Recombinant bacmid preparation system and bmnpv-raav recombinant bacmid

Abstract

Provided are a recombinant bacmid preparation system and a BmNPV-recombinant adeno-associated virus (rAAV) recombinant bacmid. The recombinant bacmid preparation system comprises: a) DH10pureBac competent cells containing a loss-controllable helper plasmid and a modified baculovirus genome, and b) a loss-controllable transfer vector. The BmNPV-rAAV recombinant bacmid is a single BmNPV bacmid containing an rAAV Cap gene expression cassette, a core expression element ITR-GOI, and a Rep gene expression cassette. The present application solves the problem of residual helper plasmids and transfer vectors in a Bac-to-Bac baculovirus expression system and involves a simple technique process.

Description

A recombinant bacmid preparation system and BmNPV-rAAV recombinant bacmid Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a recombinant bacmid preparation system and a BmNPV-rAAV recombinant bacmid. Background Art

[0002] The silkworm (Bombyx mori) is an important economic insect with distinct Chinese characteristics. Its life cycle progresses through four stages: egg, larva, pupa, and moth (adult). While the primary goal of silkworm rearing has been to produce high-yield, high-quality cocoons to provide raw materials for the silk industry and its derivatives, silkworm larvae and pupae are also highly efficient bioreactors for expressing exogenous proteins. Since Motohashi et al. established the Bac-to-Bac expression system based on silkworms in 2005, hundreds of recombinant proteins have been successfully expressed using the Bombyx mori baculovirus system, encompassing a wide range of applications, including vaccine production and pharmaceutical protein applications.

[0003] The Bombyx mori baculovirus (BmNPV) expression system operates on a similar principle to the commercialized Autographa californica baculovirus (AcMNPV) Bac-to-Bac system. Both rely on site-specific transposition of the Tn7 transposon to simplify and enhance the production of recombinant bacmid DNA, enabling rapid and efficient production of recombinant baculovirus. However, due to the inherent properties of the Bac-to-Bac system, the resulting recombinant bacmids often contain residual helper plasmids and transfer vectors, which inevitably compromises the rescue of daughter BEVs. Despite being a promising eukaryotic expression system, the BmNPV expression system has yet to be further developed, and obtaining high-purity recombinant bacmids remains a challenge. Although the temperature-sensitive element-based strategy developed by the applicant in the early stage (a recombinant bacmid and its preparation method, patent application number: 202310643354.7) solved the problem of residual auxiliary plasmids and transfer vectors in the Bac-to-Bac AcMNPV expression system, the technical process is cumbersome and time-consuming (cultivation at 30°C for 12 hours → picking white recombinants at 48 hours → culturing at 37°C for 16 hours → picking single colonies at 37°C overnight → expanding culture → extracting recombinant bacmids), so it cannot be directly transferred to the BmNPV expression system and needs further improvement.

[0004] Summary of the Invention

[0005] In view of the above-mentioned deficiencies of the prior art, the present invention provides a recombinant bacmid preparation system and a BmNPV-rAAV recombinant bacmid, the purpose of which is to: 1) introduce a temperature-sensitive replicon and a counter-selection element into the helper plasmid and the transfer vector, respectively, and quickly obtain a high-purity recombinant bacmid by controlling the temperature and using a counter-selection method. The temperature-sensitive replicon can control the presence of the plasmid in the host. When the temperature is low, the plasmid can exist in the host, but when the temperature is increased, the plasmid is lost. The counter-selection element can be the streptomycin-sensitive gene rpsL. When the rpsL element is present, the DH10B strain is sensitive to the streptomycin. 1) The strain is sensitive to streptomycin, whereas the strain is resistant to streptomycin; 2) The mini-attTn7 element is moved from the Polh site of the baculovirus genome to the ODV-E56 site using Red / ET recombination technology, or to any site other than the ODV-E56 site that does not affect baculovirus packaging. The bacterial replication regulatory element is still placed at the Polh position, and non-essential genes are further knocked out to increase the expression of the exogenous protein; 3) The helper plasmid containing the temperature-sensitive replicon and streptomycin-sensitive element in 1) and the modified baculovirus genome in 2) are transformed into MegaX DH10B T1R competent cells were electroporated to produce DH10pureBac competent cells, which were then combined with the transfer vector prepared in 1) to form a novel recombinant bacmid production system, named PureBac, free of helper plasmid and transfer vector residues. 4) A PureBac system based on Bombyx mori nuclear polyhedrosis virus (BmNPV) was established, in which the three elements required for AAV production (serotype Cap gene, Rep gene, and ITR core expression element) were simultaneously mounted on the transfer vector of this system to obtain high-purity BmNPV-rAAV recombinant bacmids.

[0006] The specific technical solutions of the present invention are as follows:

[0007] The present invention provides a recombinant bacmid preparation system, comprising: a) DH10pureBac competent cells containing a controllable loss helper plasmid and a modified baculovirus genome, b) a controllable loss transfer vector;

[0008] The method for preparing the DH10 pureBac competent cells containing the controllable loss helper plasmid and the modified baculovirus genome comprises the following steps:

[0009] (1) Preparation of a controllable loss helper plasmid: The initial replicon Ori in the helper plasmid encoding the Tn7 transposase was replaced with a temperature-sensitive replicon and a reverse selection element to obtain a controllable loss helper plasmid;

[0010] (2) Modification of the baculovirus genome: Isolate the baculovirus genome from the Bac-to-Bac system, and use Red / ET recombination technology to move the mini-attTn7 in the genome from the Polh site to the ODV-E56 site while the bacterial replication regulatory elements are still placed at the Polh site, or move the mini-attTn7 to any site other than the ODV-E56 site that does not affect baculovirus packaging while the bacterial replication regulatory elements are still placed at the Polh site to obtain a modified baculovirus genome;

[0011] (3) The auxiliary plasmid with controlled loss in step (1) and the baculovirus genome transformed in step (2) were transformed into MegaX DH10B T1 R DH10pureBac competent cells containing a controllable loss of helper plasmid and a modified baculovirus genome were prepared at 30°C.

