A recombinant adenovirus type 5 vector with reduced reverse mutation frequency and its construction method

Abstract

The present invention belongs to the field of bioengineering technology. To reduce the probability of homologous recombination between a recombinant adenovirus and host cells, a recombinant adenovirus type 5 vector and a method for constructing the vector are provided, which reduces the frequency of reverse mutations. By knocking out the E1B region gene of human adenovirus type 5, and knocking out the gene at base 4090, the length of the homologous sequence between adenovirus type 5 and HEK293 genomic DNA is shortened to 254 base pairs. The deleted E1 region gene fragment includes the pIX-encoding sequence Ad5 3551-4070. The pIX-encoding sequence Ad5 3551-4070 is then reversely inserted into the E4 region, i.e., Ad5 35771. This reduces the frequency of homologous recombination during packaging and amplification of the recombinant adenovirus, thereby reducing the frequency of RCA. This method not only prevents reverse homologous recombination between the recombinant adenovirus vector and HEK293 cell genomic DNA, but also ensures the stability of the recombinant adenovirus particle structure.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering technology, and in particular relates to a recombinant adenovirus type 5 vector capable of reducing the frequency of reverse mutation and a construction method thereof. Background Art

[0002] Gene therapy involves the introduction of exogenous normal or therapeutically effective genes into target cells to correct or compensate for genetic defects and abnormalities, achieving therapeutic goals. In recent years, recombinant adenoviral vectors have played a significant role in gene therapy. Recombinant adenoviral vectors, characterized by their ability to recombine in culture and high transgene insertion capacity, have important applications in malignant tumors, central nervous system diseases, and genetic disorders.

[0003] Although recombinant adenovirus vectors are widely used, they also have potential risks. During the production process of recombinant adenovirus, there is a probability that the recombinant adenovirus will undergo homologous recombination with the host cell, that is, reverse mutation (RCA). Reverse mutation not only leads to the loss of exogenous normal genes or genes with therapeutic effects, but also produces adenoviruses with replication ability, which will have potential side effects on the human body. Regulatory authorities in various countries have set very high requirements for the proportion of reverse mutations in gene therapy drugs using recombinant adenovirus vectors. For example, the US Food and Drug Administration (FDA) requires that no more than 1 copy of reverse mutation be found in 3×1010 copies of adenovirus particles. Therefore, it is crucial to construct a recombinant adenovirus vector that can reduce the frequency of reverse mutations. Summary of the Invention

[0004] In order to reduce the probability of homologous recombination between the recombinant adenovirus and the host cell, the present invention provides a recombinant adenovirus type 5 vector with reduced back mutation frequency and a construction method thereof.

[0005] The present invention is achieved by the following technical solution: a recombinant adenovirus type 5 vector with reduced reversion mutation frequency, wherein the recombinant adenovirus type 5 vector with reduced reversion mutation frequency is prepared by the following method: by knocking out the E1B region gene of human adenovirus type 5, and knocking out to base 4090, the length of the homologous sequence between adenovirus type 5 and HEK293 genomic DNA is shortened to 254bp, and the deleted E1 region gene segment includes the sequence Ad5 3551-4070 encoding pIX; then, the sequence Ad5 3551-4070 encoding pIX is reversely inserted into the E4 region, i.e., Ad5 35771.

[0006] The method for preparing the recombinant adenovirus type 5 vector with reduced back mutation frequency comprises the following steps:

[0007] (1) Construction of the left plasmid: retain the left terminal repeat sequence (ITR) and E1A region, delete the E1B region, including the sequence encoding pIX, insert the foreign gene at the original position of the E1B region, and retain the adenovirus type 5 4091-5196 sequence;

[0008] (2) Construct the right plasmid: retain the rest of the adenovirus genome except the E3 region, retain the sequence encoding the adenovirus death protein ADP in the E3 region, and insert the sequence encoding pIX in the E4 region in reverse direction;

