HEK293 derived cell strain capable of stably expressing adenovirus ADP, and construction method and application of HEK293 derived cell strain

Abstract

The invention discloses an HEK293 derived cell strain capable of stably expressing adenovirus ADP as well as a construction method and application of the HEK293 derived cell strain. The preservation number of the cell strain is GDMCC No.65235. According to the cell strain, an ADP gene complete ORF of an Ad5E3 region and an expression cassette of the ADP gene complete ORF are stably inserted into an AAVS1 site of HEK293. Compared with an HEK293 cell, the derivative cell strain can promote the formation of replication-deficient Ad5 vector plaques, increase the copy number of the replication-deficient Ad5 vector and improve the specific activity of the replication-deficient Ad5 vector, can be applied to the production of Ad5 vector vaccines and gene therapy products, increase the yield and reduce the industrialization cost, and has transformation application value.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a HEK293-derived cell line stably expressing adenovirus ADP and a construction method and application thereof. Background Art

[0002] Adenovirus (Ad) is a double-stranded DNA virus with a genome length of about 35-40kb. Currently, more than 114 types have been found, which are divided into 7 subgroups (A~G). Adenovirus has been widely developed and applied in vaccine vectors and gene therapy, among which Ad5 of subgroup C is the most mature. Commonly used adenovirus vectors are replication-defective vectors with E1 and E3 genes knocked out. The genes in the E1 region of adenovirus are essential genes for its replication, and the genes in the E3 region are key genes against host immune responses. At present, the commonly used cell line for the production of replication-defective adenovirus vectors is HEK293, which stably expresses the 1-4344nt genes in the E1 region of adenovirus Ad5, which plays a trans-complementary role in the growth and replication of Ad5. However, this cell line has the disadvantages of producing replication-defective Ad5 vectors, such as contamination with adenovirus (RCA) with replication ability, low production efficiency, and low specific activity. Studies have shown that the E3 region of adenovirus mainly encodes 5 protein products, among which the main function of the ADP gene is to promote cell lysis and promote viral release and spread. Based on the biological function of ADP in promoting cell lysis, in the study of oncolytic viruses, it was attempted to be recombined into the E3-knocked Ad5 vector, and A549 cells were used to produce and amplify normally. There are also studies that insert the ADP gene of Ad5, Ad41, etc. back into the E3 region of the adenovirus, and the replicative adenovirus vector obtained can improve viral growth in cell lines such as 293TE32. However, the existing technical strategies all replenish ADP to the viral vector genome itself, and only study the replicative adenovirus vector, and no direct experimental comparison of the effect of ADP on adenovirus production has been conducted. Moreover, the early strategy requires the individual modification of each applied viral vector, which has the disadvantages of multiple promoters and reduced load, and the application is limited. It is necessary to develop a more generally applicable industrial strategy to increase the production of replication-defective adenoviruses. The transformation and improvement of cell lines can be widely applied to the production of all Ad5 vectors. Therefore, starting with cells is the preferred strategy.

[0003] Therefore, it is necessary to develop a cell line that can improve the production and specific activity of replication-defective adenoviral vectors. Summary of the invention

[0004] The object of the present invention is to provide a HEK293-derived cell strain stably expressing adenovirus ADP and a construction method and application thereof. Compared with HEK293 cells, the derived cell strain can promote the formation of replication-defective Ad5 vector plaques, increase the copy number of replication-defective Ad5 vectors, and enhance the specific activity of replication-defective Ad5 vectors. It can be applied to the production of Ad5 vector vaccines, increase the yield, reduce the industrialization cost, and has transformation application value.

[0005] In order to achieve the above object, the present invention adopts the following technical solution:

[0006] In a first aspect of the present invention, a HEK293-derived cell line stably expressing adenovirus ADP is provided, and the deposit number of the cell line is GDMCC No.65235.

