Recombinant bacmid and preparation method therefor

Abstract

Disclosed in the present invention are a recombinant bacmid and a preparation method therefor. A heterologous functional gene expression element is linked to a donor plasmid, and then a first temperature-sensitive replicon control element is linked to the donor plasmid to obtain a first shuttle plasmid; before the first shuttle plasmid is converted into DH10Bac bacteria, a second temperature-sensitive replicon control element is linked to a helper plasmid; so that the expression and replication of the shuttle plasmid and the helper plasmid can be controlled by temperature; after Tn7 transposase-mediated recombination is completed, the temperature is elevated, so that the first shuttle plasmid and the helper plasmid can be automatically lost in the bacteria, thereby improving the purity of the separated recombinant bacmid, and obtaining a high-purity recombinant bacmid.

Description

A recombinant bacmid and its preparation method Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a recombinant bacmid and a preparation method thereof. Background Art

[0002] Insect baculovirus expression systems such as Bac-to-Bac and MultBac are widely used in heterologous protein expression, vaccine and antibody production, and large-scale preparation of recombinant adeno-associated viruses.

[0003] The Bac-to-Bac baculovirus expression system includes DH10Bac bacteria and a pFastBac donor plasmid. The pFastBac donor plasmid contains two target sites for the E. coli Tn7 transposon, with a multiple cloning site and a resistance selection marker gene between the target sites. The DH10Bac bacteria also contain a baculovirus shuttle plasmid (bacmid) and a helper plasmid. A bacmid is a plasmid containing the baculovirus genome that can shuttle between bacteria and insect cells. The helper plasmid provides the Tn7 transposase required for transposition, helping to complete the transposition process. In the Bac-to-Bac baculovirus expression system, the pFastBac donor plasmid carrying the target gene is introduced into the DH10Bac bacteria to obtain a recombinant bacmid. The specific steps are as follows:

[0004] First, the exogenous gene is cloned into the multiple cloning site of the pFastBac donor plasmid; then, the pFastBac donor plasmid is transformed into DH10Bac bacteria containing a baculovirus shuttle plasmid and a helper plasmid using the conventional CaCl2 method; under the action of the Tn7 transposase provided by the helper plasmid, the exogenous gene is transposed onto the gene fragment of the baculovirus shuttle plasmid, and the recombinant bacmid is obtained through blue-white spot screening.

[0005] The MultBac system consists of a donor plasmid, a recipient plasmid, and DH10MultiBac bacteria. The donor and recipient plasmids can be recombined in vitro to construct a first shuttle plasmid containing multiple heterologous gene expression cassettes. The shuttle plasmid is then transferred into DH10MultiBac bacteria to complete Tn7-mediated recombination and prepare a recombinant bacmid.

[0006] Since the helper plasmid only provides Tn7 transposase and does not directly participate in recombination, the extracted recombinant bacmid mixture contains a large amount of helper plasmid; and, the later transformed shuttle plasmid can also replicate in DH10Bac bacteria, so in addition to the recombinant bacmid, the mixed plasmid also contains the helper plasmid and the shuttle plasmid that has not undergone recombination.

[0007] Existing insect baculovirus expression systems produce mixed plasmids containing a variety of mixtures, including recombinant bacmids. This results in extremely low transfection efficiency when used in subsequent cell transfection experiments. Therefore, to improve transfection efficiency in cell transfection experiments, the recombinant bacmid must be separated from the mixed plasmids, but this separation process is complex and tedious. For example, pulsed-field gel electrophoresis (PFGE) can be used to separate the recombinant bacmid from the mixed plasmids and directly use it for transfection. PFGE uses two alternating electric fields, switching them on and off alternately, causing the electrophoretic direction of DNA molecules to change with the changes in the electric field. This alternating change in the electric field direction allows the separation of large DNA molecules. However, the PEGE method has significant limitations. For example, the experimental process is cumbersome and time-consuming, and the electrophoretic band pattern is easily affected by various factors, including human factors, requiring a high level of technical expertise. Therefore, the PEGE separation process for recombinant bacmids is cumbersome and difficult.

[0008] The recombinant bacmids obtained by the current insect baculovirus expression system have low purity, resulting in low transfection efficiency in cell transfection experiments.

[0009] Summary of the Invention

[0010] In response to the above-mentioned deficiencies or improvement needs of the prior art of insect baculovirus expression systems, the present invention aims to provide a recombinant bacmid and a method for preparing the same, thereby resolving the problems of the mixed plasmids in the above-mentioned background art containing helper plasmids and shuttle plasmids, resulting in low purity of the recombinant bacmid in the mixed plasmids and low transfection efficiency in cell transfection experiments using the mixed plasmids.