[0009] (3) Virus packaging: The left and right plasmids were linearized using restriction endonuclease Pac I, respectively, and digested at 37°C for 30-60 min; then incubated at 65°C for 30-60 min to inactivate the restriction endonuclease Pac I; the linearized plasmids were co-transfected with HEK293 cells using PEI transfection reagent when the degree of polymerization was 80%-90%. 10-14 days after transfection, HEK293 cells showed pathological effects, with more than 50% of the cells falling off from the bottom of the culture flask; at this time, the cell suspension was collected, frozen and thawed three times, and then centrifuged at 3000 rpm for 5-10 min to collect the supernatant to obtain the recombinant virus suspension.

[0010] The specific method for constructing the left plasmid in step (1) is as follows: using pfg140 as a template to amplify the Ad5 1-1668 fragment, using pBHG10 as a template to amplify the Ad5 4091-5196 fragment, inserting the CMV-hGM-CSF-SV40 fragment in the middle as a linker, and ligating the fragments to the pGEM-7Zf(+) vector to construct the pGEM-7Zf(+)-Ad5(1-1668)-CMV-hGM-CSF-SV40-Ad5(4091-5196) plasmid, i.e., the left plasmid.

[0011] The specific method for constructing the right side plasmid in step (2) is as follows: using plasmid pBHG10 as a template to amplify the Ad53551-4070 fragment; using plasmid pBHG10 as a template to amplify pBHG10 2682-2866 and pBHG105612-7286 fragments, using plasmid pFG140 as a template to amplify Ad5 29485-29771, and using pBHG10 as a template to amplify pBHG10 26924-547; inserting the amplified fragments pBHG102682-2866, pBHG10 5612-7286, Ad5 29485-29771, and pBHG10 26924-547 into the pBHG10 vector, and then reversely inserting the Ad53551-4070 amplified fragment to obtain a complete right side plasmid.

[0012] The amplified fragment pBHG10 5612-7286 and Ad5 4091-5196 fragment are homologous sequences, and homologous recombination occurs here during subsequent transfection.

[0013] The amplified fragment Ad5 29485-29771 encodes adenovirus death protein ADP.

[0014] First-generation recombinant adenoviral vectors delete the E1 and E3 regions. Deletion of the E1 region renders the recombinant adenovirus incapable of replication, so it can only be packaged in cell lines that can provide the proteins necessary for its proliferation. The HEK293 cell line has been modified to contain a 4kb adenoviral DNA sequence, Ad5(1-4344), on chromosome 19, which meets the requirements for recombinant adenovirus proliferation. Therefore, recombinant adenoviral vectors can be packaged and propagated in HEK293 cells.

[0015] When first-generation recombinant adenoviruses delete the E1 region, they typically delete up to Ad5 (3512), creating a homologous sequence of approximately 830 bp with the HEK293 cell genomic DNA. The presence of this homologous sequence allows for homologous recombination between the recombinant adenovirus and the HEK293 cell genome, resulting in the product of homologous recombination, known as RCA.

[0016] The frequency of homologous recombination is positively correlated with the length of the homologous sequence. In the present invention, the recombinant adenovirus vector was redesigned to delete the E1 region up to base 4090. The length of the homologous sequence between the recombinant adenovirus and the HEK293 genomic DNA was shortened to 254 bp. This shortening of the homologous sequence length reduces the frequency of homologous recombination. The deleted segment includes the Ad5 sequence encoding pIX (3551-4070). pIX stabilizes the hexon lattice arrangement and plays an important role in the stability of the adenovirus particle structure. Therefore, the pIX segment was reversely inserted into the E4 region to ensure the stability of the viral structure.

[0017] This leaves open the possibility that the recombinant adenovirus and HEK293 cell genomic DNA could undergo homologous recombination in the reverse direction. If reverse homologous recombination occurs, the E1 region is incorporated into the recombinant product, which still has the ability to replicate in target cells, posing a biosafety risk. To prevent reverse homologous recombination between the recombinant adenovirus vector and HEK293 cell genomic DNA, the pIX-encoding sequence is inserted in the reverse direction into the E4 region, i.e., at the site of Ad5 (35771).

