WO2025129367A1 - 一种重组杆粒制备系统及BmNPV-rAAV重组杆粒 - Google Patents

一种重组杆粒制备系统及BmNPV-rAAV重组杆粒 Download PDF

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WO2025129367A1
WO2025129367A1 PCT/CN2023/000125 CN2023000125W WO2025129367A1 WO 2025129367 A1 WO2025129367 A1 WO 2025129367A1 CN 2023000125 W CN2023000125 W CN 2023000125W WO 2025129367 A1 WO2025129367 A1 WO 2025129367A1
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recombinant bacmid
bmnpv
preparation system
competent cells
transfer vector
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French (fr)
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文鹏杰
王培培
徐富强
朱续涛
韩丹
朱珍香
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Shenzhen Institute of Advanced Technology of CAS
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/866—Baculoviral vectors

Definitions

  • 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.
  • the silkworm (Bombyx mori) is an important economic insect with Chinese characteristics. It goes through four forms in its life: egg, larva, pupa, and moth (adult). Although the main goal of silkworm breeding is to obtain high-yield and high-quality cocoons to provide raw materials for the silk industry and its derivative industries, in fact, silkworm larvae/pupae are also efficient bioreactors for expressing foreign 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 silkworm baculovirus system, widely involved in vaccine production, medical drug proteins and other fields.
  • the Bombyx mori baculovirus (BmNPV) expression system works similarly to the commercialized Autographa californica baculovirus (AcMNPV) Bac-to-Bac system, both of which are based on site-specific transposition of the Tn7 transposon to simplify and enhance the production of recombinant bacmids (Bacmid DNA), thereby quickly and efficiently producing recombinant baculoviruses.
  • AcMNPV Autographa californica baculovirus
  • Bac-to-Bac system both of which are based on site-specific transposition of the Tn7 transposon to simplify and enhance the production of recombinant bacmids (Bacmid DNA), thereby quickly and efficiently producing recombinant baculoviruses.
  • the obtained recombinant bacmids generally contain residual auxiliary plasmids and transfer vectors, which inevitably affects the rescue of the next generation of BEV.
  • the BmNPV expression system has not been further developed so far, and it is still impossible to obtain high-purity recombinant bacmids.
  • the temperature-sensitive element-based strategy developed by the applicant in the early stage 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 (culture at 30°C for 12h ⁇ pick out white recombinants at 48h ⁇ culture at 37°C for 16h ⁇ pick out single colonies at 37°C overnight ⁇ expand culture ⁇ extract recombinant bacmid), so it cannot be directly transferred to the BmNPV expression system and needs further improvement.
  • the present invention provides a recombinant bacmid preparation system and a BmNPV-rAAV recombinant bacmid, the purposes of which are: 1) introducing a temperature-sensitive replicon and a reverse screening element into a helper plasmid and a transfer vector, respectively, and quickly obtaining a high-purity recombinant bacmid by controlling the temperature and reverse screening - the temperature-sensitive replicon can control the presence of the plasmid in the host, and the plasmid can exist in the host when the temperature is low, and the plasmid is lost when the temperature is increased, and the reverse screening element can be the streptomycin-sensitive gene rpsL, and when the rpsL element is present, the DH10B strain is sensitive to streptomycin, and vice versa, the strain is resistant to streptomycin; 2) using Red/ET recombination technology to move the mini-attTn7 element from the
  • the present invention provides a recombinant bacmid preparation system, which comprises: a) DH10pureBac competent cells containing a controllable loss helper plasmid and a modified baculovirus genome, b) a controllable loss transfer vector;
  • the method for preparing DH10pureBac competent cells containing a controllably lost helper plasmid and a modified baculovirus genome comprises the following steps:
  • controllable loss helper plasmid replacing the initial replicon Ori in the helper plasmid encoding the Tn7 transposase with a temperature-sensitive replicon and a reverse selection element to obtain a controllable loss helper plasmid;
  • Modifying 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;
  • step (3) transforming 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 preparing DH10pureBac competent cells containing the helper plasmid with controllable loss and the modified baculovirus genome at 30° C.;
  • 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.
  • the temperature-sensitive replicon in step (1) is selected from pSC101 or a mutant thereof, or an expression cassette composed of oriV and trfA gene (235G synonymous mutation) or a mutant thereof; the temperature-sensitive replicon has a higher replication efficiency at low temperatures, and the replication efficiency decreases or is even inhibited as the temperature rises; the reverse screening element is a reverse screening marker gene;
  • the temperature-sensitive replicon is selected from an expression cassette consisting of oriV and trfA genes (235G synonymous mutation);
  • the counter-screening element is selected from the streptomycin sensitive element rpsL;
  • the helper plasmid encoding Tn7 transposase in step (1) is pMON7124;
  • the nucleotide sequence of the controllably lost auxiliary plasmid in step (1) is as shown in SEQ ID NO.1 or SEQ ID NO.2;
  • the nucleotide sequence of the controllably lost auxiliary plasmid in step (1) is as shown in SEQ ID NO.2.
  • the isolated baculovirus genome in step (2) is selected from BmNPV or AcMNPV;
  • the modified baculovirus genome further comprises knocking out non-essential genes of the baculovirus genome
  • 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;
  • nucleotide sequence of the modified baculovirus genome is shown as SEQ ID NO.11;
  • the DH10 pureBac competent cells in step (3) are chemical competent cells or electroporation competent cells.
  • the promoter of the exogenous protein gene expression frame 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 before the promoter;
  • 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;
  • the temperature-sensitive replicon in the controllable loss transfer vector is selected from the pSC101 element, and the controllable loss transfer vector also comprises a reverse screening element; it is worth noting that if the temperature-sensitive replicon is pSC101, it needs to be used in combination with the reverse screening element, and if it is oriV and trfA gene (235G synonymous mutation), it can be used alone, and the combination with the reverse screening element is more efficient;
  • the counter-selection element is a counter-selection marker gene
  • the counter-selection element is selected from the streptomycin sensitive element rpsL.
  • the present invention also provides a method for preparing a recombinant bacmid, which comprises the following steps: transforming the above-mentioned controllable loss transfer vector into the above-mentioned DH10pureBac competent cells, activating them at 37°C, and screening positive monoclones to obtain a high-purity recombinant bacmid without helper plasmid and transfer vector residues.
  • the present invention also provides a BmNPV-rAAV recombinant bacmid preparation system, which comprises: I) the DH10pureBac competent cells comprising a controllable loss helper plasmid and a modified baculovirus genome, wherein the baculovirus genome is a Bombyx mori nuclear polyhedrosis virus genome; 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.
  • DH10pureBac competent cells are selected from DH10pureBmBac2.1 competent cells or DH10pureBmBac3.1 competent cells;
  • 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;
  • the Bombyx mori nuclear polyhedrosis virus genome transformed in the DH10pureBmBac3.1 competent cells is the Bm3.3 version, in which 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;
  • nucleotide sequence of Bm3.2 is as shown in SEQ ID NO.11.
  • the serotype of AAV is selected from AAV1-13 and derived serotypes thereof;
  • Cap gene and Rep gene of the AAV are modified and placed between different baculovirus promoters and transcriptional and post-transcriptional regulatory sequences, respectively;
  • the baculovirus promoter is derived from AcMNPV or BmNPV, and is selected from Pp10, Pph, Pp6.9, Pgp64, Pie-1, and a constitutive promoter formed by their mutual chimeras or by adding an enhancer before the promoter;
  • the transcriptional and post-transcriptional regulatory sequences are selected from TK polyA or SV40 polyA;
  • 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;
  • the ITR-GOI element is placed between the AAV Cap gene expression frame and the Rep gene expression frame;
  • nucleotide sequence of the controllable loss transfer vector containing the Cap gene, Rep gene and ITR-GOI element of AAV is as shown in SEQ ID NO.15 or SEQ ID NO.16.