[0011] To achieve the above object, the present invention provides the following technical solutions:

[0012] One aspect of the present invention provides a method for preparing a recombinant bacmid, comprising the following steps:

[0013] S1, connecting the heterologous functional gene expression element with the donor plasmid to obtain the first shuttle plasmid;

[0014] S2. Transforming the first shuttle plasmid into DH10Bac bacteria to obtain a recombinant bacmid mixture, wherein the DH10Bac bacteria contain the second shuttle plasmid and the helper plasmid;

[0015] S3, separating the recombinant bacmid from the recombinant bacmid mixture;

[0016] In step S1, after the heterologous functional gene expression element is connected to the donor plasmid, the first temperature-sensitive replicon control element is connected to the donor plasmid to obtain a first shuttle plasmid;

[0017] In step S2, before transforming the first shuttle plasmid into DH10Bac bacteria, the second temperature-sensitive replicon control element is ligated to the helper plasmid;

[0018] The temperature of step S1 and step S2 is lower than the inactivation temperature of the first temperature-sensitive replicon control element and the second temperature-sensitive replicon control element.

[0019] Because the first shuttle plasmid contains a first temperature-sensitive replicon control element and the helper plasmid contains a second temperature-sensitive replicon control element, both the first shuttle plasmid and the helper plasmid can be eliminated by temperature regulation. When the ambient temperature is too high, the temperature-sensitive replicon control element in the bacteria activates, causing the first shuttle plasmid and the helper plasmid to be lost, thereby obtaining a highly pure recombinant bacmid. This high-purity recombinant bacmid increases transfection efficiency and ultimately improves the yield of progeny recombinant baculovirus, thereby enhancing the expression efficiency of the exogenous gene product.

[0020] Furthermore, the first temperature-sensitive replicon control element and / or the second temperature-sensitive replicon control element include an RK2 replicon derived from an RK2 plasmid.

[0021] The RK2 plasmid is a temperature-sensitive, low-copy plasmid whose replication is affected by ambient temperature. It is often used as a tool for traceless elimination in genetic modification. This protocol uses the RK2 plasmid to prepare temperature-sensitive shuttle and helper plasmids, thereby eliminating these shuttle and helper plasmids from recombinant products.

[0022] Furthermore, the RK2-derived replicon includes the oriV and trfA gene expression cassettes. The replicon sequence is SEQ ID NO. 1, or a sequence with at least 90% homology to SEQ ID NO. 1, or its complement. The protein expressed by the trfA gene on the RK2 plasmid acts in trans on the oriV region to control plasmid replication. The mutated trfA protein is temperature-sensitive and is not expressed at high temperatures. Therefore, when the ambient temperature is too high, the RK2 plasmid in the bacteria is gradually lost.

[0023] Furthermore, the expression cassette of the trfA gene is a mutant version at position 235. Mutated trfA proteins, such as the 235G synonymous mutation, are temperature sensitive and have the characteristic of not being expressed at high temperatures.

[0024] Furthermore, the first temperature-sensitive replicon control element and the second temperature-sensitive replicon control element are the same, so the same temperature can be set to eliminate the first shuttle plasmid and the helper plasmid, making the experimental process more convenient.

[0025] Furthermore, the donor plasmid is either a pFastBac donor plasmid or a donor plasmid. The pFastBac donor plasmid is a donor plasmid for the Bac-to-Bac system and is suitable for the Bac-to-Bac insect baculovirus expression system. The donor plasmid is a donor plasmid for the MultBac system and is suitable for the MultBac insect baculovirus expression system. This method can be applied to prepare high-purity recombinant bacmids in insect baculovirus expression systems such as Bac-to-Bac and MultBac.

[0026] Furthermore, the heterologous functional gene expression element comprises a heterologous functional gene fragment expression cassette. The heterologous functional gene fragment expression cassette can directly transform the heterologous gene into the multiple cloning site of the donor plasmid, making the transformation process more concise.

[0027] Furthermore, the heterologous functional gene expression element includes a Rep gene expression cassette and a Cap gene expression cassette. The Rep gene expression cassette, the Cap gene expression cassette, and the ITR-GOI are connected to the donor plasmid using homologous recombination to construct the first shuttle plasmid. This process is easy to operate and has a good transformation effect.

[0028] Furthermore, in step S1, the original replicon on the donor plasmid is a pBR322 plasmid replicon; a heterologous gene expression cassette is first constructed on the donor plasmid, and then the constructed replicon of the donor plasmid is replaced with a temperature-sensitive replicon to obtain a temperature-sensitive first shuttle plasmid.

[0029] The pBR322 plasmid is an important artificially constructed plasmid, known as a universal plasmid. It is constructed through a complex recombination process from three parental plasmids: pSF2124, pMB1, and pSC101. The pBR322 plasmid offers several advantages. First, its molecular weight is relatively small. Second, it carries a replication origin, ensuring that the plasmid replicates only in Escherichia coli cells. Furthermore, it contains two antibiotic resistance genes—ampicillin and tetracycline—that serve as selectable markers for transformants. Furthermore, its high copy number, accumulating 1,000 to 3,000 copies per cell after chloramphenicol amplification, greatly facilitates the preparation of recombinant DNA.

