genes.

In a further aspect of the invention there is provided the use of a recombinant baculovirus vector as described in any one of the preceding for the preparation of a recombinant baculovirus; preferably, the recombinant baculovirus vector (bacmid) is transfected into a virus producing cell to produce the recombinant baculovirus; or for inserting an exogenous gene to express the exogenous gene; preferably, a recombinant baculovirus vector (bacmid) into which a foreign gene is inserted is transfected into a virus-producing cell, and the recombinant baculovirus is produced, which expresses a foreign protein.

In another aspect of the invention there is provided a recombinant baculovirus obtained from the recombinant baculovirus vector packaging as described in any one of the preceding; or it is obtained by packaging the recombinant baculovirus vector of any one of the foregoing inserted with a foreign gene, which is capable of expressing a foreign protein.

In another aspect of the invention, there is provided a method of preparing a recombinant baculovirus, the method comprising:

(A) Preparing a recombinant baculovirus vector as described in any one of the preceding;

(B) Transfecting the recombinant baculovirus vector of (A) into a virus-producing cell, thereby packaging to obtain the recombinant baculovirus.

In another preferred example, a recombinant baculovirus vector (bacmid) into which a foreign gene is inserted is transfected into a virus-producing cell to produce the recombinant baculovirus, which expresses a foreign protein.

In another aspect of the invention there is provided the use of said recombinant baculovirus for: expressing the exogenous gene to obtain an exogenous protein; preferably, the exogenous protein includes: immunogenic proteins, pharmaceutically active proteins, diagnostically active proteins, structural proteins, enzymes, fusion peptides, reporter proteins.

In another aspect of the invention, there is provided a kit comprising: the recombinant baculovirus vector of any one of the preceding claims, or the recombinant baculovirus; preferably, virus-producing cells are also included.

Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.

Drawings

FIG. 1, ac15KO, ac47KO, ac61KO, ac69KO, ac85KO, ac91KO, ac149KO, were used as controls for expression of hTPO protein from baculovirus expression vectors deleted for 7 individual nonessential genes, which did not raise the expression level of the foreign protein.

FIG. 2, schematic representation of a knockout strategy for a multiple gene deletion on a baculovirus vector of the invention.

FIG. 3, fluorescent particle figures of the invention after transfection of three polygene deleted baculovirus vectors of Bac5.0-1, bac5.0-2 and Bac5.0-3 into Sf9 cells, with WT controls, bac5.0-1, bac5.0-2 and Bac5.0-3 had normal viral infection spreading capacity and expression capacity of the ph promoter.

FIG. 4, western blot detection after sampling using Bac5.0-1, bac5.0-2 and Bac5.0-3 polygene deleted baculovirus vectors of the present invention for hTPO expression. As a result, it was found that Bac5.0-1, bac5.0-2 and Bac5.0-3 significantly increased the expression level of the foreign protein, and that the expression levels of hTPO were increased by 1.9, 1.8 and 1.5 times, respectively, by gray scale scan analysis.

FIG. 5, acMNPV gene distribution and region division map.

Detailed Description

In the optimization research on baculovirus expression vectors, the inventor identifies a series of genes on the AcMNPV as nonessential genes for baculovirus infection diffusion in the early analysis work, but deletion of a single gene has the priority of deleted bases on one hand and has no obvious improvement on the expression level of exogenous proteins on the other hand. In order to improve the practicability and performance of the baculovirus expression vector (comprising improving the virus infection diffusion capability and improving the expression of exogenous genes), the inventor further optimizes the vector, and prepares a large number of combined gene deletion vectors to obtain the recombinant baculovirus expression vector (Bac5.0s: bac5.0-1, bac5.0-2 and Bac5.0-3) with stable and simple genes, high virus infection diffusion capability and efficient expression of exogenous genes.

Terminology

As used herein, the term "operably linked" or "operably linked" refers to a functional spatial arrangement of two or more nucleic acid regions or nucleic acid sequences. For example: the promoter region is placed in a specific position relative to the nucleic acid sequence of the gene of interest such that transcription of the nucleic acid sequence is directed by the promoter region, whereby the promoter region is "operably linked" to the nucleic acid sequence.

As used herein, the term "element" refers to a series of functional nucleic acid sequences useful for the expression of proteins. The sequences of the "elements" may be those provided in the present invention, and include variants thereof, as long as the variants substantially retain the function of the "elements" obtained by inserting or deleting some bases (e.g., 1 to 50bp; preferably 1 to 30bp, more preferably 1 to 20bp, still more preferably 1 to 10 bp), or performing random or site-directed mutation, etc.

As used herein, the term "exogenous" or "heterologous" refers to a relationship between two or more nucleic acid or protein sequences from different sources, or a relationship between a nucleic acid from different sources and a host cell. For example, if the combination of nucleic acid and host cell is not normally naturally occurring, the nucleic acid is heterologous to the host cell. The particular sequence is "exogenous" to the cell or organism into which it is inserted.

As used herein, the term "expression cassette" refers to a gene expression system comprising all the necessary elements necessary for expression of a gene of interest, typically comprising the following elements: a promoter, a target gene sequence, and a terminator; in addition, signal peptide coding sequences and the like can be optionally included. These elements are operatively connected.

As used herein, an "effective amount" or "effective dose" refers to that amount which is functional or active in and acceptable to a human and/or animal as used herein.