[0012] The controllable loss transfer vector at least comprises an antibiotic resistance marker, a temperature-sensitive replicon, a Tn7 transposable element and a foreign protein gene expression frame.

[0013] Furthermore, the temperature-sensitive replicon in step (1) is selected from pSC101 or a mutant thereof, or an expression cassette consisting of oriV and trfA genes (235G synonymous mutation) or a mutant thereof; the temperature-sensitive replicon has a high replication efficiency at low temperatures, and the replication efficiency decreases or is even inhibited as the temperature rises; the counter-selection element is a counter-selection marker gene;

[0014] Preferably, the temperature-sensitive replicon is selected from an expression cassette consisting of oriV and trfA genes (235G synonymous mutation);

[0015] Preferably, the counter-selection element is selected from the streptomycin sensitive element rpsL;

[0016] Preferably, the helper plasmid encoding Tn7 transposase in step (1) is pMON7124;

[0017] Preferably, the nucleotide sequence of the controllably lost helper plasmid in step (1) is as shown in SEQ ID NO.1 or SEQ ID NO.2;

[0018] Preferably, the nucleotide sequence of the controllably lost helper plasmid in step (1) is as shown in SEQ ID NO.2.

[0019] Furthermore, the baculovirus genome isolated in step (2) is selected from BmNPV or AcMNPV;

[0020] Preferably, the modified baculovirus genome further comprises knocking out non-essential genes of the baculovirus genome;

[0021] Preferably, the non-essential genes of the baculovirus genome are selected from one or more of Bm103, Bm104, Bm106, Bm114, Ac29-Ac33, Ac126, Ac127, Ac129, and Ac137;

[0022] Preferably, the nucleotide sequence of the modified baculovirus genome is shown as SEQ ID NO.11;

[0023] Preferably, the DH10 pureBac competent cells in step (3) are chemically competent cells or electroporation competent cells.

[0024] Furthermore, the promoter of the exogenous protein gene expression cassette in the controllable loss transfer vector is selected from one or more of Pp10, Pph, Pp6.9, Pgp64, Pie-1, and their mutual chimeras or constitutive promoters formed by adding enhancers in front of the promoters;

[0025] Preferably, the temperature-sensitive replicon in the controllable-loss transfer vector is selected from an expression cassette consisting of oriV and trfA genes or a pSC101 element;

[0026] Preferably, the temperature-sensitive replicon in the controllable-loss transfer vector is selected from the pSC101 element, and the controllable-loss transfer vector also contains a counter-selection element; it is worth noting that if the temperature-sensitive replicon is pSC101, it needs to be used in combination with the counter-selection element, while if it is oriV and trfA genes (235G synonymous mutation), they can be used alone, and the combination with the counter-selection element is more efficient;

[0027] Preferably, the counter-selection element is a counter-selection marker gene;

[0028] Preferably, the counter-selection element is selected from the streptomycin sensitive element rpsL.

[0029] The present invention also provides a method for preparing a recombinant bacmid, comprising the steps of: transforming the above-mentioned transfer vector with controllable loss into the above-mentioned DH10pureBac competent cells, activating them at 37°C, and screening positive monoclonal clones to obtain a high-purity recombinant bacmid free of helper plasmid and transfer vector residues.

[0030] The present invention also provides a BmNPV-rAAV recombinant bacmid preparation system, which comprises: I) DH10pureBac competent cells containing a controllable loss helper plasmid and a modified baculovirus genome, wherein the baculovirus genome is a Bombyx mori nuclear polyhedrosis virus genome; and II) the controllable loss transfer vector, wherein the exogenous protein gene in the controllable loss transfer vector comprises the Cap gene, Rep gene, and ITR-GOI element of AAV.

[0031] Furthermore, the DH10pureBac competent cells are selected from DH10pureBmBac2.1 competent cells or DH10pureBmBac3.1 competent cells;

[0032] The Bombyx mori nuclear polyhedrosis virus genome transformed in the DH10pureBmBac2.1 competent cells is the Bm3.2 version, in which the mini-attTn7 in the Bombyx mori nuclear polyhedrosis virus genome is moved from the Polh site to the ODV-E56 site, and the bacterial replication regulatory element is still placed at the Polh position;

[0033] The Bombyx mori nuclear polyhedrosis virus genome transformed in the DH10pureBmBac3.1 competent cells is the Bm3.3 version, in which the mini-attTn7 in the Bombyx mori nuclear polyhedrosis virus genome is moved from the Polh site to the ODV-E56 site, the bacterial replication regulatory element is still placed at the Polh position, and the Bm103 and Bm104 genes are knocked out;

[0034] Preferably, the nucleotide sequence of Bm3.2 is shown as SEQ ID NO.11.

[0035] Furthermore, the AAV serotype is selected from AAV1-13 and derived serotypes thereof;

[0036] Preferably, the Cap gene and Rep gene of the AAV are modified and placed between different baculovirus promoters and transcriptional and post-transcriptional regulatory sequences respectively;

[0037] Preferably, the baculovirus promoter is derived from AcMNPV or BmNPV, and is selected from Pp10, Pph, Pp6.9, Pgp64, Pie-1, and their mutual chimeras or constitutive promoters formed by adding enhancers before the promoters;

[0038] Preferably, the transcriptional and post-transcriptional regulatory sequences are selected from TK polyA or SV40 polyA;

[0039] Preferably, the ITR-GOI element, the Cap gene expression cassette, and the Rep gene expression cassette are placed on three plasmids respectively, and freely assembled into a transfer vector with controllable loss through a one-step Golden Gate according to the AAV production requirements;

[0040] Preferably, the ITR-GOI element is placed between the AAV Cap gene expression cassette and the Rep gene expression cassette;

[0041] Preferably, the nucleotide sequence of the controllable loss transfer vector comprising the Cap gene, Rep gene and ITR-GOI element of AAV is shown as SEQ ID NO.15 or SEQ ID NO.16.