[0030] Another aspect of the present invention provides a recombinant bacmid prepared according to the above method.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] The present invention provides a recombinant bacmid and a method for its preparation. A shuttle plasmid containing a first temperature-sensitive replicon control element and a helper plasmid containing a second temperature-sensitive replicon control element are constructed, allowing the shuttle plasmid and helper plasmid to be temperature-controlled. After Tn7 transposase-mediated recombination, increasing the temperature causes the shuttle plasmid and helper plasmid to spontaneously disappear in bacteria, thereby increasing the purity of the isolated recombinant bacmid. The high-purity recombinant bacmid increases transfection efficiency in subsequent cell transfection experiments and enhances the expression efficiency of exogenous gene products. Furthermore, the preparation method of this solution is simple, rapid, time-saving, and labor-saving, and effectively removes the shuttle plasmid and helper plasmid from mixed plasmids, making it suitable for large-scale, engineered production. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0034] FIG1 is a flow chart of a method for preparing a recombinant bacmid;

[0035] FIG2A is a schematic diagram of the structure of a shuttle plasmid of a recombinant bacmid; FIG2B is a schematic diagram of the structure of a helper plasmid of a recombinant bacmid; FIG2C is a schematic diagram of the strategy for constructing a recombinant bacmid;

[0036] FIG3 is a graph showing the growth effect of DH5α bacteria containing the first shuttle plasmid at different temperatures;

[0037] Figure 4 contains pMON7124 RK2-ts The growth effect diagram of DH5α bacteria with temperature-sensitive helper plasmid at different temperatures;

[0038] FIG5 is a graph showing the growth effect of DH10 bacteria containing only the second shuttle plasmid on different resistance plates;

[0039] Figure 6 shows the positive recombinants that underwent recombination after blue-white screening;

[0040] Figure 7 shows the identification of recombinants by PCR;

[0041] FIG8 is a graph showing the growth effect of bacteria containing only recombinant bacmids on different resistance plates;

[0042] FIG9 is a diagram of recombinant bacmid DNA separated by pulsed-field gel electrophoresis. DETAILED DESCRIPTION

[0043] The technical solution of this patent will be further described in detail below in conjunction with specific embodiments. It should be noted that the following detailed description is exemplary and is intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this application belongs.

[0044] Conventional methods for preparing recombinant bacmids contain a large number of helper plasmids and shuttle plasmids in the extracted mixed plasmids. If the isolated recombinant bacmid contains helper plasmids and shuttle plasmids, the purity of the isolated recombinant bacmid will be low. When it is used to transfect host cells, the helper plasmids and shuttle plasmids will affect the transfection efficiency of the recombinant bacmid. At the same time, the expression of non-target proteins will also reduce cell productivity, ultimately reducing the quality and yield of the progeny recombinant baculovirus, and further reducing the expression amount of the exogenous gene product.

[0045] To address the above-mentioned issues, this solution improves the existing methods for preparing recombinant bacmids and proposes a recombinant bacmid and its preparation method. In this approach, a temperature-sensitive element is linked to a shuttle plasmid and a helper plasmid, allowing the shuttle plasmid and the helper plasmid to be automatically lost in bacteria after completing Tn7 transposase-mediated recombination. This improves the purity of the isolated recombinant bacmid, increases transfection efficiency, and ultimately increases the yield of progeny recombinant baculovirus, thereby enhancing the expression efficiency of the exogenous gene product.

[0046] The technical solution of this patent will be further described in detail below in conjunction with specific embodiments. Wherein, the methods used in the examples are all conventional methods unless otherwise specified.

[0047] Example 1:

[0048] The RK2 plasmid is a temperature-sensitive, low-copy plasmid whose replication is affected by ambient temperature. It is often used as a tool for traceless elimination in genetic modification. The protein expressed by the trfA gene on the RK2 plasmid acts in trans on the oriV region to control plasmid replication. Mutations in the trfA protein, such as the synonymous 235G mutation, are temperature-sensitive and are not expressed at high temperatures. Therefore, when the ambient temperature is too high, the RK2 plasmid is gradually lost from bacteria.

[0049] This example provides a method for preparing a recombinant bacmid. To make the shuttle plasmid and helper plasmid temperature-sensitive, the nucleotide sequences of the oriV and trfA genes from the temperature-sensitive RK2 plasmid are selected as replicons, replacing the existing replicons on the shuttle plasmid and helper plasmid to prepare temperature-sensitive shuttle plasmids and helper plasmids, thereby eliminating the shuttle plasmid and helper plasmid from the recombinant product.