As used herein, a "pharmaceutically acceptable" ingredient is a substance that is suitable for use in humans and/or mammals without undue adverse side effects (such as toxicity, irritation, and allergic response), commensurate with a reasonable benefit/risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents.

As used herein, "comprising" means that the various ingredients can be applied together in a mixture or composition of the invention. Thus, the terms "consisting essentially of and" consisting of are encompassed by the term "containing.

Carrier body

There are many uncertain factors in the modification of baculovirus, the genome of which is known, but the genes of which are numerous and complex, and particularly how to actually improve the expression efficiency of the exogenous genes on the basis of maintaining the stability of the genome and high virus infection spreading capability remains a bottleneck in the field. Although the present inventors analyzed a series of genes of interest in previous studies, there was no significant effect on how to increase the expression of exogenous genes. The related research of combining deletion such as the gene of the invention and exploring multi-gene deletion on baculovirus vectors on baculovirus expression systems has not been reported at present. In the invention, by deleting genes irrelevant to baculovirus packaging replication production, the size of baculovirus genome is reduced to improve the stability of baculovirus genome and optimize the expression of exogenous proteins, a group of baculovirus expression vectors is provided, and the invention can adapt to the production of various biological products and has application prospect.

The invention aims at respectively carrying out superposition deletion on each gene on the AcMNPV, constructing a series of multi-gene deleted baculovirus expression vectors and optimizing a baculovirus system. In the specific embodiment of the invention, a series of high-yield baculovirus expression vectors with multiple gene deletions are prepared, which are called Bac5.0-1, bac5.0-2 and Bac5.0-3 by the inventor, and the genome of the vectors is relatively simple, but the vectors have good Virus infection diffusion capability, remarkably improve the expression level of exogenous proteins, and have wide application prospects in the biological product industry fields of subunit vaccines, virus LIKE PARTICLES (VLPs) vaccines, recombinant adeno-associated Virus (rAAV) vaccines and the like.

In the early stage of research, the inventor firstly tries a modification scheme of single gene deletion, uses baculovirus expression vectors deleted by various single genes (Ac 15KO, ac47KO, ac61KO, ac69KO, ac85KO, ac91KO, ac149KO and the like) to express recombinant human thyroid peroxidase (Human thyroid peroxidase, hTPO), uses a wild type baculovirus expression vector EmBacY (WT), and does not increase the expression level of exogenous proteins through Western blot detection.

Subsequently, the inventors, facing complex viral genomes, roughly divided their genes into 10 regions based on AcMNPV genome, performed different superposition combinations, successively reduced AcMNPV genome, and constructed a series of polygene deleted baculovirus vectors by iterative deletion method. In a preferred embodiment, ac1-7 is designated as region II, ac15-Ac22 is designated as region III, ac27-33 is designated as region IV, ac42-44 is designated as region I, ac47-49 is designated as region V, ac55-61 is designated as region VI, ac69-72 is designated as VII, ac84-91 is designated as region VIII, ac105-124 is designated as region IX, ac148-150 is designated as region X, and the 10 regions above are each deleted by superposition. Information on the Ac1-154 gene has been disclosed in NCBI (GeneBank: KM 667940.1).

Wherein, the Bac5.0-1 vector is deleted for up to Ac15,Ac16,Ac20/21,Ac22,Ac42,Ac43,Ac44,Ac47,Ac48,Ac49,Ac55,Ac56,Ac57,Ac58/59,Ac60,Ac61,Ac69,Ac70,Ac72, genes at the same time.

Wherein, the Bac5.0-2 vector is deleted for Ac15,Ac16,Ac20/21,Ac22,Ac42,Ac43,Ac44,Ac55,Ac56,Ac57,Ac58/59,Ac60,Ac61,Ac69,Ac70,Ac72,Ac84,Ac85,Ac86,Ac87,Ac88 and Ac91 simultaneously, and up to 22 genes.

Wherein, the Bac5.0-3 vector lacks Ac15, ac16, ac20/21, ac22, ac42, ac43, ac44, ac55, ac56, ac57, ac58/59, ac60, ac61, ac69, ac70, ac72, ac148, ac149 and Ac150 simultaneously for up to 19 genes.

Three bac5.0-1, bac5.0-2 and bac5.0-3 bac vectors reduce the genome of baculovirus by 14-18 kb, improve the expression level of exogenous protein by 1.9, 1.8 and 1.5 times respectively, and optimize the baculovirus system to meet the production requirement of diversification of different biological products in the biological product industry fields of subunit vaccine, virus LIKE PARTICLES (VLPs) vaccine, recombinant adeno-associated Virus (rAAV) vaccine and the like, and have wide application prospect.

In order to investigate the replication and proliferation infectivity of the three polygene-deleted baculovirus-carried baculovirus, in the embodiment of the invention, transfection experiments of Sf9 cells are respectively carried out by taking WT as a control to prepare P1 generation recombinant viruses, the P1 generation recombinant viruses are harvested to carry out reinfection of Sf9 cells, and fluorescent particle condition observation is carried out in the infection and transfection processes. Three multi-gene deleted baculovirus expression vectors of Bac5.0-1, bac5.0-2 and Bac5.0-3 are identified to have normal virus replication infection capacity and ph promoter expression capacity. Bac5.0-1, bac5.0-2 and Bac5.0-3 reduce baculovirus genome by about 14-17 kb and significantly increase expression level of foreign proteins.