[0042] The present invention also provides a method for preparing a BmNPV-rAAV recombinant bacmid, comprising transforming a corresponding controllable loss transfer vector into corresponding DH10pureBac competent cells, activating the cells at 37°C, and screening positive monoclonal clones to obtain a high-purity BmNPV-rAAV recombinant bacmid free of helper plasmid and transfer vector residues.

[0043] Preferably, the activation time at 37°C is 6 hours.

[0044] The present invention also provides the above-mentioned BmNPV-rAAV recombinant bacmid, which is a single BmNPV bacmid comprising a recombinant adeno-associated virus Cap gene expression cassette, a core expression element ITR-GOI, and a Rep gene expression cassette.

[0045] Compared with the existing technology, the following beneficial effects can be achieved:

[0046] (1) The high-purity recombinant bacmid preparation system provided by the present invention is achieved by isolating the baculovirus genome from the Bac-to-Bac baculovirus expression system, using Red / ET recombination technology to move the mini-attTn7 element from the Polh site to the ODV-E56 site, or to any site other than the ODV-E56 site that does not affect baculovirus packaging. Furthermore, non-essential baculovirus genes are knocked out and integrated into the helper plasmid and transfer vector, respectively, by introducing a temperature-sensitive replicon and a reverse selection element strategy. This achieves a simple and rapid preparation of recombinant bacmids without helper plasmid and transfer vector residues, thereby improving the quality and preparation efficiency of the recombinant bacmids.

[0047] (2) The BmNPV-rAAV recombinant bacmid provided by the present invention establishes a PureBac system based on Bombyx mori nuclear polyhedrosis virus (BmNPV) through the above-mentioned "high-purity recombinant bacmid preparation system", and uses the Goldengate assembly strategy to quickly load the AAV serotype Cap gene expression cassette, Rep gene expression cassette and ITR-GOI three elements onto the transfer vector of the BmNPV-PureBac system, and then transform the competent cells of the BmNPV-PureBac system to achieve high-purity BmNPV-rAAV recombinant bacmid preparation, laying the foundation for the large-scale and extremely low-cost production of rAAV vectors using silkworm larvae / pupae as bioreactors with Chinese characteristics. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] FIG1 is a process for preparing BmNPV-rAAV recombinant bacmid;

[0049] FIG2 is a diagram of the preparation of a loseable helper plasmid and verification of its lossability in Example 1;

[0050] FIG3 is a diagram showing the loss of the auxiliary plasmid prepared and verified in Example 1 in PureBac1.0 and PureBac2.0 strains;

[0051] FIG4 shows the preparation of a disposable transfer vector and verification of its disposability in Example 2;

[0052] FIG5 shows the loss of AAV transfer vector and helper plasmid after the AAV transfer vector prepared in Example 2 was transformed into PureBac competent cells and recombined;

[0053] Figure 6 is a schematic diagram of the BmNPV genome transformation in Example 3;

[0054] FIG7 is a diagram showing the establishment of the Bombyx mori baculovirus (BmNPV) PureBac system and rescue of recombinant BEV in Example 3;

[0055] FIG8 is a diagram illustrating the compatibility of the BmNPV-PureBac system verified in Example 3. DETAILED DESCRIPTION

[0056] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0057] Example 1 Preparation of Lossable Helper Plasmid

[0058] The transposase provided by the helper plasmid pMON7124 mediates transposition of exogenous genes into the mini-attTn7 site of the baculovirus genome. However, after transposition, the helper plasmid can still replicate in DH10 Bac bacteria, inevitably resulting in residual pMON7124 vector in the extracted recombinant bacmid. To address this, the present invention replaces the initial replicon Ori in the helper plasmid with a temperature-sensitive replicon and a counter-selection element in an attempt to controllably eliminate the helper plasmid.

[0059] (1) Construction of a helper plasmid containing a temperature-sensitive replicon and a reverse selection element (Ts&SMs Helper);

[0060] The pMON7124 plasmid was isolated from DH10Bac competent cells (Thermo Fisher Scientific, 10361012), and the pMON7124 vector was amplified using a high-fidelity PCR enzyme (Takara, R050A) to remove the Ori origin replicon. The counter selection element rpsL and the temperature-sensitive replicons pSC101 Ori-rapA and OriV-trfA were fused and introduced into homology arms by PCR. The rpsL gene and pSC101 Ori-rapA element were derived from the Counter Selection BAC Modification Kit (Gene Bridges, K002), and the OriV-trfA element was derived from the patent "A recombinant bacmid and its preparation method, patent application number: 202310643354.7". The two linear DNA fragments were ligated using Gibson assembly technology (NEB, E2621L) to obtain a helper plasmid containing rpsL-pSC101 Ori-rapA (nucleotide sequence shown in SEQ ID NO: 1). NO.1), the rpsL-OriV-trfA helper plasmid (nucleotide sequence is SEQ ID NO.2), as shown in Figure 2a.

[0061] (2) Verify the loss of Ts&SMs Helper plasmid;

[0062] Stbl3 glycerol bacteria containing the Ts&SMs Helper plasmid were spread on tetracycline-resistant LB solid culture medium and cultured at 30°C overnight. Single clones were selected and inoculated into LB liquid culture medium containing tetracycline resistance and expanded at 30°C. After 3 hours, 200 μL of the bacterial solution was inoculated into 700 μL of LB culture medium without resistance and cultured at 37°C for 4 hours in an attempt to lose the helper plasmid in the bacteria. Then, 100 μL of the bacterial solution was spread on tetracycline-resistant, streptomycin-resistant and tetracycline / streptomycin dual-resistant LB solid culture media and cultured at 37°C overnight, as shown in Figure 2b. The loss of the Ts&SMs Helper plasmid and whether the strain losing the Ts&SMs Helper plasmid could be screened were determined by observing the growth of the bacteria on the plate and colony PCR identification, as shown in Figure 2c. The results showed that no colonies were produced when the bacterial liquid was spread on LB solid culture medium with tetracycline / streptomycin dual resistance, but it could grow on LB solid culture medium with tetracycline resistance and streptomycin resistance. Strains from tetracycline-resistant plates and streptomycin-resistant plates were selected for PCR identification respectively, and it was observed that there was no Ts&SMs Helper plasmid signal in the strain derived from the streptomycin plate, while it could be detected in the strain derived from the tetracycline plate, indicating that both Ts&SMs Helper plasmids can be lost and the strain that lost the Ts&SMs Helper plasmid can be screened out.