[0050] First, prepare a temperature-sensitive shuttle plasmid and a temperature-sensitive auxiliary plasmid. The temperature-sensitive Bac-to-Bac shuttle plasmid contains the control elements of the temperature-sensitive replicon and the expression cassette of the heterologous functional gene fragment; or contains the control elements of the temperature replicon, the recombinant adeno-associated virus core expression element ITR-GOI with the heterologous functional gene fragment and the expression cassette of the functional protein components necessary for the production of the recombinant adeno-associated virus, and the expression cassette includes the Rep gene and the Cap gene. The donor plasmid used in this embodiment is the pFast.Bac.Dual (pFD) donor plasmid, and the pFD shuttle plasmid contains the RK2 temperature-sensitive replicon RK2-ts, the rAAV core expression element and the Rep-Cap gene expression cassette of AAV. The Rep gene is the Rep gene of type 2 AAV, which is regulated by the Ph promoter; the Cap gene is the Cap gene of type 5 AAV, which is regulated by the P10 promoter. The ITR core expression element uses the ITR nucleic acid sequence of AAV type 2 and is connected to a green fluorescent protein (GFP) expression cassette. GFP expression is controlled by the CMV promoter to facilitate the detection of rAAV activity. The fluorescent protein expression cassette facilitates the screening of shuttle plasmids carrying heterologous genes.

[0051] The temperature-sensitive Bac-to-Bac helper plasmid contains a temperature-sensitive replicon and a Tn7 transposase expression cassette. In this example, the pMON7124 helper plasmid from the Bac-to-Bac baculovirus expression system was used, and the nucleotide sequences of the oriV and trfA genes from the temperature-sensitive RK2 plasmid were selected as the replicon.

[0052] DH10Bac bacteria contain a replicon, a kanamycin resistance gene, and a baculovirus shuttle plasmid. The baculovirus shuttle plasmid is amplified in kanamycin-resistant DH10Bac bacteria and, in the presence of a chromogenic substance such as X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside) and the inducer IPTG, complements the chromosomal LacZ deletion, forming blue plaques. DH10Bac bacteria also contain a helper plasmid encoding a transferase and a tetracycline resistance gene, providing Tn7 transformation capabilities.

[0053] Subsequently, a recombinant plasmid is prepared using a temperature-sensitive shuttle plasmid and a temperature-sensitive helper plasmid. The resulting recombinant bacmid is screened using the blue-white screening method to select white recombinants, indicating that they contain the recombinant bacmid. β-galactosidase produced by wild-type E. coli cleaves the colorless compound X-gal into galactose and the dark blue substance 5-bromo-4-indigo. 5-bromo-4-indigo turns the entire colony blue. After artificially inserting a foreign gene, the β-galactosidase gene in the mutant E. coli is interrupted by the inserted foreign gene, preventing the production of a complete β-galactosidase enzyme. Consequently, the enzyme cannot cleave the colorless compound X-gal, resulting in white colonies. Blue-white screening allows for direct identification of recombinant bacmids based on the difference in colony color. On indicator medium, colonies harboring untransformed plasmids will not grow due to lack of antibiotic resistance. Colonies harboring recombinant bacmids will appear white, while those harboring non-recombinant bacmids will appear blue.

[0054] Then, by increasing the colony culture temperature, the temperature-sensitive shuttle plasmid and the temperature-sensitive helper plasmid are lost in the bacteria. Finally, the recombinant bacmid is extracted from the colony using a plasmid extraction kit.

[0055] In a further embodiment, the donor plasmid is replaced with a donor plasmid, and the shuttle plasmid is replaced with a DH10 MultiBac TM / DH10EMBacY, at this point, this method can be applied to insect baculovirus expression systems such as MultBac to obtain high-purity recombinant bacmids.

[0056] In further embodiments, the donor plasmid and shuttle plasmid are adaptively modified, and the present method can be applied to various insect baculovirus expression systems to obtain high-purity recombinant bacmids.

[0057] This example describes a technique for preparing recombinant bacmids. A temperature-sensitive replicon is used to generate a temperature-sensitive shuttle plasmid and a temperature-sensitive helper plasmid, which are then used to prepare the recombinant bacmid. Prior to plasmid extraction, the bacteria containing the recombinant bacmid, the temperature-sensitive shuttle plasmid, and the temperature-sensitive helper plasmid are treated within a suitable temperature range to remove the temperature-sensitive shuttle plasmid and the temperature-sensitive helper plasmid, thereby extracting a high-purity recombinant bacmid free of interfering plasmids. This method improves the purity of the recombinant bacmid, increases the transfection efficiency of the recombinant bacmid in subsequent cell transfection experiments, and thus enhances the expression efficiency of exogenous gene products. This method can be applied to insect baculovirus expression systems such as Bac-to-Bac and MultBac.

[0058] Example 2:

[0059] This example provides a method for preparing a recombinant bacmid. The experimental process is shown in FIG1 , and specifically includes the following steps:

[0060] S1. Preparation of temperature-sensitive shuttle plasmid and temperature-sensitive helper plasmid. The detailed steps are as follows:

[0061] S11. Use homologous recombination to connect the pFast.Bac.Dual donor plasmid with the Rep gene expression cassette, Cap gene expression cassette, and ITR-GOI to construct the pFD-Cap-(ITR-GOI)-Rep donor plasmid. Use PCR to amplify the first temperature-sensitive replicon control element and assemble it into the pFD-Cap-(ITR-GOI)-Rep donor plasmid by Gibson assembly (DNA seamless cloning) to obtain pFD RK2-ts -ITR(GOI)-Rep temperature-sensitive shuttle plasmid is the first temperature-sensitive shuttle plasmid. Please refer to Figure 2A for a schematic diagram of the first shuttle plasmid.