In the embodiment of the invention, DH10Bac strain containing EmBacY stem grains is used as a genetic operation object, emBacY stem grains are artificially modified AcMNPV genome, a mini-F replicon, a kana (Kan) resistance screening marker and an attTn7 transposition site are integrated at a polyhedron site, and two gene sites of chiA, v-cath are replaced by an eYFP fluorescent marker expressed by a ph promoter, so that the visualization of the transfection and infection process is ensured.

In the embodiment of the invention, gene deletion is realized through a lambda red/I-SceI traceless marker system, and the method mainly comprises two steps:

(1) The resistance gene replaces the target region. Amplifying a spectinomycin (Spec) resistance screening marker expression cassette with I-SceI enzyme cleavage recognition sites at two ends by using a linear targeting primer carrying 50bp homology arms at the upstream and downstream of a target region, performing ethanol precipitation purification on the obtained PCR product, then electrically transforming the PCR product into a DH10Bac strain containing EmBacY rod grains, and obtaining an intermediate deletion strain of a resistance gene replacement target region through lambda red homologous recombination and Spec resistance screening.

(2) Traceless substitution of the resistance gene. To meet the overlapping deletions of the regions on the AcMNPV genome, a Spec-resistant gene expression cassette of the middle deleted strain was subjected to traceless substitution. First, it is necessary to construct different make-up Donor plasmids, and the construction of Donor plasmids is divided into two cases depending on whether or not the target region contains an essential gene. When all of the target region is a non-essential gene, such as region I, only about 500bp of the sequence at the upstream and downstream of the target region I needs to be connected to pKSI-1 plasmids containing I-SceI recognition sites at the two ends of the MCS to construct a make-up Donor plasmid; when the target region species contains an essential gene, e.g., region II, it is necessary to construct a make-up Donor plasmid by adding an essential gene sequence between the 500bp homology arms upstream and downstream of region II. And then the mending Donor plasmid is transformed into an intermediate deletion strain, IPTG induces and expresses I-SceI enzyme, homologous recombination is carried out under the assistance of a lambda red system recombinase, the traceless replacement of Spec resistance genes is realized, and three multi-gene deleted baculovirus expression vectors, namely Bac5.0-1, bac5.0-2 and Bac5.0-3, are finally obtained.

In order to investigate the viral replication and infection capacity of three polygenic deletion type baculovirus vectors, transfection experiments of Sf9 cells were performed to prepare P1 generation recombinant viruses with wild type EmBacY (WT) as a control, and P1 generation viral supernatants were re-infected with Sf9 cells after 96 hours to prepare P2 and P3 generation viruses, respectively. As proved by detection, bac5.0-1, bac5.0-2 and Bac5.0-2 have strong virus infection replication capacity.

As a preferred mode of the invention, the vector further comprises an element or gene selected from the group consisting of the following operably linked: elements required for expressing a foreign gene (gene of interest); preferably, the elements include, but are not limited to: promoters, replicons, transposition sites, foreign gene insertion sites (e.g., cleavage sites); optionally, the element further comprises: resistance selection markers, reporter genes. It will be appreciated that the design of the reporter gene, cleavage site etc. elements is sometimes intended to provide a convenient way of observation or detection when studied, but is not necessary when the recombinant baculovirus vector or recombinant baculovirus of the invention is used in the clinical pharmaceutical field; or may be replaced with a gene capable of expressing a foreign protein having pharmaceutical activity (protein of interest) at the position of the reporter gene.

The expression vector will typically also contain an origin of replication and/or other marker genes, etc. According to the teachings of the present invention, methods well known to those skilled in the art can be used to construct the expression vectors required for the present invention. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence may be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. Furthermore, the expression vector preferably comprises one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells.

The expression vector of the invention can be inserted with an expression cassette of an exogenous gene, thereby expressing the exogenous protein of interest. The baculovirus expression vector can be used as an expression vector platform, is suitable for expressing various exogenous proteins, and is not particularly limited. As some alternative embodiments, the exogenous proteins include (but are not limited to): immunogenic proteins, pharmaceutically active proteins (e.g., antibodies), diagnostically active proteins (e.g., antibodies), structural proteins, enzymes, fusion peptides, and reporter proteins. The foreign protein may be a single or two or more, so long as the length of the encoding gene is within the packaging capacity which the baculovirus can accommodate.

The present invention allows for the reduction of genes, allowing for the accommodation of a greater length, variety of exogenous genes, which may be one or more. The foreign gene may be single-copy or multi-copy.

In the expression vector of the present invention, the insertion position of the foreign gene expression cassette may not be unique, and may be any suitable position on the vector.

The elements of the expression vectors of the invention are operably linked to facilitate packaging of the virus or efficient expression of the foreign protein.

In addition to engineering against wild-type to construct the novel baculovirus vectors of the invention, synthetic biology-based techniques can also be used to construct the novel baculovirus vectors of the invention. In contrast to the research direction of traditional biology, synthetic biology is to build parts from the most basic elements step by step, design and modify genetic materials in the process, and finally obtain ideal new-born objects. However, it should be understood that in addition to the synthetic biological techniques, other methods of constructing the baculovirus vectors of the present invention are also contemplated by the present invention after the technical solutions of the present invention are obtained.