[0063] Further, the Bombyx mori nuclear polyhedrosis virus genome was isolated from the DH10Bac (BmNPV) Escherichia coli strain (Shanghai Jiachu Bioengineering Co., Ltd., SHBCC D24962) and introduced into MegaX DH10B T1R electroporation competent cells (Thermo Fisher Scientific, C640003) to prepare electroporation competent cells BmDH10B containing the Bombyx mori nuclear polyhedrosis virus genome. Subsequently, the above two helper plasmids containing rpsL-pSC101 Ori-rapA and rpsL-OriV-trfA were introduced into BmDH10B to prepare PureBac1.0 and PureBac2.0 electroporation competent cells, as shown in Figures 3a-b. The Bombyx mori nuclear polyhedrosis virus genome and Ts&SMs were then identified by colony PCR. Whether the Helper plasmid exists in PureBac1.0 and PureBac2.0 electrocompetent cells, as shown in Figures 3c and e, colony PCR showed that both PureBac1.0 and PureBac2.0 electrocompetent cells contained the BmNPV genome and Ts&SMs Helper plasmid. The loss of Ts&SMs Helper plasmids in PureBac1.0 and PureBac2.0 strains was verified using the method shown in Figure 2b. The PureBac1.0 and PureBac2.0 strains were activated at 37°C for 4 hours and respectively spread on LB solid medium containing kanamycin / streptomycin double resistance and cultured at 37°C overnight. PCR of selected monoclonal colonies showed that the rpsL-pSC101 Ori-rapA helper plasmid could be detected in most PureBac1.0 strains (Figure 3d), while only a small part of the PureBac2.0 strains contained rpsL-OriV-trfA helper plasmid was detected (Figure 3f). This indicates that the loss efficiency of the rpsL-OriV-trfA helper plasmid at 37°C is higher than that of the rpsL-pSC101 Therefore, in subsequent experiments, the helper plasmid containing the Ori-rapA element was preferred and the helper plasmid containing rpsL-OriV-trfA was used and the activation time at 37°C was extended to 6 hours. It is worth noting that: 1) a monoclonal strain was obtained using kanamycin / tetracycline dual-resistance LB solid medium, 2) the monoclonal strain was inoculated in LB liquid medium containing kanamycin / tetracycline dual-resistance and expanded at 30°C for 3 hours, 3) 100 μL of the bacterial liquid was spread on LB solid medium containing kanamycin / streptomycin dual-resistance, and 4) the Bombyx mori nuclear polyhedrosis virus genome and Ts&SMs Helper plasmid in the colony were identified by PCR respectively.

[0064] Example 2 Preparation of Lossable Transfer Vector

[0065] Under the action of Tn7 transposase, the donor plasmid (i.e., transfer vector) can recombine with the baculovirus shuttle vector to form an expression bacmid containing the exogenous gene. However, the transfer vector that has not undergone recombination after transformation into DH10 Bac bacteria can still replicate, which causes the transfer vector to remain in the extracted recombinant bacmid. In addition, recombinant adeno-associated virus (rAAV) has been proven to be safe and effective in preclinical and clinical trials as a highly efficient gene delivery vector, and the baculovirus-based rAAV production system has attracted much attention due to its scalability, low cost, and predictable biosafety. In summary, the present invention uses a transfer vector containing three AAV elements as an example to introduce a temperature-sensitive replicon and a counter-selection element into it to demonstrate controllable loss.

[0066] (1) Loading the rpsL-pSC101 Ori-rapA element onto the AAV-Rep backbone plasmid and verifying its loss;

[0067] According to the invention patent “A method for preparing and applying a transfer vector pAAV-Donor in a baculovirus-rAAV production system”, an AAV-Rep backbone plasmid T79B00-0-0 (pBACKBONE_Bm-AAV-Rep-BsaI-ccdB-Gen, with a nucleotide sequence such as SEQ ID NO. 3) containing the Bombyx mori nuclear polyhedrosis virus ph promoter was constructed. Considering that the temperature-sensitive plasmid containing the pSC101 Ori-rapA element has a low loss efficiency when cultured at 37°C and the growth rate of bacteria at 37°C is also faster than that at 30°C, the present invention loads the rpsL-pSC101 Ori-rapA element onto the AAV-Rep backbone plasmid T79B00-0-0 to construct the plasmid tsT79B00-0-0. Specifically, T79B00-0-0 was double-digested with restriction endonucleases AgeI and BspQI to obtain a DNA fragment without the Ori initiator replicon. The rpsL-pSC101 Ori-rapA gene sequence was amplified by PCR using the "helper plasmid containing rpsL-pSC101 Ori-rapA" in Example 1 as a template and introduced into the homologous arms. The two DNA fragments were homologously recombined and ligated and transformed into DB3.1 chemically competent cells to screen positive clones to obtain the AAV-Rep backbone plasmid tsT79B00-0-0 (pBACKBONE_tsBm-AAV-Rep-BsaI-ccdB-Gen, nucleotide sequence is SEQ ID NO.4) containing rpsL-pSC101 Ori-rapA, as shown in Figure 4a.