[0062] S12 and DH10Bac bacteria contain shuttle plasmids and auxiliary plasmids; the second temperature-sensitive replicon control element was amplified by PCR, and the pMON7124 auxiliary plasmid was assembled with the second temperature-sensitive replicon control element and the Tn7 transposase expression cassette by Gibson assembly to obtain pMON7124. RK2-ts Temperature-sensitive auxiliary plasmid. In this embodiment, the bacteria used to construct the first shuttle plasmid and the temperature-sensitive auxiliary plasmid are all DH5α bacteria. Please refer to Figure 2B, which is pMON7124 RK2-ts Schematic diagram of a temperature-sensitive helper plasmid. The temperature in step S1 is below the inactivation temperature of the first and second temperature-sensitive replicon control elements. When the ambient temperature is too high, the RK2 plasmid in the bacteria is gradually lost, leading to loss of the helper plasmid, thereby increasing the purity of the recombinant bacmid in the recombinant product.

[0063] S2. In this step, a recombinant bacmid containing the exogenous gene expression cassette (GOI) is constructed. The detailed strategy is shown in Figure 2C. The detailed steps for constructing the recombinant bacmid are as follows:

[0064] S21. The DH10Bac bacteria in the Bac-to-Bac baculovirus expression system contain the pMON7124 helper plasmid and a shuttle plasmid of the baculovirus genome, i.e., the second shuttle plasmid; the second shuttle plasmid contains the kanamycin resistance gene (Kanamycin + ), pMON7124 helper plasmid contains tetracycline resistance gene (Tet + ).

[0065] At 37 ° C, shake the DH10Bac bacteria; then, use the precipitation method to extract the plasmids. The obtained mixed plasmid contains the second shuttle plasmid and the auxiliary plasmid. The mixed plasmid is transformed into DH10 bacteria without any plasmids by electroporation. The bacterial solution is spread on a plate with Kan + After a single colony grows, randomly pick some single colonies and spread them on Kan + / Tet+ double resistance plates were used for screening and bacterial growth was observed; + / Tet+ double resistance plate can not grow but on Kan + The bacteria that can grow on the resistance plate are DH10-shuttle plasmid bacteria containing only the second shuttle plasmid, and are used to prepare DH10-shuttle plasmid electroporation competent bacteria;

[0066] In a further embodiment, the above screening steps are repeated for DH10-shuttle plasmid bacteria containing only the second shuttle plasmid to further ensure purity, and then used to prepare DH10-shuttle plasmid electroporation competent bacteria.

[0067] S22, pMON7124 RK2-ts The temperature-sensitive auxiliary plasmid was electroporated into DH10-shuttle plasmid electroporation competent bacteria, and Kan + / Tet + The bacteria that were successfully transformed were screened on double-resistance plates and used to prepare the bacteria containing pMON7124. RK2-ts DH10Bac electroporation of temperature-sensitive helper plasmid into competent bacteria;

[0068] S23, electrotransform the first shuttle plasmid into DH10Bac electrotransformation competent bacteria to obtain a plasmid containing the first shuttle plasmid and pMON7124 RK2-ts Competent bacteria DH10Bac containing temperature-sensitive helper plasmids RK2-ts The competent bacteria DH10Bac RK2-ts Add to SOC medium and culture at 30℃ for 12 hours. RK2-ts Under the action of Tn7 transposase provided by the temperature-sensitive helper plasmid, transposition occurs between the first shuttle plasmid and the second shuttle plasmid, and the exogenous gene is introduced into the second shuttle plasmid to produce a recombinant bacmid; then, the bacterial solution is diluted and spread on Kan at a temperature of 30°C. + / Gen + / Tet + Blue-white screening was performed on / IPTG / X-Gal resistance plates.

[0069] The bacterial solution contains the first shuttle plasmid, the second shuttle plasmid, pMON7124 RK2-tsTemperature-sensitive helper plasmid and recombinant bacmid; colonies containing the first shuttle plasmid, the second shuttle plasmid and the pMON7124RK2-ts temperature-sensitive helper plasmid are blue spots, and only colonies containing the recombinant bacmid are white recombinants, i.e. white spots.

[0070] After 48 hours, pick out the white spots and apply them on Kan + / Gen + / Tet + Culture the cells on a 100 μg / 50 μg / 10 ...

[0071] The temperature in step S2 is below the inactivation temperature of the first temperature-sensitive replicon control element and the second temperature-sensitive replicon control element.

[0072] S3. Extract the recombinant bacmid. The detailed steps are as follows:

[0073] The white spots selected above were inoculated into a + In the antibiotic culture medium, shake the bacteria at 37℃ for 16 hours; after shaking, dilute the bacteria solution 10000 times and apply it on the culture medium containing Kan + / Tet + / Gen + / IPTG / X-Gal resistance plates and Kan + The cells were cultured on resistant plates at 37°C overnight.