  • the present invention also provides a method for preparing a BmNPV-rAAV recombinant bacmid, wherein the corresponding controllable loss transfer vector is transformed into the corresponding DH10pureBac competent cells, activated at 37°C, and positive monoclones are screened to obtain a high-purity BmNPV-rAAV recombinant bacmid without helper plasmid and transfer vector residues;
  • the 37°C activation time is 6 hours.
  • the present invention also provides the above-mentioned BmNPV-rAAV recombinant bacmid, wherein the BmNPV-rAAV recombinant bacmid 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.
  • the high-purity recombinant bacmid preparation system is to separate the baculovirus genome from the Bac-to-Bac baculovirus expression system, and use the 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, and further knock out the non-essential baculovirus genes, and integrate them into the helper plasmid and the transfer vector to introduce the temperature-sensitive replicon and the reverse selection element strategy, respectively.
  • the simple and rapid preparation of recombinant bacmids without helper plasmid and transfer vector residues is realized, thereby improving the quality and preparation efficiency of recombinant bacmids
  • the BmNPV-rAAV recombinant bacmid provided by the present invention is used to establish a PureBac system based on Bombyx mori nuclear polyhedrosis virus (BmNPV) through the above-mentioned "high-purity recombinant bacmid preparation system", and the AAV serotype Cap gene expression cassette, Rep gene expression cassette and ITR-GOI three elements are quickly loaded onto the transfer vector of the BmNPV-PureBac system using the Goldengate assembly strategy, and then the competent cells of the BmNPV-PureBac system are transformed to achieve high-purity BmNPV-rAAV recombinant bacmid preparation, laying a foundation for the large-scale and extremely low-cost production of rAAV vectors using silkworm larvae/pupae as bioreactors with Chinese characteristics.
  • BmNPV Bombyx mori nuclear polyhedrosis virus
  • FIG1 is a process for preparing the BmNPV-rAAV recombinant bacmid
  • Fig. 2 is a diagram of the preparation of a loseable auxiliary plasmid and the verification of its lossability in Example 1;
  • FIG3 is a diagram showing the loss of the auxiliary plasmid prepared and verified in Example 1 in PureBac1.0 and PureBac2.0 strains;
  • FIG4 is a diagram of the preparation of a loseable transfer vector and verification of its loseability in Example 2;
  • FIG5 is a diagram showing the loss of AAV transfer vector and auxiliary plasmid after the AAV transfer vector prepared in Example 2 is transformed into PureBac competent cells and recombined;
  • Figure 6 is a schematic diagram of the BmNPV genome transformation in Example 3.
  • FIG7 is a diagram of the establishment of the Bombyx mori baculovirus (BmNPV) PureBac system and the rescue of recombinant BEV in Example 3;
  • BmNPV Bombyx mori baculovirus
  • FIG. 8 is a diagram verifying the compatibility of the BmNPV-PureBac system in Example 3.
  • the transposase provided by the auxiliary plasmid pMON7124 can mediate the transposition of exogenous genes to the mini-attTn7 site of the baculovirus genome, but the auxiliary plasmid can still replicate in DH10 Bac bacteria after the transposition is completed, which inevitably causes the pMON7124 vector to remain in the extracted recombinant bacmid. For this reason, the present application replaces the initial replicon Ori in the auxiliary plasmid with a temperature-sensitive replicon and a reverse screening element in an attempt to control the loss of the auxiliary plasmid.
  • Ts&SMs Helper Construction of a helper plasmid containing a temperature-sensitive replicon and a reverse selection element
  • 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 initiator replicon.
  • the reverse selection element rpsL and the temperature-sensitive replicons pSC101 Ori-rapA and OriV-trfA were fused and introduced into homology arms by PCR, where the rpsL gene and pSC101 Ori-rapA element were derived from the Counter Selection BAC Modificat ion Kit (Gene Bridges, K002), 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 connected by Gibson assembly technology (NEB, E2621L) to obtain the auxiliary plasmid containing rpsL-pSC101 Ori-rapA (nucleotide sequence such as SEQ ID NO.1) and the auxiliary plasmid containing rpsL-OriV-trfA (nucleotide sequence such as SEQ ID NO.2), as shown in Figure 2a.
  • Stbl3 glycerol bacteria containing 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 ⁇ L of bacterial solution was inoculated into 700 ⁇ L 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.
  • the Bombyx mori nuclear polyhedrosis virus genome was isolated from the DH10Bac (BmNPV) Escherichia coli strain (Shanghai Jiachu Biotechnology Co., Ltd., SHBCC D24962) and introduced into MegaX DH10B T1R electrocompetent cells (Thermo Fisher Scientific, C640003) to prepare electrocompetent cells BmDH10B containing the Bombyx mori nuclear polyhedrosis virus genome. Then, the two auxiliary plasmids containing rpsL-pSC101 Ori-rapA and rpsL-OriV-trfA were separated and cloned into the Bombyx mori nuclear polyhedrosis virus genome.
  • helper plasmid containing rpsL-OriV-trfA was preferred and the activation time at 37°C was extended to 6h.
  • the monoclonal strain was obtained using kanamycin/tetracycline dual resistance LB solid medium
  • the monoclonal strain was inoculated in LB liquid medium containing kanamycin/tetracycline dual resistance and expanded at 30°C for 3h
  • 100 ⁇ L of bacterial solution was spread on LB solid medium containing kanamycin/streptomycin dual resistance
  • the Bombyx mori nuclear polyhedrosis virus genome and Ts&SMs Helper plasmid in the colony were identified by PCR respectively.
  • the donor plasmid i.e., transfer vector
  • the transfer vector can recombinate with the baculovirus shuttle vector to form an expression bacmid containing exogenous genes, but 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.
  • recombinant adeno-associated virus rAAV
  • rAAV adeno-associated virus
  • the present invention here takes a transfer vector containing three AAV elements as an example to introduce temperature-sensitive replicons and reverse screening elements into it to demonstrate controllable loss.
  • the rpsL-pSC101 Ori-rapA element is loaded onto the AAV-Rep backbone plasmid T79B00-0-0 in the present invention to construct the plasmid tsT79B00-0-0.
  • T79B00-0-0 was double-digested with restriction endonucleases AgeI and BspQI to obtain a DNA fragment without the Ori initiator replicon, and the rpsL-pSC101 Ori-rapA gene sequence was amplified by PCR using the "auxiliary plasmid containing rpsL-pSC101 Ori-rapA" in Example 1 as a template to introduce the homologous arms, and the two DNA fragments were homologously recombined and connected 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.
  • the loss of the tsT79B00-0-0 plasmid was verified using the method of “Verifying 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, so the tetracycline resistance 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.
  • a method for preparing and using a transfer vector pAAV-Donor in a baculovirus-rAAV production system 1) construct 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) AAV-Core backbone plasmid 2001 is derived from the patent, 3) Cap backbone plasmid 9B00, Core backbone plasmid 2001 and Rep backbone plasmid tsT79B00-0-0 in step (1) are assembled to prepare pAAV-tsDonor plasmid, as shown in Figure 4b.
  • AAV-Cap backbone plasmid 9B00 pBACKBONE_Bm-AAV-Cap-BsaI-SmR, nucleotide sequence such as SEQ ID NO.5
  • Rep backbone plasmid tsT79B00-0-0 contains the temperature-sensitive replicon pSC101Ori-rapA, the 37°C overnight culture condition can still be used in the preparation of the pAAV-tsDonor plasmid.
  • 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 identifying the colony by PCR.
  • 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 Figure 5a, and activated at 37°C for 6 h. 100 ⁇ L of the bacterial solution was spread on LB solid culture plates containing kanamycin/gentamicin/streptomycin/IPTG/X-Gal and cultured at 37°C overnight. White colonies were selected for PCR identification. The results showed that the white colonies originating from the PureBac1.0 and PureBac2.0 plates were all positive recombinants (Figure 5b).