[0018] The maximum loading capacity of the human adenovirus type 5 capsid is 36 kb; if the maximum loading capacity is exceeded, stable viral particles cannot be formed. Because the total DNA sequence of the adenovirus vector designed for this study is 33,018 bp, if reverse homologous recombination occurs between the recombinant adenovirus vector and HEK293 cell genomic DNA, the recombinant viral DNA sequence will be 36,696 bp long, exceeding the maximum loading capacity of the human adenovirus type 5 capsid and preventing the formation of stable viral particles. This, in turn, reduces the frequency of reverse mutations.

[0019] In summary, the present invention modifies the E1B region of human adenovirus type 5 to shorten the length of the homologous sequence between the recombinant adenovirus vector and the HEK293 cell genomic DNA, thereby reducing the frequency of homologous recombination during packaging and amplification of the recombinant adenovirus, thereby reducing the frequency of RCA. Simultaneously, the sequence Ad5 (3551-4070), encoding code pIX, is reversely inserted into the E4 region, preventing reverse homologous recombination between the recombinant adenovirus vector and the HEK293 cell genomic DNA while ensuring the stability of the recombinant adenovirus particle structure. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Schematic diagram of the structures of RD rAd5, R rAd5, and Low-RCA rAd5; in the figure: RD rAd5: replication-defective recombinant human Ad5, replication-defective recombinant human adenovirus type 5; RrAd5: replicative recombinant human Ad5, replication-competent recombinant human adenovirus type 5; Low-RCA rAd5: Low-RCA recombinant human Ad5, low RCA recombinant human adenovirus type 5;

[0021] Figure 2 Schematic diagram of the structure of the left and right plasmids and the recombination process; in the figure: Left-arm: left plasmid; Right-arm: right plasmid;

[0022] Figure 3 Schematic diagram of electrophoresis of the products after enzyme digestion; in the figure: A is a schematic diagram of electrophoresis of the products after the left plasmid is digested with restriction endonucleases SnaB I and MfeI; B is a schematic diagram of electrophoresis of the products after the right plasmid is digested with restriction endonuclease EcoR I;

[0023] Figure 4 Figures 1 and 2 show normal HEK293 cells and HEK293 cells with pathological effects. In the figure: A shows normal HEK293 cells; B shows HEK293 cells with pathological effects. The magnification is 10×10.

[0024] Figure 5 This is the electrophoresis of the product of low RCA recombinant human adenovirus type 5 digested with BstZ17 I and Pme I;

[0025] Figure 6 This is the electrophoresis diagram of the PCR product using low RCA recombinant human adenovirus type 5 as template;

[0026] Figure 7 Figure 5. Changes in the RCA levels of replication-defective recombinant human Ad5, replication-competent recombinant human Ad5, and low-RCA recombinant human Ad5 after serial passage. RD rAd5: replication-defective recombinant human Ad5; R rAd5: replicative recombinant human Ad5; Low-RCA rAd5: low-RCA recombinant human Ad5.

[0027] Figure 8 The results of recombinant virus sequencing are shown. DETAILED DESCRIPTION

[0028] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, not all the embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0029] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0030] Technical equivalents to the specific embodiments described that are apparent to those skilled in the art using no more than routine experimentation are intended to be encompassed by this application.

[0031] The experimental methods in the following examples, unless otherwise specified, are all conventional methods. The instruments and equipment used in the following examples, unless otherwise specified, are all conventional laboratory instruments and equipment; the experimental materials used in the following examples, unless otherwise specified, are all purchased from conventional biochemical reagent stores.