The invention provides a new method for constructing a novel baculovirus vector, is beneficial to expanding the application of the baculovirus vector, has more flexible length of the exogenous gene which can be accommodated by the baculovirus vector, and provides a foundation for expressing more kinds of proteins by applying the baculovirus vector.

The vector of the invention can be applied to various basic researches and clinical applications, such as being used as an expression vector of various genes, expressing exogenous proteins, and also being applied to drug screening, vaccine research and development, gene therapy and the like. Has the characteristics of safety, high efficiency and stability.

The viral plasmids exemplified in the examples of the present invention use a specific promoter (ph promoter). It will be appreciated that other types of promoters are also useful. Such as the early promoter p6.9, p10 or vp39 promoter. Other early promoters are also for example 39K or gp64. Promoters pSeL from asparagus caterpillar nuclear polyhedrosis virus (Spodoptera exigua multicapsid nucleopolyhedrovirus, seMNPV) are also useful. A promoter named pB2 from High Five cells may also be used. Very early promoter OpIE from the yellow fir moth nuclear polyhedrosis virus (Orygia pseudotsugata multicapsid nucleopolyhedrovirus, opMNPV) can also be used. The Ac46 promoter has higher activity, can be used in combination with the ph or p10 promoter, and has a superposition effect on the expression of a target gene so as to further improve the expression level of the exogenous protein. Enhancers are DNA elements that increase the expression level of a certain gene and can be used in combination with a promoter to increase its activity. hr1 can increase the expression level of exogenous proteins in both Sf21 and High Five cells. In addition to promoters, other operational elements on the engineered baculovirus vectors of the invention may be replaced with other homofunctional elements, and are included within the scope of the invention.

Baculovirus

After the baculovirus expression vector is obtained, the baculovirus expression vector is transfected into virus-producing cells to reproduce viruses. After a period of time following transfection, the virus may be harvested. The virus-producing cells may be any of a variety of cells known in the art capable of propagating baculoviruses, preferably Sf9 cells and the like.

As a preferred mode of the invention, the harvested virus may be repeatedly infected with virus-producing cells and passaged continuously. The determination of viral titer (TCID ₅₀) can be performed according to methods conventional in the art. The recombinant baculovirus obtained is also encompassed by the present invention.

The baculovirus obtained by expression of the baculovirus vector can generate high-titer viruses in a short time; the exogenous gene loading is high. According to the results of the present inventors, the exogenous gene loading amount was high.

Compositions, kits or kits

The invention also provides a composition comprising an effective amount of said baculovirus, and a pharmaceutically acceptable carrier. Preferably, the baculovirus expresses a foreign protein of interest. For example, the exogenous proteins include, but are not limited to: immunogenic proteins, pharmaceutically active proteins (e.g., antibodies), diagnostically active proteins (e.g., antibodies), structural proteins, enzymes, fusion peptides.

Typically, the baculovirus is formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5-8, preferably about 6-8.

Based on the new findings of the present invention, a kit/kit is also provided comprising said baculovirus expression vector or said baculovirus. The kit/kit may also include virus-producing cells (e.g., sf9 cells), culture medium, and the like.

In addition, the kit/kit may further comprise instructions for use of the baculovirus following the expression method.

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedures, which are not specifically noted in the examples below, are generally carried out according to conventional conditions such as those described in J.Sam Brookfield et al, molecular cloning guidelines, third edition, scientific Press, or according to the manufacturer's recommendations.

Materials and methods

The DH10Bac strain containing EmBacY rod grains is derived from the national center of protein science in Shanghai, and three plasmids, namely pREDTKI, pMDISI and pKSI-1, in a lambda red/I-SceI traceless editing system are derived from the ProbeInstructions of the university of North China, namely the university of biological engineering; the resistance of both pREDTKI and pKSI-1 plasmids was replaced, with pREDTKI replaced by Kan resistance with hygromycin (Hph) resistance and pKSI-1 replaced by ampicillin (Amp) resistance with bleomycin (Zeocin) resistance. Unless otherwise stated, the subsequent examples were run with plasmid after resistance change.

The primers used in the examples are shown in Table 1.

TABLE 1 primers

In the table, primer sequences that bind to the resistance gene template plasmid pDMISI are underlined.

Example 1 AcMNPV Gene distribution, region division and traceless deletion of Gene regions

Based on the alfalfa silver vein moth nuclear polyhedrosis virus (AcMNPV) genome (GeneBank: KM 667940.1), the inventor performs analysis and research on nonessential genes, and performs various superposition deletion analysis on the basis so as to construct a multi-gene deletion baculovirus expression vector with a simplified sequence.

For convenient observation of transfection and infection, DH10Bac strain (originated from Shanghai national protein science center) containing EmBacY bacmid was used as a genetic manipulation object, emBacY bacmid was an artificially modified AcMNPV genome, and the following modifications were performed on the basis of the AcMNPV genome: the polyhedron locus integrates a mini-F replicon, a kana (Kan) resistance screening marker and an attTn7 transposition locus, and two gene loci of the chiA and v-cath are replaced by coding genes of an eYFP fluorescent marker expressed by a ph promoter, so that the visualization of the transfection and infection process is ensured; two sites, chiA, v-cath, also introduce a loxp site and Amp resistance.

