Novel helper plasmid for producing recombinant adeno-associated virus as well as construction method and application of novel helper plasmid

Abstract

The invention discloses a novel helper plasmid for producing a recombinant adeno-associated virus as well as a construction method and application of the novel helper plasmid. The novel helper plasmid is constructed by inserting a UL12 element of an I-type herpes simplex virus HSV-1 and an NS2 element of a human bola virus HBoV into a basic pHelper plasmid, the constructed novel helper plasmid UL12-NS2-pHelper can remarkably improve the titer of rAAV produced by HEK293 cells, the proportion of solid rAAV is increased, immunoreaction and toxicity caused by empty capsid can be reduced, and the helper plasmid can be used for preparing the rAAV. The treatment dosage is reduced, and the curative effect and safety are improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of gene and cell therapy drug carriers, and specifically relates to a novel auxiliary plasmid for producing recombinant adeno-associated virus and a construction method thereof. Background Art

[0002] Recombinant adeno-associated virus (rAAV) is an essential vector in gene therapy. It is valued for its low immunogenicity and ability to conduct proliferating cells and quiescent cells, thereby ensuring stable long-term gene expression. rAAV exhibits a wide range of tissue tropism and can accurately deliver genes to different tissues, including the central nervous system, liver, and skeletal muscle. Its non-pathogenic nature and site-specific genomic integration further improve safety. Transient transfection (abbreviated as "TT transient transfection") of HEK293 cells using a helper virus-free system is currently the mainstream large-scale rAAV production method. Among them, the pGOI plasmid carries the therapeutic gene, the pRep-Cap plasmid expresses the Rep protein and capsid protein required for rAAV production, and the pHelper plasmid carries the minimum replication unit separated from the helper virus. However, the current TT transient transfection production efficiency is limited by the plasmid transfection method and cell state, resulting in low rAAV yield and a high proportion of empty shell viruses (usually more than 80% are empty shell viruses), which poses great challenges to downstream virus purification and empty capsid removal. In addition, the high cost of plasmid preparation and transfection reagents makes this system economically inefficient for large-scale production, especially in meeting clinical-grade purity and endotoxin-free standards. Therefore, improving the existing plasmid structure and production model to more efficiently achieve high-quality AAV production is becoming a key direction to meet clinical needs. Summary of the invention

[0003] In view of the deficiencies of the prior art, the present invention provides a novel auxiliary plasmid for producing recombinant adeno-associated virus and its construction method and application. The novel auxiliary plasmid of the present invention is constructed by inserting the UL12 element of herpes simplex virus HSV-1 type I and the NS2 element of human bora virus HBoV into the basic pHelper plasmid. The constructed novel auxiliary plasmid UL12-NS2-pHelper can significantly improve the titer of rAAV virus produced by HEK293 cells, increase the proportion of solid rAAV virus, help reduce the immune response and toxicity caused by empty capsids, reduce the therapeutic dose, and improve the efficacy and safety.

[0004] The technical solution of the present invention is as follows:

[0005] A novel helper plasmid for producing recombinant adeno-associated virus, the novel helper plasmid comprising a UL12 gene derived from HSV-1 and a NS2 gene derived from HBoV1; the nucleotide sequence of the UL12 gene is shown in SEO ID No.1; the nucleotide sequence of the NS2 gene is shown in SEO ID No.2.

[0006] Furthermore, the sequence of the novel auxiliary plasmid is shown as SEO ID No.3.

[0007] A method for constructing the novel auxiliary plasmid, the method comprising the following steps:

[0008] (1) synthesizing the UL12 gene and the NS2 gene according to the nucleotide sequences of the UL12 gene and the NS2 gene, respectively, and then connecting the synthesized UL12 gene and the NS2 gene to the pcDNA3.1 vector to obtain the UL12-pcDNA3.1 cloning vector and the NS2-pcDNA3.1 cloning vector;

[0009] (2) Using the UL12-pcDNA3.1 cloning vector as a template, primers UL12-F1 and UL12-R1 were used to amplify the UL12 gene fragment; using the NS2-pcDNA3.1 cloning vector as a template, primers NS2-F1 and NS2-R1 were used to amplify the NS2 gene fragment;

[0010] (3) using restriction endonucleases NdeI and SalI to digest the pHelper plasmid to obtain a linearized pHelper plasmid;

[0011] (4) homologously recombining the UL12 gene fragment and the NS2 gene fragment obtained in step (2) with the linearized pHelper plasmid obtained in step (3) by Gibson assembly to obtain a novel auxiliary plasmid UL12-NS2-pHelper;

[0012] (5) The novel auxiliary plasmid UL12-NS2-pHelper was transformed into Escherichia coli DH5α competent cells to obtain recombinant bacteria. After resistance screening, the plasmid was extracted for PCR verification and sequencing verification to obtain the novel auxiliary plasmid UL12-NS2-pHelper with correct sequencing.