[0032] Example 1: A recombinant adenovirus type 5 vector with reduced reversion mutation frequency is prepared by the following method: the E1B region gene of human adenovirus type 5 is deleted at base 4090, shortening the homologous sequence between adenovirus type 5 and HEK293 genomic DNA to 254 bp. The deleted E1 region gene segment includes the pIX encoding sequence Ad5 3551-4070; the pIX encoding sequence Ad5 3551-4070 is then inserted in reverse order into the E4 region, i.e., at position 35771 of Ad5. The complete nucleotide sequence of the resulting vector is shown in SEQ ID NO: 1.

[0033] The specific method is as follows:

[0034] Cell culture: HEK293 cells were cultured in Gibco's DMEM medium with 10% fetal bovine serum added. The cells were cultured in a 37°C, 5% CO2 incubator. When the HEK293 cell confluence reached 80-90%, 1×10 6 Cells / well were seeded into 6-well plates (bottom area 96 cm 2 ) for virus packaging.

[0035] Construction of the left plasmid: Retain the left terminal repeat (ITR) and E1A region, delete the E1B region, including the sequence encoding pIX. Insert human granulocyte-macrophage colony-stimulating factor (hGM-CSF) and its promoter in the original E1B region, and retain the adenovirus type 5 4091-5196 sequences for recombination during co-transfection.

[0036] Specifically, the Ad5(1-1668) fragment was amplified using pfg140 as a template, the Ad5(4091-5196) fragment was amplified using pBHG10 as a template, the CMV-hGM-CSF-SV40 fragment was inserted in the middle as a linker, and the fragments were connected to the pGEM-7Zf(+) vector.

[0037] The pGEM-7Zf(+)-Ad5(1-1668)-CMV-hGM-CSF-SV40-Ad5(4091-5196) plasmid, the left plasmid, was constructed.

[0038] Identification of the left plasmid: The left plasmid was digested with restriction endonucleases SnaB I and Mfe I using Cutsmart digestion buffer at 37°C for 30 min. The digestion products were run on a 1% agarose gel to observe the bands. The identification results are as follows: Figure 3 As shown in A, two bands of 821 and 6241 bp were observed, which was consistent with expectations. The plasmid was subsequently sequenced completely, and the successful construction of the plasmid was confirmed based on the sequencing results.

[0039] The right plasmid construct retains most of the adenoviral genome except for the E3 region, where the sequence encoding the adenoviral death protein (ADP) is retained. The sequence encoding pIX is inserted in reverse orientation into the E4 region. pIX is involved in stabilizing the lattice arrangement of hexon molecules in the adenoviral capsid.

[0040] Specifically, the Ad5 (3551-4070) fragment, encoding the pIX protein, was amplified using plasmid pBHG10 as a template. The pBHG10 (2682-2866) and pBHG10 (5612-7286) fragments were also amplified using plasmid pBHG10 as a template. The pBHG10 (5612-7286) fragment and the Ad5 (4091-5196) fragment share homologous sequences, allowing for homologous recombination during subsequent transfection. The Ad5 (29485-29771) fragment, encoding the adenovirus death protein (ADP), was amplified using plasmid pFG140 as a template, and the pBHG10 (26924-547) fragment was also amplified using pBHG10 as a template. Insert pBHG10 (2682-2866), pBHG10 (5612-7286), Ad5 (29485-29771) and pBHG10 (26924-547) fragments into pBHG10 vector, and then insert Ad5 (3551-4070) fragment in reverse direction to obtain the complete right plasmid. Figure 2 shown.

[0041] Identification of the right plasmid: The right plasmid was digested with restriction endonuclease EcoR I using Cutsmart digestion buffer at 37°C for 30-60 min. The digestion product was run on a 0.8% agarose gel to observe the bands. The results are as follows Figure 3 As shown in Figure B, the results showed that two bands, 25531 bp and 776 bp, were observed, which was consistent with expectations. The plasmid was subsequently fully sequenced, and the plasmid construction was confirmed to be successful based on the sequencing results.

[0042] The samples were sent for sequencing, and the left and right plasmids were sequenced using the Sanger sequencing method. The sequencing results were consistent with the map, indicating that the left and right plasmids were successfully constructed.