  • Example 3 Modification of the baculovirus genome and establishment of a PureBac line based on Bombyx mori nuclear polyhedrosis virus (BmNPV) System
  • the pRedET plasmid was introduced into the BmDH10B electrocompetent cells prepared in step (2) of Example 1 to prepare electrocompetent cells Bm3.0/pRedET containing the pRedET plasmid and the BmNPV genome.
  • bacteria containing the BmNPV genome (Bm3.1-0) with the Cm R resistance gene inserted were prepared into electroporation competent cells Bm3.1-0/pRedET; 2) After introducing 50 bp homology arms (nucleotide sequence such as SEQ ID NO.7) on both ends of the rpsL-Amp R element by PCR, Bm3.1-0/pRedET competent cells were transformed, and the LacZ ⁇ -Kan in the Bm3.1-0 genome was replaced by Red/ET recombination technology.
  • the BmNPV genome after replacement was named Bm3.1-1, and electroporation competent cells Bm3.1-1/pRedET were prepared; 3) a gene sequence containing homologous arms on both sides of the rpsL-Amp R element (nucleotide sequence as SEQ ID NO.8) was introduced into Bm3.1-1/pRedET competent cells to knock out the rpsL-Amp R element as shown in FIG6c, and the BmNPV genome with the rpsL-Amp R element knocked out in Bm3.1-1 was named Bm3.1-2 as shown in FIG6d, and electroporation competent cells Bm3.1-2/pRedET were prepared; 4) 50 bp homologous arms (nucleotide sequence as SEQ ID NO.9) were introduced at both ends of the rpsL-Kan R element by PCR, and then Bm3.1-2/pRedET competent cells were transformed, and rpsL-Kan was transformed into R
  • the R element was inserted into the ODV-e56 site of the Bm3.1-2 genome as shown in FIG6e, and the BmNPV genome in which the rpsL-Kan R element was inserted into the ODV-e56 site of Bm3.1-2 was named Bm3.1-3, and electroporation competent cells Bm3.1-3/pRedET were prepared; 5) 50 bp homology arms (nucleotide sequence as SEQ ID NO.10) were introduced at both ends of the LaeZ ⁇ gene containing the mini-attTn7 element by PCR, and then the Bm3.1-3/pRedET competent cells were transformed, and the rpsL-Kan R element in the Bm3.1-3 genome was replaced by Red/ET recombination technology as shown in FIG6f, and the BmNPV genome in which the rpsL-Kan R element in Bm3.1-3 was replaced was named Bm3.2 as shown in FIG6g, and the whole genome of Bm3.2 was sequenced (nucleotide sequence
  • the present invention introduces the auxiliary plasmid containing the rpsL-OriV-trfA element into the Bm3.2 competent cells prepared in step (1) to prepare chemical competent cells DH10pureBmBac2.1 (abbreviated as pureBmBac2.1) as shown in Figure 7a.
  • Example 2 AAV Rep (tsT79B00-0-0) and Cap (9B00) backbone plasmids were constructed.
  • 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 frame 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 frames 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.
  • 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.
  • the two pAAV-tsDonor plasmids mentioned above were transformed into pureBmBac2.1 chemically competent cells as shown in Figure 7b. After transposition at 37°C for 6 hours, the cells were spread on LB solid culture plates containing gentamicin/streptomycin/IPTG/X-Gal and cultured at 37°C overnight. Then, white colonies were selected and streaked on LB solid culture plates with gentamicin/streptomycin/IPTG/X-Gal and cultured at 37°C overnight. Colony PCR identified the strain 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, and 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 the newly cultured BmN cells.
  • the BmN cells were observed under a fluorescence microscope and were detected to be illuminated by green fluorescence, indicating that the BmNPV-rAAV recombinant bacmid transfected BmN cells can rescue the recombinant BEV as shown in Figures 7d-e.