[0074] The results show that + / Tet + / Gen + No colonies grew on the / IPTG / X-Gal resistance plate, but on the Kan + Bacteria on the resistance plate grow normally. This bacterium is a single colony containing only the recombinant bacmid. Pick a single colony of the recombinant bacmid for expansion culture and isolate the recombinant bacmid using a plasmid isolation kit.

[0075] In this embodiment, the first temperature-sensitive replicon control element and the second temperature-sensitive replicon control element are both RK2 replicons. The sequence of the RK2 replicon is SEQ ID NO. 1, or a sequence having 90% or greater homology to SEQ ID NO. 1 (preferably 92%, more preferably 95%, and even more preferably 98% homology), or a complementary sequence thereof.

[0076] The sequence of the RK2 replicon is as follows:

[0077] In a further embodiment, in step S11, the heterologous functional gene expression element includes a heterologous functional gene fragment expression cassette, or an expression cassette comprising a recombinant adeno-associated virus ITR core expression element carrying a heterologous functional gene fragment and a functional protein component necessary for producing the recombinant adeno-associated virus.

[0078] Further, in step S11, a fluorescent protein expression cassette or a luciferase expression cassette is connected to the ITR core expression element. Fluorescent proteins include green fluorescent protein, yellow fluorescent protein, red fluorescent protein, etc. The fluorescent protein expression cassette is connected to the ITR core expression element, and the expression of the fluorescent protein is controlled by the CMV promoter, which is convenient for detecting the activity of rAAV carrying a heterologous gene, thereby helping to screen the shuttle plasmid carrying the heterologous gene. Luciferase includes firefly luciferase, Gaussia luciferase, Renilla luciferase, etc. The luciferase expression cassette is connected to the ITR core expression element, and the activity of luciferase can be detected using luciferin as a substrate, thereby screening the shuttle plasmid carrying a heterologous gene.

[0079] In a further embodiment, in step S1, the original replicon on the pFD donor plasmid is a pBR322 type plasmid replicon; first, a heterologous gene expression cassette is constructed on the pFD donor plasmid, and then the replicon of the constructed pFD donor plasmid is replaced with a temperature-sensitive replicon to obtain a temperature-sensitive first shuttle plasmid.

[0080] pBR322 is an important artificially constructed plasmid, known as a universal plasmid. It is constructed through a complex recombination process from three parental plasmids: pSF2124, pMB1, and pSC101. The pBR322 plasmid vector offers several advantages. First, its relatively low molecular weight; second, it carries a replication origin, ensuring that the plasmid replicates only in Escherichia coli cells; and third, it contains two antibiotic resistance genes—ampicillin and tetracycline—that serve as selectable markers for transformants. Furthermore, its high copy number, accumulating 1,000 to 3,000 copies per cell after chloramphenicol amplification, greatly facilitates the preparation of recombinant DNA.

[0081] Compared with the Bac-to-Bac recombinant bacmid preparation method in the prior art, the present method utilizes the Bac-to-Bac recombinant bacmid production system containing a temperature-sensitive replicon. RK2-ts, effectively producing high-purity recombinant bacmids free of interfering plasmids such as temperature-sensitive shuttle plasmids and temperature-sensitive helper plasmids. Therefore, simply raising the temperature can eliminate these and other contaminants from the plasmid mixture, resulting in a purer recombinant bacmid free of helper and shuttle plasmid contamination and higher transfection efficiency. This method is technically low-level, simple, and easy to perform, and the resulting recombinant bacmids are of high purity, making it suitable for large-scale, engineered production.

[0082] Example 3:

[0083] In this example, multiple experiments were performed to verify the feasibility of preparing high-purity recombinant bacmids using the method described in Example 2.

[0084] In step S12 of Example 2, the first shuttle plasmid was obtained. Please refer to Figure 3, which shows the growth of DH5α bacteria containing the first shuttle plasmid at different temperatures. The left side of Figure 3 shows that DH5α bacteria containing the first shuttle plasmid grow at 30°C, while the right side shows that DH5α bacteria containing the first shuttle plasmid do not grow at 37°C. Figure 3 demonstrates that increasing ambient temperature causes the temperature-sensitive plasmid in bacteria to be gradually lost, resulting in loss of the shuttle plasmid, thereby improving the purity of the recombinant bacmid in the recombinant product.

[0085] pMON7124 was obtained in step S13 of Example 2. RK2-ts Temperature-sensitive helper plasmid. Please refer to Figure 4 for a diagram containing pMON7124 RK2-ts The growth effect of DH5α bacteria containing temperature-sensitive helper plasmids at different temperatures; Figure 4 on the left shows the growth effect of DH5α bacteria containing pMON7124 RK2-ts DH5α bacteria containing temperature-sensitive helper plasmids grown at 30°C, with pMON7124 shown on the right. RK2-ts DH5α bacteria carrying a temperature-sensitive helper plasmid do not grow at 37°C. Figure 4 demonstrates that increasing ambient temperature leads to the gradual loss of the temperature-sensitive plasmid in the bacteria, which in turn causes the loss of the helper plasmid, further increasing the purity of the recombinant bacmid in the recombinant product.