[0043] The plasmid on the left and the plasmid structure and recombination process on the right are shown in the figure. Figure 2 The specific preparation process of the carrier is as follows:

[0044] 1. Specific process of left-side plasmid construction:

[0045] A. PCR amplify the Ad5(1-1668) fragment using H101 virus as a template, with the downstream primer containing an EcoRV enzyme site. PCR amplify the Ad5(4091-5196) fragment using pBHG10 as a template, with the upstream primer containing an EcoRV enzyme site. Amplify the Ad5(1-1668)-Ad5(4091-5196) fragment using the Ad5(1-1668) and Ad5(4091-5196) fragments as templates; the reaction system is shown in Table 1; the reaction schedule is: 98°C for 10 s; 55°C for 15 s; 72°C for 45 s; and 30 cycles.

[0046] Table 1: Reaction system

[0047]

[0048]

[0049] B. Amplify the functional gene element CMV-hGM-CSF-SV40 by PCR. Recover the tapped rubber and set aside. The reaction system for amplifying the functional gene element CMV-hGM-CSF-SV40 is shown in Table 1. The reaction procedure is: 98°C for 10 seconds; 55°C for 15 seconds; 72°C for 20 seconds; and 30 cycles.

[0050] C. Insert the Ad5(1-1668)-Ad5(4091-5196) fragment into the pGEM-7Zf(+) vector digested with EcoRI / BamHI by homologous recombination. Screen positive clones, extract plasmids, and send for sequencing. PCR screen positive clones, extract plasmids, and send for sequencing. The PCR reaction system is shown in Table 2. Ligate at 50°C for 50 min.

[0051] Table 2: PCR screening positive clone reaction system

[0052]

[0053] D. Insert the CMV-hGM-CSF-SV40 fragment into the pGEM-7Zf(+)-Ad5(1-1668)-Ad5(4091-5196) vector linearized with EcoRV using homologous recombination. Screen positive clones using PCR, extract plasmids, and send for sequencing. The reaction system is shown in Table 3; ligation is performed at 50°C for 50 min.

[0054] Table 3: Reaction system

[0055]

[0056] pGEM-7Zf(+)-Ad5(1-1668)-CMV-hGM-CSF-SV40-Ad5(4091-5196) is the complete left end plasmid.

[0057] 2. The specific process of constructing the right-side plasmid is as follows:

[0058] A. Using pBHG10 as a template, PCR amplify fragment 10(26924-547), retaining the upstream PacI site and adding a downstream ClaI site. Insert fragment 10(26924-5476) into pGEM-7Zf(+) vector digested with EcoRI / ClaI and transform into DH5a. Screen positive clones and extract plasmids for sequencing. The reaction system is shown in Tables 1 and 4. The reaction procedure is: 98°C for 10 s; 55°C for 15 s; 72°C for 90 s; 30 cycles. Ligation is then performed at 50°C for 50 min.

[0059] Table 4: Reaction system

[0060]

[0061] B. Using pBHG10 as a template, PCR amplify the Ad5(3551-4070) fragment and insert it in reverse order into the SnaBI-linearized pGEM-7Zf(+)-10(26924-547) fragment. Positive clones were screened and plasmids were isolated for sequencing. The reaction system is shown in Tables 1 and 5. The reaction schedule was 98°C for 10 seconds, 55°C for 15 seconds, and 72°C for 15 seconds, followed by 30 cycles. Ligation was then performed at 50°C for 50 minutes.

[0062] Table 5: Reaction system

[0063]

[0064] C. The sequenced pGEM-7Zf(+)-10(26924-547)-reverseAd5(3551-4070) plasmid was digested with PacI / ClaI to recover the large fragment. This fragment was ligated with the pBHG10 vector digested with PacI / ClaI. The resulting plasmid was designated Plasmid A. The reaction system is shown in Tables 6 and 7. Ligation was carried out at room temperature for 2 hours.