[0007] In the second aspect of the present invention, a method for constructing the HEK293-derived cell line stably expressing adenovirus ADP is provided, the method comprising:

[0008] The ADP gene fragment was obtained, digested with HindIII+NotI, and then ligated with the pEGFP-N1 vector digested with the same enzymes to obtain the pAS1-5ADP plasmid;

[0009] The sgRNA targeting AAVS1 was annealed to synthesize a double-stranded fragment and connected to the pX330 vector after being digested by bBSI to obtain the pX330-AAVSI plasmid;

[0010] The pAS1-5ADP plasmid and the pX330-AAVSI plasmid were co-transfected into HEK293 cells, and then pressure screening and selection and identification were performed in a medium containing puromycin to obtain the HEK293-derived cell line stably expressing adenovirus ADP.

[0011] Furthermore, the ADP fragment is obtained by PCR using the Ad5 genome as a template and the primers shown in the sequences SEQ ID NO.1 and SEQ ID NO.2.

[0012] Furthermore, the nucleotide sequence of the ADP gene fragment is shown as SEQ ID NO.5, and the amino acid sequence is shown as SEQ ID NO.6.

[0013] Furthermore, the nucleotide sequence of the sgRNA is shown in SEQ ID NO.3-SEQ ID NO.4.

[0014] Furthermore, the pressure screening of the puromycin-containing medium includes:

[0015] The pAS1-5ADP plasmid and the pX330-AAVSI plasmid are co-transfected into HEK293 cells, and then the cells are replaced with DMEM containing 10% FBS after 16-18 hours. After 24-25 hours, the cells are pressure-screened by replacing the culture medium containing puromycin, and control wells are set at the same time, and the HEK293 cells in the control wells are not transfected;

[0016] When the HEK293 cells in the control wells were almost all dead, the HEK293 cells in the co-transfection wells were passaged and continuously pressure-screened with DMEM medium containing puromycin, and monoclonal cells were picked by limiting dilution method, and multiple rounds of monoclonal selection were performed.

[0017] In the third aspect of the present invention, there is provided the use of the HEK293-derived cell line stably expressing adenovirus ADP in the production of replication-deficient Ad5 vector vaccines.

[0018] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0019] 1. Compared with HEK293 cells, the HEK293-derived cell line (293-ADP) stably expressing adenovirus type 5 ADP of the present application can promote the formation of replication-defective Ad5 vector plaques, increase the copy number of replication-defective Ad5 vectors, increase the titer of replication-defective Ad5 vaccines, and thus improve the specific activity of replication-defective Ad5 vectors. It can be applied to the production of replication-defective Ad5 vector vaccines, increase production, reduce industrialization costs, and has transformation application value.

[0020] 2. The HEK293-derived cell line stably expressing adenovirus ADP of the present application is used for the production of replication-defective Ad5 vector vaccines or Ad5 gene therapy vectors, and its yield is higher than that of the HEK293 cell line under the same conditions. If used in industrial production, it can reduce the production cost of replication-defective Ad5 vectors and has significant economic value.

[0021] The deposit date of the HEK293-derived cell line stably expressing adenovirus ADP of the present invention is October 10, 2024, the deposit number is GDMCC No.65235, and the taxonomic name is human embryonic kidney cell (HEK293) 293-ADP. The name of the depository is Guangdong Microbiological Culture Collection Center, and the address is Institute of Microbiology, Guangdong Academy of Sciences, Guangzhou, Guangdong, China, Postal Code: 510075. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0023] Figure 1 The plasmid map used to construct the cell line of the present invention, wherein: Figure 1 A is the map of pAS1-5ADP plasmid, Figure 1 B is the map of pX330-AAVSI plasmid.

[0024] Figure 2 This is the identification result of 293-ADP cell line, among which, Figure 2 A is the result of extracting the whole genome of 293-ADP cells of different generations, using primers Ad5ADPF+Ad5ADPR2 PCR to obtain the specific ADP target gene in 293-ADP cells. Figure 2 B is the PCR identification of the ADP target gene band in the total mRNA of cells of different generations. Figure 2 C is the result of QPCR test proving the existence of high level of ADP mRNA transcription in different generations of 293-ADP. Figure 2 D is the result of the identification of 293-ADP cell proliferation ability. Figure 2 E is the result of detecting the activity of 293-ADP cells using CCK8 kit.