  • step (1) Bm3.2/pRedET electrocompetent cells were prepared, 50 bp homology arms (nucleotide sequence such as SEQ ID NO.17) were introduced at both ends of the rpsL-Amp R element by PCR, and then the Bm3.2/pRedET competent cells were transformed, and part of the Bm103 and Bm104 gene sequences in the Bm3.2 genome were replaced by Red/ET recombination technology as shown in Figure 8a.
  • 50 bp homology arms nucleotide sequence such as SEQ ID NO.17
  • the replaced BmNPV genome was named Bm3.3-0, and electrocompetent cells Bm3.3-0/pRedET were prepared; further, a gene sequence containing homology arms on both sides of the rpsL-Amp R element (nucleotide sequence such as SEQ ID NO.18) was introduced into Bm3.3-0/pRedET competent cells to knock out the rpsL-Amp R element as shown in Figure 8b, and the rpsL-Amp in Bm3.3-0 was knocked out.
  • the BmNPV genome containing the R element was named Bm3.3. As shown in FIG8c, bacteria containing the Bm3.3 genome were prepared into electrocompetent cells Bm3.3.
  • the auxiliary 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 RK2-ts -Cap-ITR (GOI) -Rep plasmid prepared in the invention patent "A recombinant bacmid and its preparation method, patent application number: 202310643354.7" was transformed into pureBmBac2.1 and pureBmBac3.1 competent cells as shown in Figure 8e.

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Abstract

提供一种重组杆粒制备系统及BmNPV-rAAV重组杆粒，重组杆粒制备系统包括：a)包含可控丢失的辅助质粒和改造的杆状病毒基因组的DH10pureBac感受态细胞，b)可控丢失的转移载体。所述BmNPV-rAAV重组杆粒为包含重组腺相关病毒Cap基因表达框、核心表达元件ITR-GOI及Rep基因表达框的单一BmNPV杆粒。解决了Bac-to-Bac杆状病毒表达系统中辅助质粒和转移载体的残留问题，且技术流程简单。

Description

一种重组杆粒制备系统及BmNPV-rAAV重组杆粒 技术领域
本发明属于生物技术领域，具体涉及一种重组杆粒制备系统及BmNPV-rAAV重组杆粒。
背景技术
家蚕(Bombyx mori)是一种极具中国特色的重要经济昆虫，一生中要经历卵、幼虫、蛹、蛾(成虫)四种形态。虽然至今养蚕的主要目标是获得高产优质的蚕茧为丝绸工业及其衍生产业提供原料，但事实上家蚕幼虫/蛹也是表达外源蛋白的高效生物反应器。自2005年Motohashi等建立基于家蚕的Bac-to-Bac表达系统以来，已有数百种重组蛋白利用家蚕杆状病毒系统成功表达，广泛涉及疫苗生产、医用药物蛋白等领域。
家蚕杆状病毒(BmNPV)表达系统与已经商业化的苜蓿银纹夜蛾杆状病毒(AcMNPV)Bac-to-Bac系统的工作原理类似，均是基于Tn7转座子的位点特异性转座来简化和增强产生重组杆粒(Bacmid DNA)，进而快速有效地产生重组杆状病毒。然而受限于Bac-to-Bac系统的固有属性，获取的重组杆粒中普遍残留辅助质粒和转移载体，这不可避免地会影响子一代BEV的拯救。BmNPV表达系统作为具有开发价值的真核表达系统之一至今也未得到进一步发展，仍旧无法获得高纯度重组杆粒。虽然申请人前期开发的基于温度敏感元件的策略(一种重组杆粒及其制备方法，专利申请号：202310643354.7)解决了Bac-to-Bac AcMNPV表达系统中辅助质粒和转移载体的残留问题，但该技术流程繁琐且耗时长(30℃培养12h→48h挑取白色重组子→37℃培养16h→37℃过夜培养挑取单菌落→扩大培养→抽提重组杆粒)，因此不可直接平移至BmNPV表达系统，需要进一步改进。
发明内容
针对现有技术的以上缺陷，本发明提供了一种重组杆粒制备系统及BmNPV-rAAV重组杆粒，其目的在于，1)分别在辅助质粒和转移载体中引入温度敏感复制子和反向筛选元件，通过控制温度和反向筛选方式快速获得高纯度重组杆粒——温度敏感型复制子可以控制质粒在宿主中的存在，当温度较低时质粒可以存在宿主中，而温度提高后质粒发生丢失，反向筛选元件可以是链霉素敏感基因rpsL，当存在rpsL元件时DH10B菌株对链霉素敏感，反之该菌株具有链霉素抗性；2)利用Red/ET重组技术将mini-attTn7元件从杆状病毒的基因组的Polh位点移至ODV-E56位点，也可移至任何除了ODV-E56位点之外其他不影响杆状病毒包装的位点，细菌复制调控元件仍置于Polh位置，进一步再敲除非必须基因，从而提高外源蛋白表达量；3)将1)中含有温度敏感复制子和链霉素敏感元件的辅助质粒与2)中改造后的杆状病毒基因组转化MegaX DH10B T1R电转感受态细胞，以制备DH10pureBac感受态细胞，并与1)中制备的转移载体结合使用形成一套无辅助质粒和转移载体残留的新型重组杆粒制备系统，命名为PureBac；4)建立基于家蚕核型多角体病毒(BmNPV)的PureBac系统，将产生AAV所需的三元件(血清型Cap基因、Rep基因及ITR核心表达元件)同时挂载至该系统的转移载体上，进而获得高纯度BmNPV-rAAV重组杆粒。
本发明的具体技术方案如下：
本发明提供一种重组杆粒制备系统，所述重组杆粒制备系统包括：a)包含可控丢失的辅助质粒和改造的杆状病毒基因组的DH10pureBac感受态细胞，b)可控丢失的转移载体；
所述包含可控丢失的辅助质粒和改造的杆状病毒基因组的DH10pureBac感受态细胞的制备方法，包括以下步骤：
(1)制备可控丢失的辅助质粒：将编码Tn7转座酶的辅助质粒中的起始复制子Ori替换成温度敏感复制子和反向筛选元件，得到可控丢失的辅助质粒；
(2)改造杆状病毒基因组：从Bac-to-Bac系统中分离出杆状病毒基因组，利用Red/ET重组技术将基因组中mini-attTn7从Polh位点移至ODV-E56位点且细菌复制调控元件仍置于Polh位置，或者将mini-attTn7移至任何除了ODV-E56位点之外其他不影响杆状病毒包装的位点且细菌复制调控元件仍置于Polh位置，得到改造的杆状病毒基因组；
(3)将步骤(1)可控丢失的辅助质粒和步骤(2)改造的杆状病毒基因组转化至MegaX DH10B T1R感受态细胞中，在30℃条件下制备成包含可控丢失的辅助质粒和改造的杆状病毒基因组的DH10pureBac感受态细胞；
所述可控丢失的转移载体至少包含抗菌素抗性标记、温度敏感复制子、Tn7转座元件及外源蛋白基因表达框。