[0065] Table 6

[0066]

[0067]

[0068] Table 7

[0069]

[0070] D. Using pBHG10 as a template, PCR amplify the 10 fragments (2682-2866). Using pBHG10 as a template, PCR amplify the 10 fragments (5612-7286). The reaction system is shown in Table 1. The reaction program is: 98°C for 10 seconds; 55°C for 15 seconds; 72°C for 30 seconds; and 30 cycles.

[0071] E. Using fragments 10(2682-2866) and 10(5612-7286) as templates, PCR amplified the fragment 10(2682-2866)-10(5612-7286). This fragment was then inserted into transformed DH5a cells containing plasmid A linearized with BstZ17I. Positive clones were screened, and plasmids were collected for sequencing, designated as plasmid B. The reaction system is shown in Tables 1 and 8. The reaction procedure was: 98°C for 10 s; 55°C for 15 s; 72°C for 30 s; and 30 cycles. Ligation was performed at 50°C for 50 min.

[0072] Table 8

[0073]

[0074] F. Using pFG140 as a template, PCR amplify the Ad5 (29485-29771) fragment. Linearize plasmid B with PacI, and insert Ad5 (29485-29771) into the linearized plasmid B. The resulting plasmid is the intact right-end plasmid. The reaction system is shown in Tables 1 and 9, with the following procedures: 98°C for 10 s; 55°C for 15 s; 72°C for 15 s; and 30 cycles.

[0075] Table 9

[0076]

[0077] Viral packaging: Linearize the left and right plasmids using the restriction endonuclease Pac I and digest them at 37°C for 60 minutes. Inactivate the restriction endonuclease Pac I by incubating at 65°C for 30-60 minutes. Co-transfect HEK293 cells with the linearized plasmids using PEI transfection reagent when the degree of polymerization is 80%-90%, and observe the cell status daily. The specific steps are as follows:

[0078] (1) Prepare transfection solutions A and B: Add the recombinant plasmid to 100 μl of Opti-MEM medium, gently flick to mix (1 μg each of the left-end plasmid and the right-end plasmid), and then centrifuge instantly to obtain transfection solution A; add 6 μl of PEI transfection reagent to 100 μl of Opti-MEM medium, shake to mix, and then centrifuge instantly to obtain transfection solution B.

[0079] (2) Add all of Transfection Solution B to Transfection Solution A, flick gently to mix, and centrifuge immediately to obtain Transfection Solution C. Let stand at room temperature for 15 minutes.

[0080] (3) Aspirate all the culture medium in the 6-well plate and add 1 ml of serum-free Opti-MEM medium.

[0081] (4) Add all the transfection solution C to the 6-well plate, shake it crosswise to mix, and place it in a constant temperature incubator at 37°C and 5% CO2 for 4 hours.

[0082] (5) After 4 hours, aspirate all the culture medium and add 2 ml of DMEM medium containing 2% FBS to each well. Culture in a constant temperature incubator at 37°C and 5% CO2 for 10-14 days.

[0083] (6) Observe the cell status every day and add 1 ml of DMEM medium containing 2% FBS to each well every 5 days.

[0084] 10-14 days after transfection, HEK293 cells will show pathological effects, with more than 50% of cells falling off the bottom of the culture flask. At this time, the cell suspension is collected, frozen and thawed three times, and centrifuged at 1000×g for 10 minutes to obtain the recombinant virus suspension.

[0085] 10-14 days after transfection, HEK293 cells will show pathological effects, with more than 50% of cells falling off the bottom of the culture flask. At this time, the cell suspension is collected, frozen and thawed three times, and then centrifuged at 1000×g for 5-10 minutes to collect the supernatant to obtain the recombinant virus suspension. Figure 4 shown.

[0086] Virus identification: Extract viral genomic DNA from the recombinant virus suspension and digest it with restriction endonucleases BstZ17 I and Pme I using Cutsmart digestion buffer at 37°C for 2-3 hours. Observe the bands on a 0.5% agarose gel electrophoresis of the digested product. Figure 5 As shown, three bands of 20860-bp, 7490-bp, and 4722-bp were observed, which was in line with expectations.