[0013] Furthermore, in step (2), the nucleotide sequence of the UL12-F1 is shown as SEQ ID No.4; and the nucleotide sequence of the UL12-R1 is shown as SEQ ID No.5.

[0014] Furthermore, in step (2), the nucleotide sequence of the NS2-F1 is shown as SEQ ID No.6; and the nucleotide sequence of the NS2-R1 is shown as SEQ ID No.7.

[0015] An application of the novel helper plasmid or the novel helper plasmid constructed by the construction method, wherein the novel helper plasmid is used to prepare a recombinant adeno-associated virus.

[0016] Furthermore, the application is as follows:

[0017] HEK293 cells were co-transfected with the novel helper plasmid UL12-NS2-pHelper, whose sequence is shown in SEO ID No. 4, pRep2Capx plasmid and pAAV-GOI plasmid to produce recombinant adeno-associated virus.

[0018] Furthermore, the pRep2Capx plasmid encodes Rep gene and Cap gene; the cap gene includes cap1 gene, cap2 gene, cap3 gene, cap4 gene, cap5 gene, cap6 gene, cap7 gene, cap8 gene, cap9 gene and cap10 gene; the pAAV-GOI plasmid refers to a plasmid carrying a target gene, which has two inverted repeated sequences, and carries the target gene between the inverted repeated sequences at both ends.

[0019] Furthermore, the cap gene is preferably a cap5 gene.

[0020] Furthermore, the pRep2Capx plasmid is preferably a pRep2Cap5 plasmid.

[0021] The beneficial technical effects of the present invention are:

[0022] The novel helper plasmid for producing recombinant adeno-associated virus constructed by the present invention is obtained by inserting the UL12 element of type I herpes simplex virus (Herpes simplex virus, HSV-I) and the NS2 element of human bocavirus (Human Bocavirus, HBoV) into the basic pHelper plasmid. The novel helper plasmid UL12-NS2-pHelper is obtained. The constructed novel helper plasmid significantly improves the titer of rAAV virus produced by HEK293 cells by improving the replication efficiency of the viral genome, which helps to reduce production costs and improve virus preparation efficiency, which is particularly important in large-scale clinical applications.

[0023] The novel auxiliary plasmid UL12-NS2-pHelper constructed by the present invention can greatly increase the proportion of solid rAAV virus, help reduce the immune response and toxicity caused by empty capsids, thereby reducing the therapeutic dose and improving efficacy and safety. In addition, the novel auxiliary plasmid constructed in this application can be applied to a variety of serotypes, including AAV1 / AAV2 / AAV3B / AAV4 / AAV5 / AAV6 / AAV7 / AAV8 / AAV9 / AAVrh10. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the novel helper plasmid UL12-NS2-pHelper constructed in the present invention.

[0025] Figure 2 Schematic diagram of the original pHelper plasmid in the present invention.

[0026] Figure 3 Schematic diagram of the recombinant plasmid constructed in Comparative Examples 1-10 of the present invention.

[0027] In the figure: A is the pCDNA3.1(+) empty plasmid; B is the schematic diagram of the ICP0-pCDNA3.1(+) recombinant plasmid; C is the schematic diagram of the UL8-pCDNA3.1(+) recombinant plasmid; D is the schematic diagram of the UL12-pCDNA3.1(+) recombinant plasmid; E is the schematic diagram of the UL42-pCDNA3.1(+) recombinant plasmid; F is the schematic diagram of the NP1-pCDNA3.1(+) recombinant plasmid; G is the schematic diagram of the NS2-pCDNA3.1(+) recombinant plasmid; H is the schematic diagram of the E1-pCDNA3.1(+) recombinant plasmid; I is the schematic diagram of the E2-pCDNA3.1(+) recombinant plasmid; J is the schematic diagram of the E6-pCDNA3.1(+) recombinant plasmid.

[0028] Figure 4 Schematic diagram of the auxiliary plasmid constructed for Comparative Examples 11-12 of the present invention.

[0029] In the figure: A, is a schematic diagram of the auxiliary plasmid UL12-pHelper; B, is a schematic diagram of the auxiliary plasmid NS2-pHelper.

[0030] Figure 5 This is a standard curve of rAAV virus titer.

[0031] Figure 6 This is a graph of the titer of the recombinant adeno-associated virus produced by comparative examples 2-11 of the present invention.