In order to perform sequence reduction optimization, the inventor divides genes on the AcMNPV into 10 areas, performs different superposition combinations, continuously reduces the AcMNPV genome, and constructs a series of polygene deletion baculovirus vectors by adopting an iterative deletion method. Ac1-7 was designated as region II, ac15-Ac22 was designated as region III, ac27-33 was designated as region IV, ac42-44 was designated as region I, ac47-49 was designated as region V, ac55-61 was designated as region VI, ac69-72 was designated as VII, ac84-91 was designated as region VIII, ac105-124 was designated as region IX, ac148-150 was designated as region X, and the above 10 regions were each deleted by superposition. AcMNPV gene distribution and regional division map are shown in figure 5.

Gene deletion was achieved by the lambda red/I-SceI traceless marker system.

The multiple gene deletions included Spec resistance gene replacement target region and Spec resistance gene traceless elimination, deletion strategies see Step1 and Step2 of fig. 2, with primers shown in table 1.

1. Establishment of Bac1.0

DH10Bac calcium transformation competence containing EmBacY rod grains is prepared, pREDTKI plasmid encoding lambda red recombinase and I-SceI enzyme is transformed, screening is carried out on a Kan+Hph+Tet resistance plate, and positive monoclonal preparation of electric transformation competence is reserved.

The Spec resistance gene expression cassette with 50bp homology arms at the upstream and downstream of Ac42-44 and I-SceI cleavage recognition sites was amplified by PCR using the primers Spec-Ac42-F and Spec-Ac44-R and pMDISI plasmid as a template, and subjected to fragment recovery using a DNA gel recovery kit, ethanol precipitation purification and electrotransformation into pREDTKI positive DH10Bac competence. With the help of lambda red recombinase expressed by L-arabinose induction, a Spec resistance expression cassette recombination region I is screened on a Kan+Hph+Tet+spec resistance plate to obtain positive monoclonal, PCR verification and sequencing is carried out by adopting primers Ac42KO-F and Ac44KO-R to obtain a deletion strain of which the region I is replaced by a Spec resistance gene, named Ac42-44KO, and calcium transfer competence is prepared for standby.

The analysis shows that the region I is all non-essential genes, and the supplementary Donor plasmid is constructed as follows: amplifying the gene of about 500bp at the upstream of Ac42 by using a primer Ac42-D1/D2 and EmBacY rods as templates, and amplifying the gene of about 500bp at the downstream of Ac44 by using a primer Ac44-D1/D2 and EmBacY rods as templates; meanwhile, a primer p-4-F/R is used, and pKSI-1 plasmid is used as a template to obtain the pKSI-1 linearization vector.

The pKSI-1 linearization vector, up sequence and down sequence were ligated using a seamless cloning kit, transformed into pFastT competent, constructed as a make-up plasmid Donor-1, coated on Zeocin resistance plates, and after PCR-verified sequencing using primer Donor-F/R, the make-up plasmid Donor-1 was transformed into Ac42-44KO, coated on Kan+Hph+Tet+spec+Zeocin resistance plates for selection. The positive monoclonal is inoculated in 1ml Kan+Hph+Tet+Spec+Zeocin liquid culture medium and cultured overnight at 30 ℃ to be the primary bacterium. The primary bacteria are inoculated in 4ml Kan+Hph+Tet liquid culture medium according to 1% inoculation amount, 10mM L-arabinose is added at the same time, 20mM IPTG is added when the temperature is 30 ℃ and the OD is approximately equal to 0.2-0.3, and the secondary bacteria are obtained after overnight culture. The secondary bacteria are inoculated in 4ml Kan+Hph+Tet liquid culture medium according to 1% inoculation amount, 10mM L-arabinose and 20mM IPTG are added at the same time, after the secondary bacteria are cultured overnight at 30 ℃, the secondary bacteria are diluted to 10 ^-3-10^-4 and coated on Kan+Hph+Tet resistance plates, the secondary bacteria are cultured overnight at 30 ℃, and the monoclonal bacteria are selected and subjected to PCR verification by adopting Ac42-44-F/R, so that three gene-trace-free deleted pole grains of Ac42, ac43 and Ac44 in the region I are obtained and are named as Bac1.0.

2. Overlapping deletions of region VI on Bac1.0

The implementation method is similar to the construction method of Bac1.0.

Amplifying a Spec resistance expression cassette by using primers Spec-Ac55-F and Spec-Ac61-R, performing ethanol precipitation purification on a PCR product, converting into Bac1.0 electrotransformation competence, performing PCR verification sequencing by using primers Ac55KO-F and Ac61KO-R to obtain a positive monoclonal with the region VI replaced by a Spec resistance gene, named Ac55-61KO, and preparing the calcium transformation competence for later use.

Through analysis, the region VI is all nonessential genes, a primer Ac55-D1/D2 is adopted, emBacY rod grains are used as templates, an up sequence is used for amplifying about 500bp genes on the upstream of Ac55, a primer Ac61-D1/D2 is used as templates, a EmBacY rod grains are used as templates, and a down sequence is used for amplifying about 500bp genes on the downstream of Ac61 genes; meanwhile, a primer p-6-F/R and pKSI-1 plasmid are used as templates to obtain a pKSI-1 linearization vector, a seamless cloning kit is used for connection transformation, and a compensation plasmid Donor-6 is constructed.