[0086] In step S21 of Example 2, DH10-temperature-sensitive shuttle plasmid bacteria containing only the second shuttle plasmid were obtained. Please refer to Figure 5, which is a graph showing the growth effect of DH10 bacteria containing only the second shuttle plasmid on different resistance plates; the left side of Figure 5 is a graph showing the growth effect of DH10 bacteria containing only the second shuttle plasmid on different resistance plates. + Bacteria growing on the resistance plate, on the right is the bacteria grown on Kan + / Tet + Bacteria that cannot grow on double-resistant plates; + / Tet + It cannot grow on double resistance plates but grows on Kan+ The bacteria that can grow on the resistant plates are DH10-bacmid bacteria containing only the bacmid. Please refer to Figure 6, which shows the blue-white spots that screen for positive recombinants that have undergone recombination. The white spots on the plate are positive recombinants, which means that the recombinant bacmid containing the rAAV core expression element ITR-GOI and the AAV Rep-Cap gene expression cassette has been successfully constructed. The results show that pMON7124 RK2-ts It can play a normal function and mediate the recombination of the second shuttle plasmid and the first shuttle plasmid.

[0087] In step S23 of Example 2, white spots expressing the recombinant bacmid were obtained, and the recombinant bacmid obtained by separation was identified by pulsed-field gel electrophoresis. The size of the first shuttle plasmid was about 13 kb, i.e., about 13,000 bp; pMON7124 RK2-ts The size of the helper plasmid is about 12.6kb, or about 12600bp. RK2-ts The PCR graph of the colony from Bac-to-Bac is shown in the figure below. From left to right, the first column is the buffer, the second column shows the white spot of the recombinant bacmid of 8000 bp, and the third column shows the blue spot of the second shuttle plasmid of about 300 bp that has not undergone recombination. The right side of Figure 7 shows the PCR graph of the colony from Bac-to-Bac. From left to right, the first column is the buffer, the second and third columns show the blue spot of the second shuttle plasmid of about 300 bp that has not undergone recombination, and the fourth and fifth columns show the white spot of the recombinant bacmid of 8000 bp. The above results show that step S23 is effective in pMON7124. RK2-ts Under the action of Tn7 transposase provided by the helper plasmid, the first shuttle plasmid and the second shuttle plasmid undergo transposition reaction to obtain recombinant bacmid. The white spots are the colony composition of the recombinant bacmid. This experiment proves the Bac-to-Bac RK2-ts The system can function like a Bac-to-Bac system.

[0088] In step S3 of Example 2, a colony containing only the recombinant bacmid was obtained. Figure 8 shows the growth effect of bacteria containing only the recombinant bacmid on different resistance plates. The left side shows the growth effect of bacteria containing only the recombinant bacmid on Kan + Bacteria growing on the resistance plate, shown on the right + / Tet + / Gen + No colonies grew on the / IPTG / X-Gal resistance plate. + / Tet + / Gen + No colonies grew on the / IPTG / X-Gal resistance plate, while Kan + The bacteria on the resistance plate grew normally, indicating that pMON7124 in the mixed plasmid RK2-tsThe temperature-sensitive helper plasmid, the first shuttle plasmid and the second shuttle plasmid have been lost by the bacteria, resulting in the inability to grow on the corresponding resistance plates.

[0089] In step S3 of Example 2, recombinant bacmids were obtained through separation. Figure 9 shows an image of recombinant bacmid DNA separated by pulsed-field gel electrophoresis. From left to right, the first to fourth columns show DNA images of recombinant bacmids extracted from the Bac-to-Bac baculovirus expression system. These four columns show that they contain a large number of 15kb and 227.5kb bands, as well as a small number of 48.5kb and 136kb bands. The 227.5kb band represents a dimer of the recombinant bacmid, the 136kb band represents the recombinant bacmid, the 48.5kb band represents a multimer of the helper plasmid and the shuttle plasmid, and the 15kb band represents a helper plasmid or a shuttle plasmid. These experimental results indicate that the plasmids extracted from the Bac-to-Bac baculovirus expression system contain a large number of recombinant bacmids, helper plasmids, and shuttle plasmids. The sixth to ninth columns of Figure 9 show DNA images of recombinant bacmids extracted from the Bac-to-Bac baculovirus expression system. RK2-ts The four columns show the DNA of the recombinant bacmid extracted from the bacmid. The four columns show a 227.5 kb band and a small amount of 136 kb band, indicating that the recombinant bacmid was extracted from the bacmid. RK2-ts The extracted plasmid contains a large amount of recombinant bacmid, but does not contain helper plasmid and shuttle plasmid.