[0032] Figure 7 This is a graph of the titer of the recombinant adeno-associated virus produced by Application Example 1 and Comparative Examples 1, 12-13 of the present invention.

[0033] Figure 8 This is the SDS-PAGE gel electrophoresis diagram of the rAAV virus produced in Application Example 1 and Comparative Example 1 of the present invention.

[0034] Fig. 9 This is a solid graph of the rAAV virus produced by Comparative Example 1 of the present invention.

[0035] Fig.10 This is a solid graph of the rAAV virus produced in Application Example 1 of the present invention.

[0036] Fig.11 Titer graph of rAAV viruses of different serotypes produced by the novel helper plasmid UL12-NS2-pHelper constructed in Example 1 of the present invention and the original pHelper helper plasmid.

[0037] Fig.12 The solid graph shows the production of rAAV viruses of different serotypes using the novel helper plasmid UL12-NS2-pHelper constructed in Example 1 of the present invention and the original pHelper helper plasmid. DETAILED DESCRIPTION

[0038] The present invention is described in detail below in conjunction with the accompanying drawings and embodiments.

[0039] The original pHelper plasmid in the present invention carries E2A / E4 / VA RNA and is purchased from Thermo Fisher Scientific. The pHelper plasmid map is as follows: Figure 2 As shown; pRep2Cap5 plasmid was purchased from Thermo Fisher Scientific; pCDNA3.1(+) was purchased from GenScript Biotech Co., Ltd.; In the present invention, pAAV2-CMV-EGFP plasmid was used as pAAV-GOI plasmid, which was purchased from Thermo Fisher Scientific.

[0040] In the following examples of the present invention, the nucleotide sequence of the UL12 gene is shown as SEQ ID NO.1, and the nucleotide sequence of the NS2 gene is shown as SEQ ID NO.2, both of which were commissioned to be synthesized by GenScript Corporation.

[0041] The nucleotide sequence of the novel auxiliary plasmid UL12-NS2-pHelper constructed by the present invention is shown in SEO ID No.3.

[0042] SEQ ID NO.1:

[0043]

[0044] SEQ ID NO.2:

[0045]

[0046]

[0047] SEQ ID NO.3:

[0048]

[0049]

[0050]

[0051]

[0052] The primers used in the following examples are shown in Table 1:

[0053] Table 1 Primer list

[0054]

[0055]

[0056] like Figure 1 As shown, it is a schematic diagram of the novel auxiliary plasmid UL12-NS2-pHelper constructed by the present invention, and the nucleotide sequence is shown in SEO ID No.3. Based on the existing pHelper plasmid, the present invention optimizes the sequence, and the UL12 gene derived from HSV-1, NCBI sequence number NC_001806, the nucleic acid position is 24801-226888bp (specifically as shown in SEO ID No.1); the NS2 gene derived from HBoV1, NCBI sequence number NC_007455, the nucleic acid position is 253-2172bp (specifically as shown in SEO ID No.2). The novel auxiliary plasmid UL12-NS2-pHelper of the present invention, together with the pRep2Cap5 plasmid and the pAAV2-CMV-EGFP plasmid, co-transfects HEK293 cells, and compared with the commonly used pHelper plasmid carrying E2A / E4 / VA RNA, the virus packaging amount is increased by more than 2.03 times. The pRep2Cap5 plasmid encodes the Rep gene and the Cap gene; the pAAV2-CMV-EGFP plasmid refers to a plasmid carrying the target gene and the green fluorescent protein gene, and the plasmid has two inverted repeat sequences, and the target gene is carried between the inverted repeat sequences at both ends. The novel auxiliary plasmid constructed by the present invention can not only be applied to scientific research, but also to large-scale adeno-associated virus preparation, which greatly improves the production efficiency of adeno-associated virus.

[0057] The novel auxiliary plasmid constructed by the present invention can be applicable to a variety of serotypes, including AAV1 / AAV2 / AAV3 / AAV3B / AAV4 / AAV5 / AAV6 / AAV7 / AAV8 / AAV9 / AAVrh10.

[0058] Example 1

[0059] A method for constructing a novel auxiliary plasmid comprises the following steps:

[0060] (1) GenScript Corporation was commissioned to synthesize the UL12 gene and the NS2 gene according to the nucleotide sequences of the UL12 gene and the NS2 gene, respectively, and then the synthesized UL12 gene and the NS2 gene were connected to the pcDNA3.1 vector to obtain the UL12-pcDNA3.1 cloning vector and the NS2-pcDNA3.1 cloning vector.