[0068] The loss of the tsT79B00-0-0 plasmid was verified using the method of "Verifying the Ts&SMs Helper Plasmid" in Example 1. Unlike the Ts&SMs Helper plasmid, the antibiotic resistance marker of the tsT79B00-0-0 plasmid is gentamicin. Therefore, the tetracycline resistance marker needs to be replaced with gentamicin resistance, as shown in Figure 4c. Similarly, the loss of the tsT79B00-0-0 plasmid was determined by observing the growth of bacteria on the plate and colony PCR identification. The results showed that no colonies were produced when the strain was spread on LB solid culture medium with gentamicin / streptomycin dual resistance, but it could grow on both gentamicin-resistant and streptomycin-resistant LB solid culture media; strains from gentamicin-resistant plates and streptomycin-resistant plates were selected for PCR identification, and no tsT79B00-0-0 plasmid signal was observed in the strain derived from the streptomycin plate, while it could be detected in the strain derived from the gentamicin plate (Figure 4d), indicating that the tsT79B00-0-0 plasmid can be lost and strains that have lost the tsT79B00-0-0 plasmid can be screened out.

[0069] (2) Preparation of a transfer vector (pAAV-tsDonor) containing the three AAV elements for loss verification;

[0070] According to the invention patent "A method for preparing and applying a transfer vector pAAV-Donor in a baculovirus-rAAV production system": 1) constructing an AAV-Cap backbone plasmid 9B00 (pBACKBONE_Bm-AAV-Cap-BsaI-SmR, nucleotide sequence such as SEQ ID NO.5) containing the Bombyx mori nuclear polyhedrosis virus p10 promoter, 2) the AAV-Core backbone plasmid 2001 is derived from the patent, 3) the Cap backbone plasmid 9B00, the Core backbone plasmid 2001 and the Rep backbone plasmid tsT79B00-0-0 in step (1) are assembled to prepare the pAAV-tsDonor plasmid, as shown in FIG4b . In order to more efficiently obtain the transfer vector containing the three AAV elements, 5 μl of the Goldengate assembled product was taken to transform stbl3 chemically competent cells and cultured overnight at 30°C and 37°C respectively. The single colonies on the plate cultured overnight at 37°C were much larger than the single colonies cultured overnight at 30°C. The monoclonal strains from the 37°C plate and the 30°C plate were selected for PCR identification. It was observed that positive monoclonal clones could be identified regardless of whether the monoclonal clones originated from the 37°C plate or the 30°C plate (Figures 4e-f). Two positive clone strains were selected for expansion culture, plasmid extraction and sequencing, which showed that they were correct. This indicates that although the Rep backbone plasmid tsT79B00-0-0 contains the temperature-sensitive replicon pSC101Ori-rapA, 37°C overnight culture conditions can still be used in the preparation of the pAAV-tsDonor plasmid.

[0071] Furthermore, the pAAV-tsDonor plasmid prepared above was verified for loss according to the method shown in FIG4c in step (1), and the loss of the pAAV-tsDonor plasmid was determined by observing the growth of bacteria on the plate and colony PCR identification. The results showed that no colonies were produced when the strain was coated on LB solid medium with gentamicin / streptomycin resistance, but it could grow on both LB solid medium with gentamicin resistance and streptomycin resistance ( FIG4g ); strains from gentamicin-resistant plates and streptomycin-resistant plates were selected for PCR identification, and no pAAV-tsDonor plasmid signal was observed in the strain derived from the streptomycin plate, while a signal could be detected in the strain derived from the gentamicin plate ( FIG4h ), indicating that the pAAV-tsDonor plasmid can be lost and strains that have lost the pAAV-tsDonor plasmid can be screened out.

[0072] (3) After the pAAV-tsDonor plasmid is transformed into PureBac competent cells and recombination occurs, the loss of the pAAV-tsDonor and Ts&SMs Helper plasmids is verified;

[0073] The pAAV-tsDonor plasmid prepared in step (2) was transformed into the PureBac1.0 and PureBac2.0 electroporation competent cells prepared in step (2) of Example 1, respectively, as shown in FIG5a, and activated at 37°C for 6 h. 100 μL of the bacterial solution was spread on LB solid culture plates containing kanamycin / gentamicin / streptomycin / IPTG / X-Gal and cultured overnight at 37°C. White colonies were selected for PCR identification. The results showed that the white colonies from the PureBac1.0 and PureBac2.0 plates were all positive recombinants ( FIG5b ). Then, two positive recombinants were selected and re-plated on kanamycin / gentamicin / streptomycin / IPTG / X-Gal LB solid culture plates. After overnight culture at 37°C, white colonies were selected for PCR identification of the BmNPV recombinant bacmid, pAAV-tsDonor plasmid, and Ts&SMs Helper plasmid to verify the loss of pAAV-tsDonor and Ts&SMs Helper plasmids. The results showed that one colony from the monoclonal colonies derived from the PureBac1.0 plate could detect pAAV-tsDonor and a helper plasmid signal containing the rpsL-pSC101 Ori-rapA element (Figure 5c), while the monoclonal colonies from the PureBac2.0 plate only contained the BmNPV recombinant bacmid (Figure 5d), indicating that the pSC101 The pAAV-tsDonor plasmid containing the Ori-rapA element is used in combination with a helper plasmid containing rpsL-OriV-trfA to obtain high-purity recombinant bacmids free of transfer vector and helper plasmid residues.

[0074] Example 3: Modification of the baculovirus genome and establishment of the PureBac system based on Bombyx mori nuclear polyhedrosis virus (BmNPV)

[0075] When exogenous gene sequences are inserted into different loci of non-essential baculovirus genes, the expression abundance of exogenous proteins varies. Considering that 1) Gorben P et al. showed that moving mini-attTn7 to the ODV-E56 (pif-5) site of the AcMNPV genome can mediate higher levels of exogenous protein expression and recombinant BEV is more stable in continuous passages (Gorben P et al., 2020, Viruses, 12, 1448), and 2) although Bombyx mori nuclear polyhedrosis virus (BmNPV) is different from AcMNPV, the genomic homology between the two is as high as 90%, the present invention moves mini-attTn7 from the Polh site of the BmNPV genome to the ODV-E56 site.