[0087] PCR was performed using viral genomic DNA as a template to determine the success of the recombination. The primers were designed as follows: F: 5'-cccggg atc cat aat cag cca tac c-3'; R: 5'-ctc tca agt ctg tat acg ggg aca cg-3'. The PCR system for recombinant virus identification is shown in Table 10, and the PCR procedure for recombinant virus identification is shown in Table 11.

[0088] Table 10 PCR system for identification of recombinant viruses

[0089]

[0090] Table 11 PCR program for recombinant virus identification

[0091]

[0092] The PCR product was electrophoresed on 1% agarose gel to observe the bands. The electrophoresis results were as follows: Figure 6 As shown, a 1934-bp band was observed, which was in line with expectations.

[0093] The viral genomic DNA was subsequently sequenced to fully verify the structure of the recombinant virus.

[0094] The samples were sent for sequencing, and the viral genomic DNA was sequenced using the Sanger sequencing method. Figure 8 As shown, the results were consistent with the map, indicating that the recombinant virus was successfully constructed.

[0095] Virus Passaging: The harvested virus suspension is inoculated onto HEK293 cells at a confluency of 80%-90%. After 48-72 hours, the HEK293 cells show pathological changes, with more than 50% detaching from the bottom of the culture flask. The cell suspension is then harvested. The cell suspension is centrifuged at 1000×g at 4°C for 5-10 minutes. The supernatant is discarded, and the pellet is resuspended in 0.5ml of DMEM medium. After freeze-thawing three times, the harvested virus suspension is inoculated onto HEK293 cells at a confluency of 80%-90%. This completes one viral passage.

[0096] The harvested low RCA recombinant adenovirus type 5 and control virus were passaged 12 times in HEK293 cells. Figure 1 The replication-defective adenovirus type 5 with E1 and E3 regions deleted (RD rAd5) and the recombinant adenovirus with part of E1B and E3 regions deleted (R rAd5) are shown in FIG.

[0097] Virus purification: Because low-RCA recombinant adenovirus type 5 is packaged and amplified in HEK293, the extraction of viral genomic DNA may be contaminated with HEK293 cell genomic DNA, which may affect the subsequent quantitative detection of reverse mutations. Therefore, Benzonase nuclease is first added to the harvested virus suspension and incubated at 37°C for 30-60 minutes to remove some residual nucleic acids. The harvested virus suspension is then purified using the PureVirus Adenovirus Purification Kit (Cell Biolabs, USA) to remove residual proteins and nucleic acids.

[0098] Quantitative analysis of reverse mutations in the virus: Virus particles and reverse mutations were quantified using 50 μl of the harvested virus suspension from passages 2, 4, 6, 8, 10, and 12. Genomic DNA was extracted from the harvested virus suspension. Fluorescence quantitative PCR was used to quantify the number of viral particles and reverse mutations. The viral genomic DNA was diluted 100-fold for quantification of viral particle number, but not diluted for quantification of reverse mutations. The fluorescence quantitative PCR system is shown in Table 12; the fluorescence quantitative PCR procedure is shown in Table 13.

[0099] Table 12 Fluorescence quantitative PCR system

[0100]

[0101]

[0102] Table 13 Fluorescence quantitative PCR program

[0103]

[0104] The primers and probes for quantifying the number of viral particles are: F: 5'-cgg gtc aaa gtt ggc gtt t-3'; R: 5'-ctctac tcg ctg gca ctc aa-3', probe: FAM-tat acc cgg tga gtt cct caa gag gc-tamra; the primers and probes for quantifying reverse mutations are: F: 5'-tag cgg tac ggt ttt cct gg-3'; R: 5'-ccg aaccct tac atc ggt cc-3', probe: FAM-tat acc cgg tga gtt cct caa gag gc-tamra.