进一步地，步骤(1)中所述温度敏感复制子选自pSC101或其突变体，或者oriV与trfA基因(235G同义突变)组成的表达框或其突变体；所述温度敏感复制子低温时复制效率较高，随着温度升高复制效率降低甚至被抑制；所述反向筛选元件为反向筛选标记基因；
优选地，所述温度敏感复制子选自oriV与trfA基因(235G同义突变)组成的表达框；
优选地，所述反向筛选元件选自链霉素敏感元件rpsL；
优选地，步骤(1)中所述编码Tn7转座酶的辅助质粒为pMON7124；
优选地，步骤(1)中所述可控丢失的辅助质粒的核苷酸序列如SEQ ID NO.1或SEQ ID NO.2所示；
优选地，步骤(1)中所述可控丢失的辅助质粒的核苷酸序列如SEQ ID NO.2所示。
进一步地，步骤(2)中所述分离的杆状病毒基因组选自BmNPV或AcMNPV；
优选地，所述改造的杆状病毒基因组还包括敲除杆状病毒基因组的非必须基因；
优选地，所述杆状病毒基因组的非必须基因选自Bm103、Bm104、Bm106、Bm114、Ac29-Ac33、Ac126、Ac127、Ac129、Ac137中的一个或多个；
优选地，所述改造的杆状病毒基因组的核苷酸序列如SEQ ID NO.11所示；
优选地，步骤(3)中所述DH10pureBac感受态细胞为化学感受态细胞或电转感受态细胞。
进一步地，可控丢失的转移载体中所述外源蛋白基因表达框的启动子选自Pp10、Pph、Pp6.9、Pgp64、Pie-1及其相互嵌合或启动子前添加增强子形成的组成型启动子中的一种或几种；
优选地，可控丢失的转移载体中所述温度敏感复制子选自oriV与trfA基因组成的表达框或pSC101元件；
优选地，可控丢失的转移载体中所述温度敏感复制子选自pSC101元件，且所述可控丢失的转移载体同时包含反向筛选元件；值得注意地，所述温度敏感复制子若为pSC101则需要与反向筛选元件结合使用，若为oriV与trfA基因(235G同义突变)可单独使用，与反向筛选元件结合使用效率更高；
优选地，所述反向筛选元件为反向筛选标记基因；
优选地，所述反向筛选元件选自链霉素敏感元件rpsL。
本发明还提供一种重组杆粒制备方法，所述制备方法包括如下步骤：将上述可控丢失的转移载体转化至上述DH10pureBac感受态细胞，经37℃活化，筛选阳性单克隆，即得到无辅助质粒和转移载体残留的高纯度重组杆粒。
本发明还提供一种BmNPV-rAAV重组杆粒制备系统，所述BmNPV-rAAV重组杆粒制备系统包括：I)所述包含可控丢失的辅助质粒和改造的杆状病毒基因组的DH10pureBac感受态细胞，其中杆状病毒基因组为家蚕核型多角体病毒基因组；II)所述可控丢失的转移载体，其中可控丢失的转移载体中外源蛋白基因包含AAV的Cap基因、Rep基因及ITR-GOI元件。
进一步地，所述DH10pureBac感受态细胞选自DH10pureBmBac2.1感受态细胞或DH10pureBmBac3.1感受态细胞；
所述DH10pureBmBac2.1感受态细胞中改造的家蚕核型多角体病毒基因组为Bm3.2版本，其家蚕核型多角体病毒基因组中mini-attTn7从Polh位点移至ODV-E56位点，细菌复制调控元件仍置于Polh位置；
所述DH10pureBmBac3.1感受态细胞中改造的家蚕核型多角体病毒基因组为Bm3.3版本，其家蚕核型多角体病毒基因组中mini-attTn7从Polh位点移至ODV-E56位点，细菌复制调控元件仍置于Polh位置，且敲除Bm103、Bm104基因；
优选地，所述Bm3.2的核苷酸序列如SEQ ID NO.11所示。
进一步地，所述AAV的血清型选自AAV1-13及其衍生血清型；
优选地，所述AAV的Cap基因和Rep基因经修饰后分别置于不同的杆状病毒启动子与转录和转录后调控序列之间；
优选地，所述杆状病毒启动子来源于AcMNPV或BmNPV，选自Pp10、Pph、Pp6.9、Pgp64、Pie-1及其相互嵌合或启动子前添加增强子形成的组成型启动子中的一种；
优选地，所述转录和转录后调控序列选自TK polyA或SV40 polyA；
优选地，将ITR-GOI元件、Cap基因表达框、Rep基因表达框分别置于三个质粒上，根据AAV生产需求经一步Golden gate自由组装成可控丢失的转移载体；
优选地，所述ITR-GOI元件置于AAV Cap基因表达框与Rep基因表达框之间；
优选地，所述包含AAV的Cap基因、Rep基因及ITR-GOI元件的可控丢失的转移载体的核苷酸序列如SEQ ID NO.15或SEQ ID NO.16所示。
本发明还提供一种BmNPV-rAAV重组杆粒的制备方法，将对应的可控丢失的转移载体转化至对应的DH10pureBac感受态细胞中，经37℃活化，筛选阳性单克隆，即得到无辅助质粒和转移载体残留的高纯度BmNPV-rAAV重组杆粒；
优选地，所述37℃活化时间为6小时。
本发明还提供上述BmNPV-rAAV重组杆粒，所述BmNPV-rAAV重组杆粒为包含重组腺相关病毒Cap基因表达框、核心表达元件ITR-GOI及Rep基因表达框的单一BmNPV杆粒。
与现有技术相比，能够取得以下有益效果：
(1)本发明提供的高纯度重组杆粒制备系统，通过从Bac-to-Bac杆状病毒表达系统中分离出杆状病毒基因组，利用Red/ET重组技术将mini-attTn7元件由Polh位点移至ODV-E56位点，也可移至任何除了ODV-E56位点之外其他不影响杆状病毒包装的位点，进一步再敲除杆状病毒非必须基因，并整合在辅助质粒与转移载体分别引入温度敏感复制子和反向筛选元件策略， 实现了无辅助质粒和转移载体残留的重组杆粒的简便、快速制备，从而提高了重组杆粒的质量和制备效率
(2)本发明提供的BmNPV-rAAV重组杆粒，通过上述“高纯度重组杆粒制备系统”建立了基于家蚕核型多角体病毒(BmNPV)的PureBac系统，并利用Goldengate组装策略将AAV的血清型Cap基因表达框、Rep基因表达框及ITR-GOI三元件快速负载于BmNPV-PureBac系统的转移载体上，进而转化BmNPV-PureBac系统的感受态细胞实现了高纯度BmNPV-rAAV重组杆粒制备，为具有中国特色的家蚕幼虫/蛹作为生物反应器开展rAAV载体的大规模、极低成本生产奠定基础。
附图说明
图1是BmNPV-rAAV重组杆粒的制备流程；
图2是实施例1中制备可丢失的辅助质粒并进行丢失性验证；
图3是实施例1中验证制备的辅助质粒在PureBac1.0和PureBac2.0菌株中的丢失情况；
图4是实施例2中制备可丢失的转移载体并进行丢失性验证；
图5是实施例2中验证制备的AAV转移载体转化PureBac感受态细胞发生重组后AAV转移载体和辅助质粒的丢失情况；
图6是实施例3中BmNPV基因组改造示意图；
图7是实施例3中建立家蚕杆状病毒(BmNPV)PureBac系统及拯救重组BEV；
图8是实施例3中验证BmNPV-PureBac系统的兼容性。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅用以解释本发明，并不用于限定本发明。
实施例1制备可丢失的辅助质粒
辅助质粒pMON7124提供的转座酶可介导外源基因转座到杆状病毒基因组mini-attTn7位点，但在完成转座后辅助质粒仍可在DH10 Bac细菌中复制，这不可避免地造成抽提的重组杆粒中残留pMON7124载体。为此，本申请将辅助质粒中的起始复制子Ori替换成温度敏感复制子和反向筛选元件试图使辅助质粒可控丢失。
(1)构建包含温度敏感复制子和反向筛选元件的辅助质粒(Ts&SMs Helper)；
从DH10Bac感受态细胞(赛默飞世尔科技公司，10361012)中分离出pMON7124质粒，并使用高保真PCR酶(Takara，R050A)扩增pMON7124载体以去除Ori起始复制子；通过PCR分别将反向筛选元件rpsL和温度敏感复制子pSC101 Ori-rapA、OriV-trfA融合并引入同源臂，其中rpsL基因和pSC101 Ori-rapA元件来源于Counter Selection BAC Modification Kit(Gene Bridges，K002)，OriV-trfA元件来源于专利“一种重组杆粒及其制备方法，专利申请号：202310643354.7”；利用Gibson组装技术(NEB公司，E2621L)分别连接上述两个线性DNA片段获得含有rpsL-pSC101 Ori-rapA的辅助质粒(核苷酸序列如SEQ ID NO.1)、rpsL-OriV-trfA的辅助质粒(核苷酸序列如SEQ ID NO.2)，如附图2a所示。