[0076] (1) Modification of the BmNPV genome;

[0077] According to the instructions of the "Counter Selection BAC Modification Kit", the pRedET plasmid was introduced into the BmDH10B electroporation competent cells prepared in step (2) of Example 1 to prepare electroporation competent cells Bm3.0 / pRedET containing the pRedET plasmid and the BmNPV genome. 1) In the case of the chloramphenicol resistance gene (Cm R , derived from the pBeloBAC11 vector, Tianjin Zhuangmeng Biotechnology Co., Ltd., ZK130) were cloned on both sides by PCR into 50 bp homology arms (nucleotide sequence as SEQ ID NO.6) and then transformed into Bm3.0 / pRedET competent cells. The Cm R Insertion between the lef2 gene and the mini-F ori element is shown in Figure 6a, including insertion of Cm R Bacteria carrying the BmNPV genome (Bm3.1-0) with the resistance gene were prepared into electroporation competent cells Bm3.1-0 / pRedET; 2) in rpsL-Amp R The 50 bp homology arms (nucleotide sequence as SEQ ID NO.7) were introduced at both ends of the element by PCR and then transformed into Bm3.1-0 / pRedET competent cells. The LacZα-Kan in the Bm3.1-0 genome was replaced by Red / ET recombination technology. R The elements are shown in FIG6b. The replaced BmNPV genome was named Bm3.1-1, and electroporation competent cells Bm3.1-1 / pRedET were prepared; 3) a segment containing rpsL-Amp R The gene sequence of the homologous arms on both sides of the element (nucleotide sequence such as SEQ ID NO.8) was introduced into Bm3.1-1 / pRedET competent cells to knock out rpsL-Amp R The elements are shown in Figure 6c, and rpsL-Amp is knocked out in Bm3.1-1. R The BmNPV genome with the element was named Bm3.1-2 as shown in FIG6d, and electroporation competent cells Bm3.1-2 / pRedET were prepared; 4) in rpsL-Kan R The two ends of the element were introduced by PCR with 50 bp homology arms (nucleotide sequence as SEQ ID NO.9) and then transformed into Bm3.1-2 / pRedET competent cells. The rpsL-Kan was transformed into rpsL-Kan using Red / ET recombination technology. R The element was inserted into the ODV-e56 site of the Bm3.1-2 genome as shown in Figure 6e. The ODV-e56 site in Bm3.1-2 was inserted into rpsL-Kan RThe BmNPV genome containing the element was named Bm3.1-3, and electroporation competent cells Bm3.1-3 / pRedET were prepared; 5) 50 bp homology arms (nucleotide sequence such as SEQ ID NO.10) were introduced at both ends of the LaeZα gene containing the mini-attTn7 element by PCR and then transformed into Bm3.1-3 / pRedET competent cells, and the rpsL-Kan in the Bm3.1-3 genome was replaced by Red / ET recombination technology. R The components are shown in Figure 6f, completing the rpsL-Kan in Bm3.1-3 R The BmNPV genome with element replacement was named Bm3.2 as shown in Figure 6g. The whole genome of Bm3.2 was sequenced (nucleotide sequence as SEQ ID NO.11, Sangon Biotech (Shanghai) Co., Ltd.), and bacteria containing the Bm3.2 genome were prepared into electrocompetent cells Bm3.2. The pRedET plasmid was further introduced into the Bm3.2 competent cells to prepare electrocompetent cells Bm3.2 / pRedET.

[0078] (2) Establishment of the Bombyx mori baculovirus (BmNPV) PureBac system;

[0079] As shown in Example 1, compared with the rpsL-pSC101 Ori-rapA element, the helper plasmid containing the rpsL-OriV-trfA element has the best loss effect. Therefore, the present invention introduces the helper plasmid containing the rpsL-OriV-trfA element into the Bm3.2 competent cells prepared in step (1) to prepare chemically competent cells DH10pureBmBac2.1 (abbreviated as pureBmBac2.1) as shown in Figure 7a.

[0080] In Example 2, AAV Rep (tsT79B00-0-0) and Cap (9B00) backbone plasmids were constructed. According to the invention patent "A method for preparing and using a transfer vector pAAV-Donor in a baculovirus-rAAV production system", 1) the modified AAV2-Rep gene expression cassette was inserted into tsT79B00-0-0 to obtain AAV-Rep plasmid tsT79B00-0-4 (nucleotide sequence such as SEQ ID NO.12), 2) the modified AAV2 and AAV9 Cap gene expression cassettes were respectively inserted into 9B00 to obtain AAV-Cap plasmids 9B02 and 9B09 (nucleotide sequences such as SEQ ID NO.13 and SEQ ID NO.14); 3) the AAV-Core plasmid 2006 in the patent was used. Assemble tsT79B00-0-4+9B02+2006 to form tsT79B02-2006-4 (nucleotide sequence such as SEQ ID NO.15), and tsT79B00-0-4+9B09+2006 to form tsT79B09-2006-4 (nucleotide sequence such as SEQ ID NO.16), namely pAAV-tsDonor.