[0061] (2) Using the UL12-pcDNA3.1 cloning vector as a template, primers UL12-F1 and UL12-R1 were used for amplification to obtain the UL12 gene fragment; using the NS2-pcDNA3.1 cloning vector as a template, primers NS2-F1 and NS2-R1 were used for amplification to obtain the NS2 gene fragment.

[0062] (3) The pHelper plasmid was digested with restriction endonucleases NdeI and SalI to obtain a linearized pHelper plasmid.

[0063] (4) The UL12 gene fragment and the NS2 gene fragment obtained in step (2) are homologously recombined with the linearized pHelper plasmid obtained in step (3) by Gibson assembly to obtain a novel auxiliary plasmid UL12-NS2-pHelper.

[0064] (5) The novel auxiliary plasmid UL12-NS2-pHelper was transformed into Escherichia coli DH5α competent cells to obtain recombinant bacteria. After kanamycin resistance screening, the plasmid was extracted for PCR verification and sequencing verification to obtain the novel auxiliary plasmid UL12-NS2-pHelper with correct sequencing.

[0065] Comparative Example 1

[0066] This comparative example provides a commercially available pCDNA3.1(+) empty plasmid, the schematic diagram of which is shown in Figure 3 As shown in A.

[0067] Comparative Example 2

[0068] A recombinant plasmid ICP0-pCDNA3.1(+) carrying the ICP0 gene, specifically as follows:

[0069] GenScript was commissioned to synthesize the ICP0 gene based on the nucleotide sequence of the ICP0 gene (NC_001806), and then the synthesized ICP0 gene was connected to the pcDNA3.1(+) vector, with the nucleic acid position of 929-3256 bp, to obtain the ICP0-pCDNA3.1(+) recombinant plasmid. The schematic diagram of the recombinant plasmid is shown in the figure Figure 3 As shown in B.

[0070] Comparative Example 3

[0071] A recombinant plasmid UL8-pCDNA3.1(+) carrying the UL8 gene, specifically as follows:

[0072] GenScript was commissioned to synthesize the UL8 gene based on the nucleotide sequence of the UL8 gene (NC_001806), and then the synthesized UL8 gene was connected to the pcDNA3.1(+) vector, with the nucleic acid position being 929-3181 bp, to obtain the UL8-pCDNA3.1(+) recombinant plasmid. The schematic diagram of the recombinant plasmid is shown in the figure Figure 3 As shown in C.

[0073] Comparative Example 4

[0074] A recombinant plasmid UL12-pCDNA3.1(+) carrying the UL12 gene, specifically as follows:

[0075] GenScript was commissioned to synthesize the UL12 gene based on the nucleotide sequence of the UL12 gene NC_001806, and then the synthesized UL12 gene was connected to the pcDNA3.1(+) vector, with the nucleic acid position being 929-2996 bp, to obtain the UL12-pCDNA3.1(+) recombinant plasmid. The schematic diagram of the recombinant plasmid is shown in the figure Figure 3 As shown in D.

[0076] Comparative Example 5

[0077] A recombinant plasmid UL42-pCDNA3.1(+) carrying the UL42 gene, specifically as follows:

[0078] GenScript was commissioned to synthesize the UL42 gene based on the nucleotide sequence of the UL42 gene (NC_001806), and then the synthesized UL42 gene was connected to the peDNA3.1(+) vector, with the nucleic acid position being 929-2395 bp, to obtain the UL42-pCDNA3.1(+) recombinant plasmid. The schematic diagram of the recombinant plasmid is shown in the figure Figure 3 As shown in E.

[0079] Comparative Example 6

[0080] A recombinant plasmid NP1-pCDNA3.1(+) carrying the NP1 gene, specifically as follows:

[0081] GenScript was commissioned to synthesize the NP1 gene based on the nucleotide sequence of the NP1 gene (NC_007455), and then the synthesized NP1 gene was connected to the pcDNA3.1(+) vector, with the nucleic acid position of 929-3304 bp, to obtain the NP1-pCDNA3.1(+) recombinant plasmid. The schematic diagram of the recombinant plasmid is shown in the figure Figure 3 As shown in F.

[0082] Comparative Example 7

[0083] A recombinant plasmid NS2-pCDNA3.1(+) carrying the NS2 gene, specifically as follows:

[0084] GenScript was commissioned to synthesize the NS2 gene based on the nucleotide sequence of the NS2 gene NC_007455, and then the synthesized NS2 gene was connected to the pcDNA3.1(+) vector, with the nucleic acid position of 929-2851 bp, to obtain the NS2-pCDNA3.1(+) recombinant plasmid. The schematic diagram of the recombinant plasmid is shown in the figure Figure 3 As shown in G.