(2)验证Ts&SMs Helper质粒的丢失情况；
将含有Ts&SMs Helper质粒的Stbl3甘油菌分别涂布在四环素抗性的LB固体培养基上，30℃培养过夜，挑选单克隆接种在含有四环素抗性的LB液体培养基中30℃扩大培养，3h后再取200μL菌液接种在700μL无抗的LB培养中37℃培养4h试图使细菌中的辅助质粒丢失，之后再取100μL菌液分别涂布在四环素抗性、链霉素抗性以及四环素/链霉素双抗性的LB固体培养基中37℃培养过夜，如附图2b所示，通过观察细菌在平板上的生长情况、菌落PCR鉴定判断Ts&SMs Helper质粒的丢失情况及能否筛选出丢失Ts&SMs Helper质粒的菌株，如附图2c所示。结果显示，菌液涂布在四环素/链霉素双抗性的LB固体培养基上时无菌落产生，而在四环素抗性和链霉素抗性的LB固体培养基上均可生长；分别挑选四环素抗性平板、链霉素抗性平板中的菌株进行PCR鉴定，观察到链霉素平板来源的菌株中无Ts&SMs Helper质粒信号，而四环素平板来源的菌株则可以检测到，这表明两个Ts&SMs Helper质粒均可丢失而且丢失Ts&SMs Helper质粒的菌株可以被筛选出来。
进一步，从DH10Bac(BmNPV)大肠杆菌菌株(上海嘉楚生物工程有限公司，SHBCC D24962)中分离出家蚕核型多角体病毒基因组，并导入MegaX DH10B T1R电转感受态细胞(赛默飞世尔科技公司，C640003)中制备成包含家蚕核型多角体病毒基因组的电转感受态细胞BmDH10B，接着将上述两个含有rpsL-pSC101 Ori-rapA的辅助质粒、rpsL-OriV-trfA的辅助质粒分别导入BmDH10B中制备成PureBac1.0和PureBac2.0电转感受态细胞，如附图3a-b所示，之后通过菌落PCR分别鉴定家蚕核型多角体病毒基因组和Ts&SMs Helper质粒是否存在于PureBac1.0和PureBac2.0电转感受态细胞中，如附图3c，e所示，菌落PCR显示PureBac1.0和PureBac2.0电转感受态细胞中均包含BmNPV基因组和Ts&SMs Helper质粒。利用附图2b所示的方法验证PureBac1.0和PureBac2.0菌株中Ts&SMs Helper质粒的丢失情况，将PureBac1.0和PureBac2.0菌株在37℃活化4h并分别涂布在含有卡那霉素/链霉素双抗性的LB固体培养基37℃培养过夜，挑选单克隆菌落PCR显示，大多数PureBac1.0菌株中含有rpsL-pSC101 Ori-rapA的辅助质粒可被检测到(附图3d)，而PureBac2.0菌株中只有很少部分含有rpsL-OriV-trfA的辅助质粒被检测到(附图3f)，这表明含有rpsL-OriV-trfA的辅助质粒在37℃的丢失效率要高于含有rpsL-pSC101 Ori-rapA元件的辅助质粒，因此在后续实验中优先选择使用含有rpsL-OriV-trfA的辅助质粒并将37℃活化时间延长至6h，值得注意地：1)利用卡那霉素/四环素双抗性LB固体培养基获取单克隆菌株，2)单克隆株接种在含有卡那霉素/四环素双抗性的LB液体培养基中30℃扩大培养3h，3)取100μL菌液涂布在含有卡那霉素/链霉素双抗性的LB固体培养基上，4)对菌落中家蚕核型多角体病毒基因组和Ts&SMs Helper质粒分别进行PCR鉴定。
实施例2制备可丢失的转移载体
在Tn7转座酶作用下，供体质粒(即转移载体)可与杆状病毒穿梭载体发生重组形成一种包含外源基因的表达杆粒，但转化DH10 Bac细菌未发生重组的转移载体仍可复制，这造成抽提的重组杆粒中也残留转移载体。此外，重组腺相关病毒(Recombinant Adeno-associated Virus，rAAV)作为一种高效的基因递送载体已被证实在临床前和临床实验中安全有效，而基于杆状病毒的rAAV生产系统因其具有可扩展性、低成本及可预测的生物安全性而备受关注。综上，这里本发明以包含AAV三元件的转移载体为例将温度敏感复制子和反向筛选元件引入其中展示可控丢失。
(1)将rpsL-pSC101 Ori-rapA元件负载至AAV-Rep骨架质粒上并进行丢失验证；
根据发明专利“一种杆状病毒-rAAV生产系统中转移载体pAAV-Donor的制备方法及应用”构建含有家蚕核型多角体病毒ph启动子的AAV-Rep骨架质粒T79B00-0-0(pBACKBONE_Bm-AAV-Rep-BsaI-ccdB-Gen，核苷酸序列如SEQ ID NO.3)，考虑到含有pSC101 Ori-rapA元件的温度敏感型质粒在37℃培养时丢失效率较低而细菌在37℃的生长速度也比30℃快，本发明将rpsL-pSC101 Ori-rapA元件负载至AAV-Rep骨架质粒T79B00-0-0上构建质粒tsT79B00-0-0。具体地，利用限制性内切酶AgeI和BspQI双酶切T79B00-0-0获得去除Ori起始复制子的DNA片段，以实施例1中“含有rpsL-pSC101 Ori-rapA的辅助质粒”为模板通过PCR扩增rpsL-pSC101 Ori-rapA基因序列引入同源臂，将两个DNA片段同源重组连接并转化至DB3.1化学感受态细胞中筛选阳性克隆以获得含有rpsL-pSC101 Ori-rapA的AAV-Rep骨架质粒tsT79B00-0-0(pBACKBONE_tsBm-AAV-Rep-BsaI-ccdB-Gen，核苷酸序列如SEQ ID NO.4)，如附图4a所示。
利用实施例1中“验证Ts&SMs Helper质粒”的方法验证tsT79B00-0-0质粒的丢失情况，与Ts&SMs Helper质粒不同的是tsT79B00-0-0质粒的抗菌素抗性标记为庆大霉素，因此需要将四环素抗性更换成庆大霉素抗性，如附图4c所示，同样地通过观察细菌在平板上的生长情况、菌落PCR鉴定判断tsT79B00-0-0质粒的丢失情况。结果显示，菌株涂布在庆大霉素/链霉素双抗性的LB固体培养基时无菌落产生，而在庆大霉素抗性和链霉素抗性的LB固体培养基上均可生长；分别挑选庆大霉素抗性平板、链霉素抗性平板中的菌株进行PCR鉴定，观察到链霉素平板来源的菌株中无tsT79B00-0-0质粒信号，而庆大霉素平板来源的菌株则可以检测到(附图4d)，这表明tsT79B00-0-0质粒可丢失而且丢失tsT79B00-0-0质粒的菌株可以被筛选出来。
(2)制备包含AAV三元件的转移载体(pAAV-tsDonor)进行丢失验证；
根据发明专利“一种杆状病毒-rAAV生产系统中转移载体pAAV-Donor的制备方法及应用”：1)构建含有家蚕核型多角体病毒p10启动子的AAV-Cap骨架质粒9B00(pBACKBONE_Bm-AAV-Cap-BsaI-SmR，核苷酸序列如SEQ ID NO.5)，2)AAV-Core骨架质粒2001来源于该专利，3)将Cap骨架质粒9B00、Core骨架质粒2001与步骤(1)中Rep骨架质粒tsT79B00-0-0组装制备pAAV-tsDonor质粒，如附图4b所示。为了更高效地获取包含AAV三元件的转移载体，取Goldengate组装后的产物5μl转化stbl3化学感受态细胞分别在30℃和37℃过夜培养，经37℃过夜培养平板上的单菌落远大于在30℃过夜培养的单菌落，挑选37℃平板、30℃平板中的单克隆菌株进行PCR鉴定，观察到不论单克隆来源于37℃平板还是30℃平板均可鉴定出阳性单克隆(附图4e-f)，并分别各挑选两个阳性克隆菌株扩大培养、提取质粒送测序显示正确，这表明虽然Rep骨架质粒tsT79B00-0-0中包含温度敏感复制子pSC101Ori-rapA，但在制备pAAV-tsDonor质粒过程中仍可使用37℃过夜培养条件。
进一步，按照步骤(1)中如附图4c所示的方法对上述制备pAAV-tsDonor质粒进行丢失验证，通过观察细菌在平板上的生长情况、菌落PCR鉴定判断pAAV-tsDonor质粒的丢失情况。结果显示，菌株涂布在庆大霉素/链霉素双抗性的LB固体培养基时无菌落产生，而在庆大霉素抗性和链霉素抗性的LB固体培养基上均可生长(附图4g)；分别挑选庆大霉素抗性平板、链霉素抗性平板中的菌株进行PCR鉴定，观察到链霉素平板来源的菌株中无pAAV-tsDonor质粒信号，而庆大霉素平板来源的菌株则可以检测到(附图4h)，这表明pAAV-tsDonor质粒可丢失而且丢失pAAV-tsDonor质粒的菌株可以被筛选出来。
(3)pAAV-tsDonor质粒转化PureBac感受态细胞发生重组后对pAAV-tsDonor和Ts&SMs Helper质粒进行丢失验证；