[0081] The two pAAV-tsDonor plasmids were transformed into pureBmBac2.1 chemically competent cells, as shown in Figure 7b. After transposition at 37°C for 6 h, the cells were spread on LB solid culture plates containing gentamicin / streptomycin / IPTG / X-Gal and cultured overnight at 37°C. White colonies were then selected and streaked on LB solid culture plates containing gentamicin / streptomycin / IPTG / X-Gal and cultured overnight at 37°C. Colony PCR identified strains containing only the BmNPV-rAAV recombinant bacmid, as shown in Figure 7c. The BmNPV-rAAV recombinant bacmid was extracted and transfected into BmN cells (ATCC, CRL-8910). The results showed that the green fluorescence was strongest on the 4th day of transfection, among which the fluorescence intensity of the transfected shuttle vector Bm3.2-rAAV2-CMV-eGFP-hGH was higher than that of Bm3.2-rAAV9-CMV-eGFP-hGH (Figure 7d). The supernatant of BmN cells transfected with the BmNPV-rAAV recombinant bacmid was further added to newly cultured BmN cells. After 4 days, fluorescence microscopy was used to observe that the BmN cells were illuminated by green fluorescence, indicating that BmNPV-rAAV recombinant bacmid transfection into BmN cells can rescue recombinant BEV as shown in Figures 7d-e.

[0082] (3) Verify the compatibility of the BmNPV-PureBac system;

[0083] In step (1), Bm3.2 / pRedET electroporation competent cells were prepared and the rpsL-Amp RAfter 50 bp of homology arms (nucleotide sequence as SEQ ID NO. 17) were introduced at both ends of the element by PCR, the element was transformed into Bm3.2 / pRedET competent cells. The Red / ET recombination technology was used to replace part of the Bm103 and Bm104 gene sequences in the Bm3.2 genome as shown in Figure 8a. The replaced BmNPV genome was named Bm3.3-0, and electroporation competent cells Bm3.3-0 / pRedET were prepared. Furthermore, a segment containing rpsL-Amp R The gene sequence of the homologous arms on both sides of the element (nucleotide sequence such as SEQ ID NO.18) was introduced into Bm3.3-0 / pRedET competent cells to knock out rpsL-Amp R The elements are shown in Figure 8b, and rpsL-Amp is knocked out in Bm3.3-0. R The BmNPV genome containing the element was named Bm3.3. As shown in FIG8c, bacteria containing the Bm3.3 genome were prepared into electrocompetent cells Bm3.3.

[0084] The helper plasmid containing the rpsL-OriV-trfA element was introduced into the prepared Bm3.3 competent cells to prepare chemical competent cells DH10pureBmBac3.1 (abbreviated as pureBmBac3.1) as shown in Figure 8d. The pFD prepared in the invention patent "A recombinant bacmid and its preparation method, patent application number: 202310643354.7" RK2-ts -Cap-ITR(GOI)-Rep plasmids were transformed into pureBmBac2.1 and pureBmBac3.1 competent cells as shown in Figure 8e. After 6 hours of transposition at 37°C, the cells were spread on LB solid culture plates containing chloramphenicol / gentamicin / streptomycin / IPTG / X-Gal and cultured overnight at 37°C. Clearly visible white monoclonal colonies were observed as shown in Figure 8f. The white colonies were selected and used for BmNPV recombinant bacmid, pFD RK2-ts PCR identification of the OriV-trfA element shared by the -Cap-ITR(GOI)-Rep and Ts&SMs Helper plasmids was performed, as shown in Figure 8g. The results showed that one strain derived from the pureBmBac2.1 plate contained a Bm3.2 recombinant bacmid signal but lacked AAV transfer plasmid and helper plasmid signals, while three positive clones were found in the strain derived from the pureBmBac3.1 plate. Further extraction of the recombinant bacmid and sequencing of the inserted AAV Cap gene expression cassette, core expression element ITR-GOI, and Rep gene expression cassette by PCR confirmed the correctness of the results, demonstrating the compatibility of the established BmNPV-PureBac system based on Bombyx mori nuclear polyhedrosis virus.

Claims

1. A recombinant bacmid preparation system, characterized in that, The recombinant bacmid preparation system includes: a) DH10pureBac competent cells containing an auxillary plasmid with controllable loss and a modified baculovirus genome, b) a transfer vector with controllable loss; The method for preparing the DH10pureBac competent cells containing an auxillary plasmid with controllable loss and a modified baculovirus genome includes the following steps: (1) Prepare the auxillary plasmid with controllable loss: Replace the origin of replication Ori in the auxillary plasmid encoding Tn7 transposase with a temperature-sensitive replicon and a counter-selection element to obtain the auxillary plasmid with controllable loss; (2) Modify the baculovirus genome: Isolate the baculovirus genome from the Bac-to-Bac system, and use the Red / ET recombination technology to move mini-attTn7 in the genome from the Polh locus to the ODV-E56 locus while the bacterial replication regulatory element remains at the Polh position, or move mini-attTn7 to any site other than the ODV-E56 site that does not affect baculovirus packaging while the bacterial replication regulatory element remains at the Polh position, to obtain the modified baculovirus genome; (3) Transform the helper plasmid with controllable loss in step (1) and the modified baculovirus genome in step (2) into MegaX DH10B T1 R competent cells, and prepare DH10pureBac competent cells containing the helper plasmid with controllable loss and the modified baculovirus genome at 30 °C. The transfer vector with controllable loss contains at least an antibiotic resistance marker, a temperature-sensitive replicon, a Tn7 transposon element, and an exogenous protein gene expression cassette.

2. The recombinant plasmid preparation system according to claim 1, wherein In step (1), the temperature-sensitive replicon is selected from pSC101 or its mutants, or an expression cassette composed of oriV and the trfA gene or its mutants; the counter-selection element is a counter-selection marker gene.

3. The recombinant bacmid preparation system according to claim 1, characterized in that, In step (1), the temperature-sensitive replicon is selected from an expression cassette composed of oriV and the trfA gene; The counter-selection element is selected from the streptomycin-sensitive element rpsL; 4. The recombinant bacmid preparation system according to claim 1, wherein In step (1), the auxillary plasmid encoding Tn7 transposase is pMON7124.

5. The recombinant bacmid preparation system according to claim 1, characterized in that, The nucleotide sequence of the auxillary plasmid with controllable loss in step (1) is as shown in SEQ ID NO.1 or SEQ ID NO.

2.