The anaplerotic plasmid Donor-6 was transformed into Ac55-61KO to effect traceless replacement of the Spec resistance expression cassette, resulting in a bac2.0-5 with overlapping deletions of region I and region VI.

3. Overlapping deletions of region VII on Bac2.0-5

The implementation method is similar to the construction method of Bac1.0.

Amplifying a Spec resistance expression cassette by using primers Spec-Ac69-F and Spec-Ac72-R, performing ethanol precipitation purification on a PCR product, converting into Bac2.0-5 electrotransformation competence, performing PCR verification sequencing by using primers Ac69KO-F and Ac72KO-R to obtain a positive monoclonal with a region VII replaced by a Spec resistance gene, named Ac69-72KO, and preparing the calcium transformation competence for later use.

As a result of analysis, region VII contains, in addition to three nonessential genes Ac69, ac70 and Ac72, an essential gene Ac71, so that the elimination of the Spec-resistant fragment requires a back-filling of Ac 71.

The make-up plasmid was constructed as follows: amplifying the gene of about 500bp upstream of Ac69 by using a primer Ac69-D1/D2 and EmBacY rod grains as templates to obtain an up sequence; amplifying the Ac71 sequence by using a primer Ac71-D1/D2 and EmBacY rod grains as templates; amplifying a gene about 500bp downstream of Ac72 by using a primer Ac72-D1/D2 and EmBacY rod grains as templates to obtain a down sequence; meanwhile, using pKSI-1 plasmid as a template, using a primer p-7-F/R to obtain a pKSI-1 linearization vector, and performing seamless cloning, connection and transformation to obtain a compensation plasmid Donor-7.

Donor-7 converts Ac69-72KO to realize traceless elimination of the Spec resistance expression cassette and Ac71 gene complementation, and a bac3.0-4 with overlapping deletion of region I, region VI and region VII is obtained.

4. Overlapping deletions of region III on Bac3.0-4

The implementation method is similar to the construction method of Bac1.0.

The Spec resistance expression cassette is amplified by using the primers Spec-Ac15-F and Spec-Ac22-R, and the PCR product is subjected to ethanol precipitation and purification to convert Bac3.0-4 electrotransformation competence, so that a positive monoclonal with the region III replaced by the Spec resistance gene is obtained, named Ac15-22KO, and the calcium transformation competence is prepared for standby.

As a result of analysis, region III contained the essential genes Ac17, ac18 and Ac19 in addition to the five nonessential genes Ac15, ac16, ac20/21 and Ac22, and thus, the elimination of the Spec resistance fragment was accompanied by the need for back-filling of these three essential genes.

The make-up plasmid was constructed as follows: amplifying the gene of about 500bp upstream of Ac15 by using a primer Ac15-D1/D2 and EmBacY rod grains as templates to obtain an up sequence; amplifying the Ac17-19 sequence by using the primers Ac17-D1 and Ac19-D2 and EmBacY rod grains as templates; amplifying the about 500bp gene downstream of Ac22 by using a primer Ac22-D1/D2 and EmBacY rod grains as templates to obtain a down sequence; meanwhile, using pKSI-1 plasmid as a template, using a primer p-3-F/R to obtain a pKSI-1 linearization vector, and performing seamless cloning, connection and transformation to obtain a compensation plasmid Donor-3.

Donor-3 transformed Ac15-22KO to effect traceless elimination of the Spec resistance expression cassette and Ac17-19 gene complementation, resulting in a stack of deleted bacmid in region I, region III, region VI and region VII, designated Bac4.0-1 (deleted fragments in each plasmid see Table 2).

5. Overlapping deletion of region V on Bac4.0-1

The implementation method is similar to the construction method of Bac1.0.

The Spec resistance expression cassette is amplified by using primers Spec-Ac47-F and Spec-Ac49-R, bac4.0-1 electrotransformation competence is transformed after ethanol precipitation and purification of PCR products, PCR verification sequencing is performed by using primers Ac47KO-F and Ac49KO-R, positive monoclonal with the region V replaced by the Spec resistance gene is obtained, named Ac47-49KO, and calcium transformation competence is prepared for standby.

According to analysis, the region V is all nonessential genes, a primer Ac47-D1/D2 is adopted, emBacY rod grains are used as templates, an up sequence is used for amplifying about 500bp genes at the upstream of Ac47, a primer Ac49-D1/D2 is used as templates, a EmBacY rod grain is used as a template, and a down sequence is used for amplifying about 500bp genes at the downstream of Ac49 genes; meanwhile, a primer p-5-F/R and pKSI-1 plasmid are used as templates to obtain a pKSI-1 linearization vector, a seamless cloning kit is used for connection transformation, and a compensation plasmid Donor-5 is constructed.

The anaplerotic plasmid Donor-5 was transformed into Ac47-49KO to effect traceless replacement of the Spec resistance expression cassette, resulting in a stack of deleted bac, designated bac5.0-1, in region I, region III, region V, region VI and region VII.

6. Overlapping deletion of region VIII on Bac4.0-1

The implementation method is similar to the construction method of Bac1.0.