[0085] Comparative Example 8

[0086] A recombinant plasmid E1-pCDNA3.1(+) carrying the E1 gene, specifically as follows:

[0087] GenScript was commissioned to synthesize the E1 gene according to the nucleotide sequence of the E1 gene (NC_001526), ​​and then the synthesized E1 gene was connected to the pcDNA3.1(+) vector, with the nucleic acid position being 929-3016 bp, to obtain the E1-pCDNA3.1(+) recombinant plasmid. The schematic diagram of the recombinant plasmid is shown in the figure Figure 3 As shown in H.

[0088] Comparative Example 9

[0089] A recombinant plasmid E2-pCDNA3.1(+) carrying the E2 gene, specifically as follows:

[0090] GenScript was commissioned to synthesize the E2 gene based on the nucleotide sequence of the E2 gene (NC_001526), ​​and then the synthesized E2 gene was connected to the pcDNA3.1(+-) vector, with the nucleic acid position being 929-2681 bp, to obtain the E2-pCDNA3.1(+) recombinant plasmid. The schematic diagram of the recombinant plasmid is shown in the figure Figure 3 As shown in I.

[0091] Comparative Example 10

[0092] A recombinant plasmid E6-pCDNA3.1(+) carrying the E6 gene, specifically as follows:

[0093] GenScript was commissioned to synthesize the E6 gene based on the nucleotide sequence of the E6 gene (NC_001526), ​​and then the synthesized E6 gene was connected to the pcDNA3.1(+) vector, with the nucleic acid position being 929-1405 bp, to obtain the E6-pCDNA3.1(+) recombinant plasmid. The schematic diagram of the recombinant plasmid is shown in the figure Figure 3 As shown in J.

[0094] Comparative Example 11

[0095] A method for constructing an auxiliary plasmid comprises the following steps:

[0096] (1) GenScript was commissioned to synthesize the UL12 gene based on the nucleotide sequence of the UL12 gene, and then the synthesized UL12 gene was connected to the pcDNA3.1 vector to obtain the UL12-pcDNA3.1 cloning vector.

[0097] (2) Using the UL12-pcDNA3.1 cloning vector as a template, primers UL12-F2 and UL12-R2 were used for amplification to obtain the UL12-2 gene fragment.

[0098] (3) The pHelper plasmid was digested with restriction endonucleases NdeI and SalI to obtain a linearized pHelper plasmid.

[0099] (4) The UL12-2 gene fragment obtained in step (2) and the linearized pHelper plasmid obtained in step (3) are homologously recombined by Gibson assembly to obtain the auxiliary plasmid UL12-pHelper.

[0100] (5) The auxiliary plasmid UL12-pHelper was transformed into Escherichia coli DH5α competent cells to obtain recombinant bacteria. After kanamycin resistance screening, the plasmid was extracted for PCR verification and sequencing verification to obtain the auxiliary plasmid UL12-pHelper with correct sequencing. The schematic diagram of the auxiliary plasmid is shown in FIG. Figure 4 As shown in A.

[0101] Comparative Example 12

[0102] A method for constructing an auxiliary plasmid comprises the following steps:

[0103] (1) GenScript was commissioned to synthesize the NS2 gene based on the nucleotide sequence of the NS2 gene, and then the synthesized NS2 gene was connected to the pcDNA3.1 vector to obtain the NS2-pcDNA3.1 cloning vector.

[0104] (2) Using the NS2-pcDNA3.1 cloning vector as a template, primers NS2-F2 and NS2-R2 were used for amplification to obtain the NS2 gene fragment;

[0105] (3) using restriction endonucleases NdeI and SalI to digest the pHelper plasmid to obtain a linearized pHelper plasmid;

[0106] (4) homologously recombining the NS2-2 gene fragment obtained in step (2) with the linearized pHelper plasmid obtained in step (3) by Gibson assembly to obtain a novel auxiliary plasmid NS2-pHelper;

[0107] (5) The auxiliary plasmid NS2-pHelper was transformed into Escherichia coli DH5α competent cells to obtain recombinant bacteria. After kanamycin resistance screening, the plasmid was extracted for PCR verification and sequencing verification to obtain the auxiliary plasmid NS2-pHelper with correct sequencing. The schematic diagram of the auxiliary plasmid is shown in FIG. Figure 4 As shown in B.

[0108] Application Example 1

[0109] The application of a new type of auxiliary plasmid, taking AAV5 as an example, is as follows:

[0110] HEK293 cells were cultured at a density of 1.3 × 10 6 cells / mL, 7.5 μg of the novel auxiliary plasmid UL12-NS2-pHelper, 7.5 μg of the pRep2Cap5 plasmid and 7.5 μg of the pAAV2-CMV-EGFP plasmid constructed in Example 1 were co-transfected into HEK293 cells, and cultured in a 125 mL baffled shake flask for 72 h to preliminarily form recombinant adeno-associated virus.