[0105] The results are as follows Figure 7 As shown, the results showed that: the amount of reversion mutation of low RCA recombinant type 5 adenovirus during serial passage remained at a low level; the amount of reversion mutation of replication-deficient type 5 adenovirus during serial passage increased with the increase of passage number; the amount of reversion mutation of recombinant type 5 adenovirus during serial passage remained at a high level.

[0106] The experimental results show that the low RCA recombinant adenovirus type 5 constructed by the present invention can achieve the purpose of reducing the frequency of reversion mutations, and will not produce more reversion mutations during continuous passage.

[0107] 2. Verification experiment of the effect of the obtained recombinant vector

[0108] The harvested low RCA recombinant adenovirus type 5 was used to infect HEK293 cells with a polymerization degree of 80-90%. The cells were cultured in an incubator at 37°C and 5% CO2. When more than 50% of the cells showed pathological effects and fell off from the bottom of the culture flask, the cell suspension was collected and centrifuged at 4°C and 1000×g for 10 minutes. The supernatant was discarded and the cell pellet was resuspended in 500μl DMEM medium. After freezing and thawing three times, the harvested virus suspension was inoculated into HEK293 cells with a polymerization degree of 80%-90%, completing one virus passage.

[0109] The harvested low RCA recombinant adenovirus type 5 and control virus were passaged 12 times in HEK293 cells. Figure 1 The replication-defective adenovirus type 5 with E1 and E3 regions deleted (RD rAd5) and the recombinant adenovirus with part of E1B and E3 regions deleted (R rAd5) are shown in FIG.

[0110] After harvesting the 12th passage of recombinant virus, viruses from passages 2, 4, 6, 8, 10, and 12 were selected for purification. Benzonase nuclease was first added to the harvested virus suspension and incubated at 37°C for 30–60 min to remove any residual nucleic acid. The harvested virus suspension was then purified using the PureVirus Adenovirus Purification Kit (Cell Biolabs, USA) to remove residual protein and nucleic acid. Approximately 450 μl of virus suspension was obtained after purification.

[0111] 50 μl of the purified virus suspension was taken and the viral genomic DNA was extracted using the QIAamp MinElute Virus Spin Kit. The number of viral particles and the number of reverse mutations were quantified using fluorescent quantitative PCR. The fluorescent quantitative PCR system and procedure are shown in Tables 3 and 4. The viral genomic DNA was diluted 100-fold for quantification of viral particles, but not diluted for quantification of reverse mutations. The result was judged as per 1×10 8 The number of reverse mutations in the number of virus particles is as follows Figure 7 The results showed that the number of reversion mutations in the low-RCA recombinant adenovirus type 5 remained low during serial passage; the number of reversion mutations in the replication-deficient adenovirus type 5 increased with increasing passage number; and the number of reversion mutations in the replication-competent recombinant adenovirus type 5 remained high during serial passage.

[0112] According to the requirements of the U.S. Food and Drug Administration (FDA), 10 The result judgment standard of the present invention is that every 1×108 The number of reverse mutations in the number of virus particles is as follows Figure 7 The experimental results show that the low RCA recombinant adenovirus type 5 constructed by the present invention can achieve the effect of reducing the frequency of reversion mutations, and will not produce more reversion mutations during continuous passage.

[0113] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A recombinant adenovirus type 5 vector with reduced back mutation frequency, characterized in that: The recombinant adenovirus type 5 vector with reduced back mutation frequency is prepared by the following method: by knocking out the E1B region gene of human adenovirus type 5, and knocking out to base 4090, the length of the homologous sequence between adenovirus type 5 and HEK293 genomic DNA is shortened to 254 bp, and the deleted E1 region gene segment includes the sequence Ad5 3551-4070 encoding pIX; then, the sequence Ad5 3551-4070 encoding pIX is reversely inserted into the E4 region, i.e., Ad5 35771; The nucleic acid sequence of the recombinant adenovirus type 5 vector is shown in Seq no. 1.

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