将步骤(2)制备的pAAV-tsDonor质粒分别转化实施例1中步骤(2)制备的PureBac1.0和PureBac2.0电转感受态细胞，如附图5a所示，37℃活化6h，取100μL菌液分别涂布在含有卡那霉素/庆大霉素/链霉素/IPTG/X-Gal LB固体培养板上37℃培养过夜，挑选白色菌落进行PCR鉴定，结果显示源于PureBac1.0和PureBac2.0平板的白色菌落均为阳性重组子(附图5b)。接着各挑选两个阳性重组子复涂布于卡那霉素/庆大霉素/链霉素/IPTG/X-Gal LB固体培养板上，经37℃过夜培养后挑选白色菌落分别对BmNPV重组杆粒、pAAV-tsDonor质粒及Ts&SMs Helper质粒进行PCR鉴定，验证pAAV-tsDonor和Ts&SMs Helper质粒的丢失情况，结果显示，来源PureBac1.0平板的单克隆菌落中有1个菌落可检测到pAAV-tsDonor和含有rpsL-pSC101 Ori-rapA元件的辅助质粒信号(附图5c)，而来源PureBac2.0平板的单克隆菌落中仅含有BmNPV重组杆粒(附图5d)，这表明含有pSC101 Ori-rapA元件的pAAV-tsDonor质粒与含有rpsL-OriV-trfA的辅助质粒组合使用可获得无转移载体和辅助质粒残留的高纯度重组杆粒。
实施例3改造杆状病毒基因组并建立基于家蚕核型多角体病毒(BmNPV)的PureBac系 统
在杆状病毒非必须基因的不同基因座插入外源基因序列时，外源蛋白的表达丰度不尽相同。考虑到1)Gorben P等研究表明，将mini-attTn7移至AcMNPV基因组的ODV-E56(pif-5)位点可介导更高水平的外源蛋白表达且重组BEV在连续传代中更稳定(Gorben P等，2020，Viruses，12，1448)，2)虽然家蚕核型多角体病毒(BmNPV)不同于AcMNPV，但两者的基因组同源性高达90％，综上本发明将mini-attTn7从BmNPV基因组的Polh位点移至ODV-E56位点。
(1)改造BmNPV基因组；
按照“Counter Selection BAC Modification Kit”说明书，将pRedET质粒导入实施例1步骤(2)制备的BmDH10B电转感受态细胞中，制备成含有pRedET质粒和BmNPV基因组的电转感受态细胞Bm3.0/pRedET。1)在氯霉素抗性基因(CmR，源自pBeloBAC11载体，天津庄盟生物科技有限公司，ZK130)两侧通过PCR引入50bp的同源臂(核苷酸序列如SEQ ID NO.6)后转化Bm3.0/pRedET感受态细胞，利用Red/ET重组技术将CmR插入lef2基因与mini-F ori元件之间如附图6a所示，包含插入CmR抗性基因的BmNPV基因组(Bm3.1-0)的细菌被制备成电转感受态细胞Bm3.1-0/pRedET；2)在rpsL-AmpR元件两端通过PCR引入50bp的同源臂(核苷酸序列如SEQ ID NO.7)后转化Bm3.1-0/pRedET感受态细胞，利用Red/ET重组技术替换Bm3.1-0基因组中的LacZα-KanR元件如附图6b所示，完成替换的BmNPV基因组命名为Bm3.1-1，并制备电转感受态细胞Bm3.1-1/pRedET；3)将一段含有rpsL-AmpR元件两侧同源臂的基因序列(核苷酸序列如SEQ ID NO.8)导入Bm3.1-1/pRedET感受态细胞中敲除rpsL-AmpR元件如附图6c所示，敲除Bm3.1-1中rpsL-AmpR元件的BmNPV基因组命名为Bm3.1-2如附图6d所示，制备电转感受态细胞Bm3.1-2/pRedET；4)在rpsL-KanR元件两端通过PCR引入50bp的同源臂(核苷酸序列如SEQ ID NO.9)后转化Bm3.1-2/pRedET感受态细胞，利用Red/ET重组技术将rpsL-KanR元件插入Bm3.1-2基因组的ODV-e56位点如附图6e所示，Bm3.1-2中ODV-e56位点插入rpsL-KanR元件的BmNPV基因组命名为Bm3.1-3，并制备电转感受态细胞Bm3.1-3/pRedET；5)在含有mini-attTn7元件的LaeZα基因两端通过PCR引入50bp的同源臂(核苷酸序列如SEQ ID NO.10)后转化Bm3.1-3/pRedET感受态细胞，利用Red/ET重组技术替换Bm3.1-3基因组中的rpsL-KanR元件如附图6f所示，完成Bm3.1-3中rpsL-KanR元件替换的BmNPV基因组命名为Bm3.2如附图6g所示，对Bm3.2进行全基因组测序(核苷酸序列如SEQ ID NO.11，生工生物工程(上海)股份有限公司)，并且包含Bm3.2基因组的细菌被制备成电转感受态细胞Bm3.2，进一步将pRedET质粒导入Bm3.2感受态细胞中制备成电转感受态细胞Bm3.2/pRedET。
(2)建立家蚕杆状病毒(BmNPV)PureBac系统；
由实施例1可知与rpsL-pSC101 Ori-rapA元件相比，含有rpsL-OriV-trfA元件的辅助质粒丢失效果最佳，因此本发明将含有rpsL-OriV-trfA元件的辅助质粒导入步骤(1)制备的Bm3.2感受态细胞中，制备成化学感受态细胞DH10pureBmBac2.1(简称pureBmBac2.1)如附图7a所示。
实施例2中构建了AAV Rep(tsT79B00-0-0)和Cap(9B00)骨架质粒，根据发明专利“一种杆状病毒-rAAV生产系统中转移载体pAAV-Donor的制备方法及应用”，1)经修饰的AAV2-Rep基因表达框插入tsT79B00-0-0获得AAV-Rep质粒tsT79B00-0-4(核苷酸序列如SEQ ID NO.12)，2)修饰后的AAV2和AAV9 Cap基因表达框分别插入9B00获得AAV-Cap质粒9B02和9B09(核苷酸序列如SEQ ID NO.13和SEQ ID NO.14)；3)使用该专利中的AAV-Core质粒2006。组装tsT79B00-0-4+9B02+2006形成tsT79B02-2006-4(核苷酸序列如SEQ ID NO.15)、tsT79B00-0-4+9B09+2006形成tsT79B09-2006-4(核苷酸序列如SEQ ID NO.16)即pAAV-tsDonor。
将上述两个pAAV-tsDonor质粒分别转化pureBmBac2.1化学感受态细胞如附图7b所示，在37℃经6h转座后涂布在含有庆大霉素/链霉素/IPTG/X-Gal LB固体培养板上37℃培养过夜，之后挑选白色菌落在庆大霉素/链霉素/IPTG/X-Gal LB固体培养板上划线并37℃过夜培养，菌落PCR鉴定出仅含有BmNPV-rAAV重组杆粒的菌株如附图7c所示。提取BmNPV-rAAV重组杆粒并转染至BmN细胞(ATCC，CRL-8910)，结果显示，在转染第4天时绿色荧光最强，其中转染穿梭载体Bm3.2-rAAV2-CMV-eGFP-hGH的荧光强度高于Bm3.2-rAAV9-CMV-eGFP-hGH(附图7d)，进一步取转染BmNPV-rAAV重组杆粒的BmN细胞上清液加入到新培养的BmN细胞中，4天后荧光显微镜进行观察，检测到BmN细胞被绿色荧光点亮，这表明BmNPV-rAAV重组杆粒转染BmN细胞可以拯救出重组BEV如附图7d-e所示。
(3)验证BmNPV-PureBac系统的兼容性；
步骤(1)中制备了Bm3.2/pRedET电转感受态细胞，在rpsL-AmpR元件两端通过PCR引入50bp的同源臂(核苷酸序列如SEQ ID NO.17)后转化Bm3.2/pRedET感受态细胞，利用Red/ET重组技术替换Bm3.2基因组中的部分Bm103和Bm104基因序列如附图8a所示，完成替换的BmNPV基因组命名为Bm3.3-0，并制备电转感受态细胞Bm3.3-0/pRedET；进一步，将一段含有rpsL-AmpR元件两侧同源臂的基因序列(核苷酸序列如SEQ ID NO.18)导入Bm3.3-0/pRedET感受态细胞中敲除rpsL-AmpR元件如附图8b所示，敲除Bm3.3-0中rpsL-AmpR元件的BmNPV基因组命名为Bm3.3如附图8c所示，包含Bm3.3基因组的细菌被制备成电转感受态细胞Bm3.3。