[0111] Application Comparative Example 1

[0112] An application of an original pHelper plasmid carrying E2A / E4 / VA RNA, the application method is basically the same as that in Application Example 1, the difference is that in this application comparison example, a commercially purchased original pHelper plasmid carrying E2A / E4 / VA RNA is used to replace the "new helper plasmid UL12-NS2-pHelper" in Application Example 1 for transfecting HEK293 cells.

[0113] Application Comparative Example 2

[0114] The application of a commercially purchased pCDNA3.1(+) empty vector plasmid, taking AAV5 as an example, is as follows:

[0115] HEK293 cells were cultured at a density of 1.3 × 10 6cells / mL, the pCDNA3.1(+) empty plasmid, the original pHelper plasmid, the pRep2Cap5 plasmid and the pAAV2-CMV-EGFP plasmid in Comparative Example 1 were co-transfected into HEK293 cells and cultured in a 125 mL baffled shake flask for 72 h to preliminarily form recombinant adeno-associated virus.

[0116] Application Comparative Example 3

[0117] An application of a recombinant plasmid ICP0-pCDNA3.1(+) carrying an ICP0 gene, the application method is basically the same as that of application example 2, except that in this application example, the "recombinant plasmid ICP0-pCDNA3.1(+)" constructed in application example 2 is used to replace the "pCDNA3.1(+) empty plasmid" in application example 2 for transfection of HEK293 cells.

[0118] Application Comparative Example 4

[0119] An application of a recombinant plasmid UL8-pCDNA3.1(+) carrying a UL8 gene, the application method of which is basically the same as that of application example 2, except that in this application example, the "recombinant plasmid UL8-pCDNA3.1(+)" constructed in application example 3 is used to replace the "pCDNA3.1(+) empty plasmid" in application example 2 for transfection of HEK293 cells.

[0120] Application Comparative Example 5

[0121] An application of a recombinant plasmid UL12-pCDNA3.1(+) carrying a UL12 gene, the application method is basically the same as that of application example 2, except that in this application example, the "recombinant plasmid UL12-pCDNA3.1(+)" constructed in application example 4 is used to replace the "pCDNA3.1(+) empty plasmid" in application example 2 for transfection of HEK293 cells.

[0122] Application Comparative Example 6

[0123] An application of a recombinant plasmid UL42-pCDNA3.1(+) carrying the UL42 gene, the application method is basically the same as that of Application Example 2, except that in this application example, the "recombinant plasmid UL42-pCDNA3.1(+)" constructed in Application Example 5 is used to replace the "pCDNA3.1(+) empty plasmid" in Application Example 2 for transfection of HEK293 cells.

[0124] Application Comparative Example 7

[0125] An application of a recombinant plasmid NP1-pCDNA3.1(+) carrying the NP1 gene, the application method is basically the same as that of application example 2, except that in this application example, the "recombinant plasmid NP1-pCDNA3.1(+)" constructed in application example 6 is used to replace the "pCDNA3.1(+) empty plasmid" in application example 2 for transfection of HEK293 cells.

[0126] Application Comparative Example 8

[0127] An application of a recombinant plasmid NS2-pCDNA3.1(+) carrying the NS2 gene, the application method is basically the same as that of Application Example 2, except that in this application example, the "recombinant plasmid NS2-pCDNA3.1(+)" constructed in Application Example 7 is used to replace the "pCDNA3.1(+) empty plasmid" in Application Example 2 for transfection of HEK293 cells.

[0128] Application Comparative Example 9

[0129] An application of a recombinant plasmid E1-pCDNA3.1(+) carrying the E1 gene, the application method is basically the same as that of Application Example 2, except that in this application example, the "recombinant plasmid E1-pCDNA3.1(+)" constructed in Application Example 8 is used to replace the "pCDNA3.1(+) empty plasmid" in Application Example 2 for transfection of HEK293 cells.

[0130] Application Comparative Example 10

[0131] An application of a recombinant plasmid E2-pCDNA3.1(+) carrying an E2 gene, the application method is basically the same as that of Application Example 2, except that in this application example, the "recombinant plasmid E2-pCDNA3.1(+)" constructed in Application Example 9 is used to replace the "pCDNA3.1(+) empty plasmid" in Application Example 2 for transfection of HEK293 cells.