6. The recombinant plasmid preparation system according to claim 1, wherein In step (2), the isolated baculovirus genome is selected from BmNPV or AcMNPV.

7. The recombinant bacmid preparation system according to claim 1, wherein, The modified baculovirus genome further includes knocking out non-essential genes of the baculovirus genome.

8. The recombinant bacmid preparation system according to claim 7, wherein The non-essential genes of the baculovirus genome are selected from one or more of Bm103, Bm104, Bm106, Bm114, Ac29 - Ac33, Ac126, Ac127, Ac129, Ac137.

9. The recombinant bacmid preparation system according to claim 1, wherein The nucleotide sequence of the modified baculovirus genome is as shown in SEQ ID NO.

11.

10. The recombinant bacmid preparation system according to claim 1, wherein, In step (3), the DH10pureBac competent cells are chemically competent cells or electrocompetent cells.

11. The recombinant bacmid preparation system according to claim 1, characterized in that, The promoter of the exogenous protein gene expression cassette in the transfer vector with controllable loss is selected from one or more of Pp10, Pph, Pp6.9, Pgp64, Pie-1, their chimeras with each other, or constitutive promoters formed by adding enhancers in front of the promoters; The temperature-sensitive replicon in the transfer vector with controllable loss is selected from an expression cassette composed of oriV and the trfA gene or a pSC101 element; The reverse selection element is a reverse selection marker gene.

12. The recombinant bacmid preparation system according to claim 1, characterized in that, In the transfer vector with controllable loss, the temperature-sensitive replicon is selected from pSC101 elements, and the transfer vector with controllable loss contains a reverse selection element at the same time; The reverse selection element is selected from the streptomycin-sensitive element rpsL.

13. A method for preparing recombinant bacmid, characterized in that, The preparation method includes the following steps: transforming the transfer vector with controllable loss described in claim 1 into the DH10pureBac competent cell described in claim 1, activating at 37°C, and screening positive monoclonal colonies to obtain a high-purity recombinant bacmid without residual helper plasmid and transfer vector.

14. A BmNPV-rAAV recombinant bacmid preparation system, characterized in that, The BmNPV-rAAV recombinant bacmid preparation system includes: I) the DH10pureBac competent cell described in claim 1 containing a helper plasmid with controllable loss and a modified baculovirus genome, wherein the baculovirus genome is a Bombyx mori nucleopolyhedrovirus genome; II) the transfer vector with controllable loss described in claim 1, wherein the foreign protein gene in the transfer vector with controllable loss includes the Cap gene, Rep gene and ITR-GOI element of AAV.

15. The BmNPV-rAAV recombinant bacmid preparation system according to claim 14, wherein The DH10pureBac competent cell is selected from DH10pureBmBac2.1 competent cell or DH10pureBmBac3.1 competent cell; In the DH10pureBmBac2.1 competent cell, the modified Bombyx mori nucleopolyhedrovirus genome is the Bm3.2 version, in which mini-attTn7 in the Bombyx mori nucleopolyhedrovirus genome is moved from the Polh locus to the ODV-E56 locus, and the bacterial replication regulatory element is still placed at the Polh position; In the DH10pureBmBac3.1 competent cell, the modified Bombyx mori nucleopolyhedrovirus genome is the Bm3.3 version, in which mini-attTn7 in the Bombyx mori nucleopolyhedrovirus genome is moved from the Polh locus to the ODV-E56 locus, the bacterial replication regulatory element is still placed at the Polh position, and the Bm103 and Bm104 genes are knocked out.

16. The BmNPV-rAAV recombinant bacmid preparation system according to claim 15, wherein The nucleotide sequence of Bm3.2 is as shown in SEQ ID NO.

11.

17. The BmNPV-rAAV recombinant bacmid preparation system according to claim 14, characterized in that, The serotype of the AAV is selected from AAV1-13 and its derived serotypes.

18. The BmNPV-rAAV recombinant bacmid preparation system according to claim 14, wherein, The Cap gene and Rep gene of the AAV are respectively placed between different baculovirus promoters and transcription and post-transcriptional regulatory sequences after modification; The baculovirus promoter is derived from AcMNPV or BmNPV, and is selected from one or more of Pp10, Pph, Pp6.9, Pgp64, Pie-1 and their chimeras or constitutive promoters formed by adding enhancers in front of the promoter; The transcription and post-transcriptional regulatory sequences are selected from TK polyA or SV40 polyA.

19. The BmNPV-rAAV recombinant bacmid preparation system according to claim 14, characterized in that, The ITR-GOI element, Cap gene expression cassette, and Rep gene expression cassette are respectively placed on three plasmids, and are assembled into a transfer vector with controllable loss by one-step Golden gate free assembly according to the AAV production requirements.

20. The BmNPV-rAAV recombinant bacmid preparation system according to claim 14, wherein The ITR-GOI element is placed between the AAV Cap gene expression cassette and the Rep gene expression cassette.

21. The BmNPV-rAAV recombinant bacmid preparation system according to claim 14, wherein The nucleotide sequence of the transfer vector with controllable loss containing the AAV Cap gene, Rep gene, and ITR-GOI element is as shown in SEQ ID NO.15 or SEQ ID NO.

16.

22. A method for preparing a BmNPV-rAAV recombinant bacmid, characterized in that, The transfer vector with controllable loss described in claim 14 is transformed into the DH10pureBac competent cells described in claim 14, activated at 37°C, and positive monoclonal colonies are screened to obtain high-purity BmNPV-rAAV recombinant bacmids without residual helper plasmid and transfer vector.

23. The preparation method according to claim 22, characterized in that, The activation time at 37°C is 6 hours.

24. The BmNPV-rAAV recombinant bacmid prepared by the preparation method according to claim 22, characterized in that, The BmNPV-rAAV recombinant bacmid is a single BmNPV bacmid containing the recombinant adeno-associated virus Cap gene expression cassette, the core expression element ITR-GOI, and the Rep gene expression cassette.

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