Amplifying a Spec resistance expression cassette by using primers Spec-Ac84-F and Spec-Ac91-R, performing ethanol precipitation purification on a PCR product, converting into Bac4.0-1 electrotransformation competence, performing PCR verification sequencing by using primers Ac84KO-F and Ac91KO-R to obtain a positive monoclonal with a region VIII replaced by a Spec resistance gene, named Ac84-91KO, and preparing the calcium transformation competence for later use.

According to analysis, region VIII contains two essential genes Ac89 and Ac90 in addition to the six nonessential genes Ac84, ac85, ac86, ac87, ac88 and Ac91, so that the elimination of the Spec resistance fragment requires the back-filling of both essential genes Ac89 and Ac 90.

The make-up plasmid was constructed as follows: amplifying the gene of about 500bp upstream of Ac84 by using a primer Ac84-D1/D2 and EmBacY rod grains as templates to obtain an up sequence; amplifying the Ac89-90 sequence by using the primers Ac89-D1 and Ac90-D2 and EmBacY rod grains as templates; amplifying a gene about 500bp downstream of Ac91 by using a primer Ac91-D1/D2 and EmBacY rod grains as templates to obtain a down sequence; meanwhile, using pKSI-1 plasmid as a template, using a primer p-8-F/R to obtain a pKSI-1 linearization vector, and performing seamless cloning, connection and transformation to obtain a compensation plasmid Donor-8.

Donor-8 converts Ac84-91KO to achieve traceless elimination of the Spec resistance expression cassette and Ac89-90 gene complementation to obtain a bac5.0-2 with overlapping deletions of region I, region III, region VI, region VII and region VIII.

7. Overlapping deletion of region X on Bac4.0-1

The implementation method is similar to the construction method of Bac1.0.

The Spec resistance expression cassette is amplified by using primers Spec-Ac148-F and Spec-Ac150-R, bac5.0-2 electrotransformation competence is transformed after ethanol precipitation and purification of PCR products, PCR verification sequencing is carried out by using primers Ac148KO-F and Ac150KO-R, positive monoclonal with the region X replaced by the Spec resistance gene is obtained, named Ac148-150KO, and calcium transformation competence is prepared for standby.

Through analysis, the region X is all nonessential genes, a primer Ac148-D1/D2 is adopted, emBacY rod grains are used as templates, an up sequence is used for amplifying about 500bp genes at the upstream of the Ac148, a primer Ac150-D1/D2 is used as templates, a EmBacY rod grain is used as a template, and a down sequence is used for amplifying about 500bp genes at the downstream of the Ac150 gene; meanwhile, a primer p-10-F/R and pKSI-1 plasmid are used as templates to obtain a pKSI-1 linearization vector, a seamless cloning kit is used for connection transformation, and a compensation plasmid Donor-10 is constructed. The make-up plasmid Donor-10 was transformed into Ac148-150KO to effect traceless replacement of the Spec resistance expression cassette, resulting in a stack of deleted bac, designated bac5.0-3, for region I, region III, region VI, region VII and region X.

In summary, the deletion of fragments in each plasmid is shown in Table 2.

TABLE 2

Example 2 transfection and infection of Sf9 cells

To investigate the effect of three polygenic deletion baculovirus vectors of Bac5.0-1, bac5.0-2 and Bac5.0-3 on baculovirus infection spreading capacity and ph promoter expression capacity, bac5.0-1, bac5.0-2 and Bac5.0-3 were used for Bac extraction with NucleoBond Xtra Midi kit, sf9 cells were transfected with wild type EmBacY Bac according to Bac-to-Bac manual, P1 generation virus was harvested after 96h, reinfection of fresh Sf9 cells with an appropriate amount of P1 generation virus was performed, and P2 generation virus was harvested after 96 h.

Fluorescent observations were made during transfection and infection, with each fluorescent particle case shown in FIG. 3.

Example 3 protein expression level determination

To investigate the effect of different baculovirus expression vectors on the expression level of foreign proteins, recombinant human thyroid peroxidase (Human thyroid peroxidase, hTPO) (GenBank: NP-000538.3) was expressed on baculovirus expression vectors (Table 3) with 7 single gene deletions of Ac15KO, ac47KO, ac61KO, ac69KO, ac85KO, ac91KO, ac149KO, respectively, and the foreign gene was inserted into position atttn.

TABLE 3 Table 3

After extracting the bacmid, preparing P3 generation recombinant baculovirus by taking a wild type baculovirus expression vector EmBacY (WT) as a control, carrying out titer measurement, carrying out Sf9 cell shake flask infection according to MOI=1, and sampling after 96h infection for Western blot detection.

The results showed that Ac15KO, ac47KO, ac61KO, ac69KO, ac85KO, ac91KO, ac149KO were used to express the foreign proteins, respectively, and that the expression levels of the foreign proteins were not increased and some decreased as compared with the wild type, as shown in fig. 1.

2. Multiple gene deletion

The human thyroid peroxidase (hTPO) gene was transposed in three polygene deletion baculovirus expression vectors of Bac5.0-1, bac5.0-2 and Bac5.0-3, and the foreign gene was inserted into atttn sites.

After extracting the bacmid, preparing the P3 generation recombinant baculovirus by taking the wild bacmid as a control, carrying out titer measurement, carrying out Sf9 cell shake flask infection according to MOI=1, and sampling after 96h infection for Western blot detection, as shown in fig. 4.