[0132] Application Comparative Example 11

[0133] An application of a recombinant plasmid E6-pCDNA3.1(+) carrying the E6 gene, the application method is basically the same as that of Application Example 2, except that in this application example, the "recombinant plasmid E6-pCDNA3.1(+)" constructed in Application Example 10 is used to replace the "pCDNA3.1(+) empty plasmid" in Application Example 2 for transfection of HEK293 cells.

[0134] Application Comparative Example 12

[0135] The application of a new type of auxiliary plasmid, taking AAV5 as an example, is as follows:

[0136] HEK293 cells were cultured at a density of 1.3 × 10 6 cells / mL, the auxiliary plasmid UL12-pHelper 7.5 μg, pRep2Cap5 plasmid 7.5 μg and pAAV2-CMV-EGFP plasmid 7.5 μg constructed in Comparative Example 11 were co-transfected into HEK293 cells and cultured in a 125 mL baffled shake flask for 72 h to preliminarily form recombinant adeno-associated virus.

[0137] Application Comparative Example 13

[0138] The application of a new type of auxiliary plasmid, taking AAV5 as an example, is as follows:

[0139] HEK293 cells were cultured at a density of 1.3 × 10 6 cells / mL, HEK293 cells were co-transfected with 7.5 μg of the auxiliary plasmid NS2-pHelper, 7.5 μg of the pRep2Cap5 plasmid and 7.5 μg of the pAAV2-CMV-EGFP plasmid constructed in Comparative Example 12, and cultured in a 125 mL baffled shake flask for 72 h to preliminarily form recombinant adeno-associated virus.

[0140] Test Case

[0141] (1) rAAV virus titer

[0142] The pAAV2-CMV-EGFP plasmid was diluted according to the concentration in Table 2 and prepared into standard products 1-6, and a standard curve was drawn. The standard curve is as follows Figure 5 shown.

[0143] Table 2 Standard preparation concentration table

[0144]

[0145] The crude cell product after transfection and culture for 72 hours of the comparative example 2-11 of the present invention was taken, and the virus titer was detected by real-time fluorescence quantitative PCR, and the virus titer produced by the HEK293 cells after transfection and culture for 72 hours of the comparative example 2-11 was calculated by the standard curve and the PCR result. The results are as follows: Figure 6 As shown. Figure 6 The results show that compared with the application of comparative example 2 (Normal group), preliminary screening found that the auxiliary elements UL12 and NS2 can increase the rAAV production by 1.53 times and 1.21 times respectively.

[0146] HEK293 cells after transfection and culture for 72 hours in Application Example 1 and Application Comparative Examples 1 and 12-13 of the present invention were taken, and the virus titer was detected by real-time fluorescence quantitative PCR, and the virus titer produced by HEK293 cells after transfection and culture for 72 hours in Application Example 1 and Application Comparative Examples 1 and 12-13 was calculated by the standard curve and PCR results. The results are as follows Figure 7 As shown. Figure 7 The results show that the rAAV virus titer produced by Application Example 1 of the present invention is significantly higher than that of Application Comparative Examples 1 and 12-13, and the virus titer yield is 2.03 times that of Application Comparative Example 1, indicating that the new auxiliary plasmid UL12-NS2-pHelper constructed by the present invention can significantly improve the virus titer yield.

[0147] (2) rAAV virus purity

[0148] HEK293 cells were cultured for 72 h after transfection in Application Example 1 and Application Comparative Example 1 of the present invention, and the crude cell products were collected by centrifugation. After the viral particles were released by lysis, the rAAV virus produced was purified by affinity chromatography column Capture Select AAVX (Thermo Fisher Scientific), and the purity of the virus was verified by SDS-PAGE gel electrophoresis. The results are as follows: Figure 8 As shown. Figure 8 The results show that the purity of the purified rAAV is 95%, which can be used for subsequent solid rate analysis.

[0149] (3) Virus solid rate

[0150] HEK293 cells were cultured for 72 h after transfection in Application Example 1 and Comparative Example 1 of the present invention, and the crude cell products were collected by centrifugation. The viral particles were released by lysis, and the rAAV virus produced was purified by affinity chromatography column Capture Select AAVX. The purified rAAV virus was purified by anion chromatography column CIMac TM AAV full / empty (Sartorius) was used for solidity analysis. Based on the different isoelectric points, the virus containing the empty capsid was separated first, and the solid virus was separated later. The results are shown in Figure 9-10 As shown. Fig. 9 The results show that, calculated at 280 nm, pHelper solid ratio = 84425 / (68676×9+84425) = 12%. Fig.10 It can be seen that the solid rate of UL12-NS2-pHelper = 134405 / (50843*9+134405) = 23%. The results confirm that the UL12-NS2-pHelper auxiliary plasmid constructed by the present invention can greatly increase the proportion of solid rAAV virus for virus packaging.