[0090] Based on the above data, the method for preparing recombinant bacmids described in Example 2 can effectively obtain temperature-sensitive shuttle plasmids and temperature-sensitive helper plasmids, and use them to prepare high-purity recombinant bacmids. Because both the shuttle plasmid and the helper plasmid contain control elements of the temperature-sensitive replicon, when the ambient temperature is too high, the control elements of the temperature-sensitive replicon in the bacteria become activated, resulting in the loss of the shuttle plasmid and the helper plasmid, thereby obtaining a high-purity recombinant bacmid.

[0091] In a further embodiment, the ITR core expression element of the exogenous protein in the shuttle plasmid uses a green fluorescent protein, yellow fluorescent protein, or red fluorescent protein expression cassette. The fluorescent protein expression cassette helps to screen the shuttle plasmid carrying the heterologous gene.

[0092] In a further embodiment, the shuttle plasmid is a temperature sensitive replicon and pFastBac Dual, pFastBac TM 1. pFastBac TM Any of the HTA plasmids can be constructed. pFastBac Dual, pFastBac TM 1. pFastBac TMHTA are shuttle plasmids commonly used in Bac-to-Bac system, among which pFastBac Dual has double MCS region and PH and P10 dual promoters. TM 1. pFastBac TM HTA has only a single MCS region and PH promoter.

[0093] In summary, the present invention provides a recombinant bacmid and a method for preparing the same. After ligating a heterologous functional gene expression element to a donor plasmid, a first temperature-sensitive replicon control element is ligated to the donor plasmid to obtain a first shuttle plasmid. Before transforming the first shuttle plasmid into DH10Bac bacteria, a second temperature-sensitive replicon control element is ligated to a helper plasmid. This allows the shuttle plasmid and helper plasmid to be temperature-controlled. After Tn7 transposase-mediated recombination, increasing the temperature causes the shuttle plasmid and helper plasmid to spontaneously disappear from the bacteria, thereby increasing the purity of the recombinant bacmid in the plasmid mixture and the transfection efficiency of the recombinant bacmid in subsequent cell transfection experiments, thereby enhancing the expression efficiency of the exogenous gene product. This method is suitable for use in insect baculovirus expression systems, not only enabling the production of high-purity recombinant bacmids, but also offering low technical difficulty, ease of operation, and suitability for large-scale, engineered production.

[0094] The foregoing is merely a list of specific embodiments of the present application, intended to enable those skilled in the art to understand or implement the present application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application is not limited to the embodiments shown herein, but is intended to conform to the broadest scope consistent with the principles and novel features of the present application.

Claims

1. A method for preparing a recombinant bacmid, The following steps are involved: S1, connecting the heterologous functional gene expression element to the donor plasmid to obtain the first shuttle plasmid; S2, transforming the first shuttle plasmid into DH10Bac bacteria to obtain a recombinant bacmid mixture; the DH10Bac bacteria contain the second shuttle plasmid and the helper plasmid; S3, separating the recombinant bacmid from the recombinant bacmid mixture; Characterized in that, in the step S1, after the heterologous functional gene expression element is connected to the donor plasmid, the first temperature-sensitive replicon control element is connected to the donor plasmid to obtain the first shuttle plasmid; In the step S2, before transforming the first shuttle plasmid into the DH10Bac bacteria, the second temperature-sensitive replicon control element is connected to the helper plasmid; The temperature of step S1 and step S2 is below the inactivation temperature of the first temperature-sensitive replicon control element and the second temperature-sensitive replicon control element.

2. The preparation method according to claim 1, It is characterized in that The first temperature-sensitive replicon control element and / or the second temperature-sensitive replicon control element comprises an RK2 replicon.

3. The preparation method according to claim 2, It is characterized in that The sequence of the RK2 replicon is SEQ ID NO.1 or a sequence having more than 90% homology with SEQ ID NO.1, or a complementary sequence thereof.

4. The preparation method according to claim 3, It is characterized in that The expression frame of the trfA gene is a mutant version at position 235.

5. The preparation method according to claim 1 or 2, It is characterized in that The first temperature-sensitive replicon control element and the second temperature-sensitive replicon control element are identical.

6. The preparation method according to claim 1, It is characterized in that The donor plasmid is a pFastBac donor plasmid or a donor donor plasmid.

7. The preparation method according to claim 1, It is characterized in that The heterologous functional gene expression element comprises a heterologous functional gene fragment expression cassette.

8. The preparation method according to claim 1, It is characterized in that The heterologous functional gene expression element comprises a Rep gene expression cassette and a Cap gene expression cassette.

9. The preparation method according to claim 1, It is characterized in that In step S1, the original replicon on the donor plasmid is a pBR322 plasmid replicon; a heterologous gene expression cassette is first constructed on the donor plasmid, and then the temperature-sensitive replicon is used to replace the constructed replicon of the donor plasmid to obtain the first shuttle plasmid.

10. A recombinant bacmid, prepared according to the method of any one of claims 1 to 9.

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