From the experimental results, the expression of the foreign protein is not improved by the deletion of a single unnecessary gene, but the expression levels of the three polygene deleted baculovirus vectors Bac5.0-1, bac5.0-2 and Bac5.0-3 constructed by the invention are reduced by 14-18 kb in genome, have the infection replication and proliferation capacities of normal viruses and the expression capacities of a ph promoter, can be obviously improved, and the expression levels of hTPO are respectively improved by 1.9, 1.8 and 1.5 times by gray scale scanning (Table 4).

TABLE 4 Table 4

Therefore, the constructed genome is relatively simplified Bac5.0-1, and the expression level of the exogenous proteins can be remarkably improved by Bac5.0-2 and Bac5.0-3.

The foregoing examples illustrate only a few embodiments of the application and are described in detail herein without thereby limiting the scope of the application. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the application, which are all within the scope of the application. Accordingly, the scope of protection of the present application is to be determined by the appended claims. All documents referred to in this disclosure are incorporated by reference herein as if each was individually incorporated by reference.

Claims

1. A method of constructing a baculovirus vector comprising: taking the genome of the noctiluca californica nuclear polyhedrosis virus as a basis for constructing a baculovirus vector, and performing polygene deletion, wherein the polygene deletion comprises deleting Ac15, ac16, ac20/21, ac22, ac42, ac43, ac44, ac55, ac56, ac57, ac58/59, ac60, ac61, ac69, ac70 and Ac72, and deleting genes selected from the following groups:

Ac47, ac48 and Ac49;

ac84, ac85, ac86, ac87, ac88 and Ac91; or (b)

Ac148, ac149 and Ac150.

2. The method of claim 1, wherein the baculovirus vector further comprises elements required for expression of a foreign gene; preferably, the element comprises: promoters, replicators, transposition sites, foreign gene insertion sites; optionally, the element further comprises: resistance screening markers, reporter genes;

More preferably, the promoter comprises a ph promoter, the replicon comprises a mini-F replicon, the transposition site comprises an attTn7 transposition site, the resistance selection marker comprises a kana resistance selection marker, and the reporter gene comprises an eYFP fluorescent gene.

3. The method of claim 1, wherein the alfalfa silver vein moth nuclear polyhedrosis virus genomic sequence is an artificial sequence from EmBacY bacmid.

4. The method of claim 1, wherein the genomic sequence of the noctiluca californica nuclear polyhedrosis virus as a base sequence for constructing a baculovirus vector comprises the Ac1 to Ac154 genes; or (b)

Homologous recombination is carried out by utilizing lambda Red recombinase, and gene deletion is carried out; preferably, deletion of the gene is performed using the lambda Red recombinase/I-SceI enzyme traceless editing system.

5. A recombinant baculovirus vector which is an engineered baculovirus vector based on the genomic sequence of the noctiluca californica nuclear polyhedrosis virus and which has been polygenic deleted, comprising deleting Ac15, ac16, ac20/21, ac22, ac42, ac43, ac44, ac55, ac56, ac57, ac58/59, ac60, ac61, ac69, ac70 and Ac72, and deleting genes selected from the group consisting of:

Ac47, ac48 and Ac49;

ac84, ac85, ac86, ac87, ac88 and Ac91; or (b)

Ac148, ac149 and Ac150.

6. The recombinant baculovirus vector of claim 5, further comprising elements required for expression of a foreign gene; preferably, the element comprises: promoters, replicators, transposition sites, foreign gene insertion sites; optionally, the element further comprises: resistance screening markers, reporter genes;

More preferably, the promoter comprises a ph promoter, the replicon comprises a mini-F replicon, the transposition site comprises an attTn7 transposition site, the resistance selection marker comprises a kana resistance selection marker, and the reporter gene comprises an eYFP fluorescent gene;

More preferably, the alfalfa silver vein moth nuclear polyhedrosis virus genomic sequence is an artificial sequence from EmBacY bacmid.

7. Use of the recombinant baculovirus vector of any one of claims 5-6 for the preparation of a recombinant baculovirus; preferably, the recombinant baculovirus vector is transfected into a virus-producing cell to produce the recombinant baculovirus; or (b)

For inserting an exogenous gene to express the exogenous gene; preferably, the recombinant baculovirus vector into which the foreign gene is inserted is transfected into a virus-producing cell to produce the recombinant baculovirus, which expresses the foreign protein.

8. A recombinant baculovirus obtained from the recombinant baculovirus vector package of any one of claims 5-6; or it is obtained by packaging the recombinant baculovirus vector of any one of claims 5 to 6, into which a foreign gene is inserted, which is capable of expressing a foreign protein.

9. A method of producing a recombinant baculovirus, comprising:

(A) Preparing the recombinant baculovirus vector of any one of claims 5-6;

(B) Transfecting the recombinant baculovirus vector of (a) into a virus-producing cell, thereby packaging to obtain a recombinant baculovirus;

Preferably, the recombinant baculovirus vector into which the foreign gene is inserted is transfected into a virus-producing cell to produce the recombinant baculovirus, which expresses the foreign protein.

10. Use of the recombinant baculovirus of claim 8 for: expressing the exogenous gene to obtain an exogenous protein; preferably, the exogenous protein includes: immunogenic proteins, pharmaceutically active proteins, diagnostically active proteins, structural proteins, enzymes, fusion peptides, reporter proteins.