含有rpsL-OriV-trfA元件的辅助质粒导入制备的Bm3.3感受态细胞中，制备成化学感受态细胞DH10pureBmBac3.1(简称pureBmBac3.1)如附图8d所示。将发明专利“一种重组杆粒及其制备方法，专利申请号：202310643354.7”制备的pFDRK2-ts-Cap-ITR(GOI)-Rep质粒分别转化pureBmBac2.1和pureBmBac3.1感受态细胞如附图8e所示，37℃经6h转座后涂布在含有氯霉素/庆大霉素/链霉素/IPTG/X-Gal LB固体培养板上37℃培养过夜，观察到清晰可见的白色单克隆菌落如附图8f所示，挑选白色菌落分别对BmNPV重组杆粒、pFDRK2-ts-Cap-ITR(GOI)-Rep和Ts&SMs Helper质粒中共有的OriV-trfA元件进行PCR鉴定，如附图8g所示。结果显示，pureBmBac2.1平板来源的1个菌株中有Bm3.2重组杆粒信号、无AAV转移质粒和辅助质粒信号，而pureBmBac3.1平板来源的菌株有3个阳性克隆，进一步抽提重组杆粒并通过PCR插入的AAV Cap基因表达框、核心表达元件ITR-GOI及Rep基因表达框送测序显示结果正确，这表明建立的基于家蚕核型多角体病毒的BmNPV-PureBac系统具有兼容性。

Claims (24)

  1. 一种重组杆粒制备系统，其特征在于，所述重组杆粒制备系统包括：a)包含可控丢失的辅助质粒和改造的杆状病毒基因组的DH10pureBac感受态细胞，b)可控丢失的转移载体；
    所述包含可控丢失的辅助质粒和改造的杆状病毒基因组的DH10pureBac感受态细胞的制备方法，包括以下步骤：
    (1)制备可控丢失的辅助质粒：将编码Tn7转座酶的辅助质粒中的起始复制子Ori替换成温度敏感复制子和反向筛选元件，得到可控丢失的辅助质粒；
    (2)改造杆状病毒基因组：从Bac-to-Bac系统中分离出杆状病毒基因组，利用Red/ET重组技术将基因组中mini-attTn7从Polh位点移至ODV-E56位点且细菌复制调控元件仍置于Polh位置，或者将mini-attTn7移至任何除了ODV-E56位点之外其他不影响杆状病毒包装的位点且细菌复制调控元件仍置于Polh位置，得到改造的杆状病毒基因组；
    (3)将步骤(1)可控丢失的辅助质粒和步骤(2)改造的杆状病毒基因组转化至MegaX DH10B T1R感受态细胞中，在30℃条件下制备成包含可控丢失的辅助质粒和改造的杆状病毒基因组的DH10pureBac感受态细胞；
    所述可控丢失的转移载体至少包含抗菌素抗性标记、温度敏感复制子、Tn7转座元件及外源蛋白基因表达框。
  2. 如权利要求1所述的重组杆粒制备系统，其特征在于，步骤(1)中所述温度敏感复制子选自pSC101或其突变体，或者oriV与trfA基因组成的表达框或其突变体；所述反向筛选元件为反向筛选标记基因。
  3. 如权利要求1所述的重组杆粒制备系统，其特征在于，步骤(1)中所述温度敏感复制子选自oriV与trfA基因组成的表达框；
    所述反向筛选元件选自链霉素敏感元件rpsL；
  4. 如权利要求1所述的重组杆粒制备系统，其特征在于，步骤(1)中所述编码Tn7转座酶的辅助质粒为pMON7124。
  5. 如权利要求1所述的重组杆粒制备系统，其特征在于，步骤(1)中所述可控丢失的辅助质粒的核苷酸序列如SEQ ID NO.1或SEQ ID NO.2所示。
  6. 如权利要求1所述的重组杆粒制备系统，其特征在于，步骤(2)中所述分离的杆状病毒基因组选自BmNPV或AcMNPV。
  7. 如权利要求1所述的重组杆粒制备系统，其特征在于，所述改造的杆状病毒基因组还包括敲除杆状病毒基因组的非必须基因。
  8. 如权利要求7所述的重组杆粒制备系统，其特征在于，所述杆状病毒基因组的非必须基因选自Bm103、Bm104、Bm106、Bm114、Ac29-Ac33、Ac126、Ac127、Ac129、Ac137中的一个或多个。
  9. 如权利要求1所述的重组杆粒制备系统，其特征在于，所述改造的杆状病毒基因组的核苷酸序列如SEQ ID NO.11所示。
  10. 如权利要求1所述的重组杆粒制备系统，其特征在于，步骤(3)中所述DH10pureBac感受态细胞为化学感受态细胞或电转感受态细胞。
  11. 如权利要求1所述的重组杆粒制备系统，其特征在于，可控丢失的转移载体中所述外源蛋白基因表达框的启动子选自Pp10、Pph、Pp6.9、Pgp64、Pie-1及其相互嵌合或启动子前添加增强子形成的组成型启动子中的一种或几种；
    可控丢失的转移载体中所述温度敏感复制子选自oriV与trfA基因组成的表达框或pSC101元件；
    所述反向筛选元件为反向筛选标记基因。
  12. 如权利要求1所述的重组杆粒制备系统，其特征在于，可控丢失的转移载体中所述温度敏感复制子选自pSC101元件，且所述可控丢失的转移载体同时包含反向筛选元件；
    所述反向筛选元件选自链霉素敏感元件rpsL。
  13. 一种重组杆粒制备方法，其特征在于，所述制备方法包括如下步骤：将权利要求1所述可控丢失的转移载体转化至权利要求1所述DH10pureBac感受态细胞，经37℃活化，筛选阳性单克隆，即得到无辅助质粒和转移载体残留的高纯度重组杆粒。
  14. 一种BmNPV-rAAV重组杆粒制备系统，其特征在于，所述BmNPV-rAAV重组杆粒制备系统包括：I)权利要求1所述包含可控丢失的辅助质粒和改造的杆状病毒基因组的DH10pureBac感受态细胞，其中杆状病毒基因组为家蚕核型多角体病毒基因组；II)权利要求1所述可控丢失的转移载体，其中可控丢失的转移载体中外源蛋白基因包含AAV的Cap基因、Rep基因及ITR-GOI元件。
  15. 如权利要求14所述的BmNPV-rAAV重组杆粒制备系统，其特征在于，所述DH10pureBac感受态细胞选自DH10pureBmBac2.1感受态细胞或DH10pureBmBac3.1感受态细胞；
    所述DH10pureBmBac2.1感受态细胞中改造的家蚕核型多角体病毒基因组为Bm3.2版本，其家蚕核型多角体病毒基因组中mini-attTn7从Polh位点移至ODV-E56位点，细菌复制调控元件仍置于Polh位置；
    所述DH10pureBmBac3.1感受态细胞中改造的家蚕核型多角体病毒基因组为Bm3.3版本，其家蚕核型多角体病毒基因组中mini-attTn7从Polh位点移至ODV-E56位点，细菌复制调控元件仍置于Polh位置，且敲除Bm103、Bm104基因。
  16. 如权利要求15所述的BmNPV-rAAV重组杆粒制备系统，其特征在于，所述Bm3.2的核苷酸序列如SEQ ID NO.11所示。
  17. 如权利要求14所述的BmNPV-rAAV重组杆粒制备系统，其特征在于，所述AAV的血清型选自AAV1-13及其衍生血清型。
  18. 如权利要求14所述的BmNPV-rAAV重组杆粒制备系统，其特征在于，所述AAV的Cap基因和Rep基因经修饰后分别置于不同的杆状病毒启动子与转录和转录后调控序列之间；
    所述杆状病毒启动子来源于AcMNPV或BmNPV，选自Pp10、Pph、Pp6.9、Pgp64、Pie-1及其相互嵌合或启动子前添加增强子形成的组成型启动子中的一种或几种；
    所述转录和转录后调控序列选自TK polyA或SV40 polyA。
  19. 如权利要求14所述的BmNPV-rAAV重组杆粒制备系统，其特征在于，将ITR-GOI元件、Cap基因表达框、Rep基因表达框分别置于三个质粒上，根据AAV生产需求经一步Golden gate自由组装成可控丢失的转移载体。
  20. 如权利要求14所述的BmNPV-rAAV重组杆粒制备系统，其特征在于，所述ITR-GOI元件置于AAV Cap基因表达框与Rep基因表达框之间。
  21. 如权利要求14所述的BmNPV-rAAV重组杆粒制备系统，其特征在于，所述包含AAV的Cap基因、Rep基因及ITR-GOI元件的可控丢失的转移载体的核苷酸序列如SEQ ID NO.15或SEQ ID NO.16所示。
  22. 一种BmNPV-rAAV重组杆粒的制备方法，其特征在于，将权利要求14所述可控丢失的转移载体转化至权利要求14所述DH10pureBac感受态细胞中，经37℃活化，筛选阳性单克隆，即得到无辅助质粒和转移载体残留的高纯度BmNPV-rAAV重组杆粒；
  23. 如权利要求22所述的制备方法，其特征在于，所述37℃活化时间为6小时。
  24. 权利要求22所述制备方法制备得到的BmNPV-rAAV重组杆粒，其特征在于，所述BmNPV-rAAV重组杆粒为包含重组腺相关病毒Cap基因表达框、核心表达元件ITR-GOI及Rep基因表达框的单一BmNPV杆粒。
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