[0151] (4) Serotype testing

[0152] The novel auxiliary plasmid UL12-NS2-pHelper constructed in Example 1 of the present invention was used to produce rAAV of different serotypes (AAV1 / AAV2 / AAV3B / AAV4 / AAV5 / AAV6 / AAV7 / AAV8 / AAV9 / AAVrh10) according to the method of Application Example 1, and the titer and solid rate were detected. The results were as follows: Fig.11 and Fig.12 As shown. Fig.11 and Fig.12 It can be seen that the new auxiliary plasmid UL12-NS2-pHelper constructed by the present invention can be used for the rAAV virus of the above serotype.

[0153] The above is only a preferred embodiment of the present invention, and the present invention is not limited to the above embodiments. It is understood that other improvements and changes directly derived or associated by those skilled in the art without departing from the spirit and concept of the present invention should be considered to be included in the protection scope of the present invention.

Claims

1. A novel helper plasmid for producing recombinant adeno-associated virus, characterized in that: The novel helper plasmid comprises the UL12 gene derived from HSV-1 and the NS2 gene derived from HBoV1; the nucleotide sequence of the UL12 gene is shown in SEO ID No.1; the nucleotide sequence of the NS2 gene is shown in SEO ID No.

2.

2. The novel helper plasmid according to claim 1, characterized in that The sequence of the novel auxiliary plasmid is shown in SEOID No.

3.

3. A method for constructing the novel helper plasmid according to claim 1, characterized in that: The construction method comprises the following steps: (1) synthesizing the UL12 gene and the NS2 gene according to the nucleotide sequences of the UL12 gene and the NS2 gene, respectively, and then connecting the synthesized UL12 gene and the NS2 gene to the pcDNA3.1 vector to obtain the UL12-pcDNA3.1 cloning vector and the NS2-pcDNA3.1 cloning vector; (2) Using the UL12-pcDNA3.1 cloning vector as a template, primers UL12-F1 and UL12-R1 were used to amplify the UL12 gene fragment; using the NS2-pcDNA3.1 cloning vector as a template, primers NS2-F1 and NS2-R1 were used to amplify the NS2 gene fragment; (3) using restriction endonucleases NdeI and SalI to digest the pHelper plasmid to obtain a linearized pHelper plasmid; (4) homologously recombining the UL12 gene fragment and the NS2 gene fragment obtained in step (2) with the linearized pHelper plasmid obtained in step (3) by Gibson assembly to obtain a novel auxiliary plasmid UL12-NS2-pHelper; (5) The novel auxiliary plasmid UL12-NS2-pHelper was transformed into Escherichia coli DH5α competent cells to obtain recombinant bacteria. After resistance screening, the plasmid was extracted for PCR verification and sequencing verification to obtain the novel auxiliary plasmid UL12-NS2-pHelper with correct sequencing.

4. The construction method according to claim 3, characterized in that: In step (2), the nucleotide sequence of the UL12-F1 is shown as SEQ ID No.4; the nucleotide sequence of the UL12-R1 is shown as SEQ ID No.

5.

5. The construction method according to claim 3, characterized in that: In step (2), the nucleotide sequence of the NS2-F1 is shown as SEQ ID No.6; the nucleotide sequence of the NS2-R1 is shown as SEQ ID No.

7.

6. Use of the novel auxiliary plasmid according to claim 1 or the novel auxiliary plasmid constructed by the construction method according to claim 3, characterized in that: The novel helper plasmid is used for preparing recombinant adeno-associated virus.

7. The use according to claim 6, characterized in that: The application is as follows: HEK293 cells were co-transfected with the novel helper plasmid UL12-NS2-pHelper, whose sequence is shown in SEO ID No. 4, pRep2Capx plasmid and pAAV-GOI plasmid to produce recombinant adeno-associated virus.

8. The use according to claim 7, characterized in that: The pRep2Capx plasmid encodes Rep gene and Cap gene; the cap gene includes cap1 gene, cap2 gene, cap3 gene, cap4 gene, cap5 gene, cap6 gene, cap7 gene, cap8 gene, cap9 gene and cap10 gene; The pAAV-GOI plasmid refers to a plasmid carrying a target gene. The plasmid has two inverted repeat sequences, and the target gene is carried between the inverted repeat sequences at both ends.

9. The use according to claim 8, characterized in that: The cap gene is preferably a cap5 gene.

10. The use according to claim 7, characterized in that: The pRep2Capx plasmid is preferably a pRep2Cap5 plasmid.

Images