[0025] Figure 3 The plaque formation results of 293-ADP cell line infected with replication-deficient Ad5-EGFP, among which, Figure 3 A shows the plaque formation of Ad5-EGFP in HEK293 and 293-ADP cells. Figure 3 B shows that the diameter of the plaques formed in 293-ADP is significantly larger than that in HEK293 cells, and the number of plaques is significantly larger.

[0026] Figure 4 The replication-deficient Ad5 vector vaccine is produced using the 293-ADP cell line with high yield, among which, Figure 4 A is the comparison of genome copy numbers of replication-defective Ad5 vector vaccines in 293-ADP and HEK293 cells. Figure 4 B is the titer comparison result of the replication-defective Ad5 vector vaccine produced in 293-ADP cell line and HEK293 cells. DETAILED DESCRIPTION

[0027] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0028] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0029] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0030] The overall idea of ​​the present invention is:

[0031] Studies have shown that the adenovirus E3 region mainly encodes five protein products, among which the main function of the ADP gene is to promote cell lysis and promote virus release and spread. It is proved that ADP may have a positive effect in the release of packaged mature adenovirus particles and the initiation of a new round of infection. It can promote the release and spread of virus particles, shorten the intracellular packaging and replication cycle, and improve the virus production efficiency within the same production time, thereby increasing the virus yield.

[0032] Therefore, the inventors of the present application inserted the ADP gene in the E3 region of Ad5 into HEK293 cells and stably expressed it. After screening, the cell strain of the present application was obtained and preserved. Other cell strains screened by the same method were not preserved.

[0033] The inserted ADP gene sequence is:

[0034] ATGACCAACACAACCAACGCGGCCGCCGCTACCGGACTTACATCTACCACAAATACACCCCAAGTTTCTGCCTTTGTCAATAACTGGGATAACTTGGGCATGTGGTGGTTCTCCATAGCGCTTATGTTTGTATGCCTTATTAT TATGTGGCTCATCTGCTGCCTAAAGCGCAAACGCGCCCGACCACCCATCTATAGTCCCATCATTGTGCTACACCCAAACAATGATGGAATCCATAGATTGGACGGACTGAAACACATGTTCTTTTCTCTTACAGTATGA(SEQ ID NO.5);

[0035] Insert the ADP amino acid sequence as:

[0036] MTNTTNAAAATGLTSTTNTPQVSAFVNNWDNLGMWWFSIALMF VCLIIMWLICCLKRKRARPPIYSPIIVLHPNNDGIHRLDGLKHMFFSLTV*(SEQ ID NO.6);

[0037] Through the effect experiment, it was found that compared with the HEK293 cells commonly used for adenovirus vector production, the cell line 293-ADP of the present application increased the copy number of the replication-defective Ad5 genome by 7.3 times in 48 hours, and the virus titer by 5.2 times, which was the highest among the five verified monoclonal 293-ADP cells. This shows that the HEK293-derived cell line stably expressing adenovirus ADP deposited in the present application has produced unexpected technical effects.

[0038] The effects of the present application will be described in detail below in conjunction with the examples and experimental data. Unless otherwise specified, the molecular cloning methods, cell culture methods and various detection methods mentioned in the following schemes are all traditional experimental methods and can be obtained by consulting the literature; the relevant reagents used can be purchased from the corresponding reagent suppliers.

[0039] Example 1. Obtaining a HEK293-derived cell line that stably expresses adenovirus ADP

[0040] 1. Construction of plasmid

[0041] The HEK293 cell line overexpressing ADP was constructed using CRISPR-Cas9 technology, so the double plasmid was constructed as follows:

[0042] 1. Donor plasmid construction:

[0043] Using the Ad5 genome as a template, the ADP gene sequence was obtained by PCR with the following primers, digested with HindIII+NotI, and ligated with the pEGFP-N1 vector digested with the same enzymes to obtain the pAS1-5ADP plasmid ( Figure 1 A).

[0044] Table 1

[0045]

[0046] 2. Construction of Cas9 plasmid containing sgRNA:

[0047] 1) Design and synthesize gRNA (as shown in Table 2), and anneal to synthesize double-stranded products;

[0048] Table 2

[0049] name Sequence (5'-3') gRNA1 CACCGGGGCCACTAGGGACAGGAT(SEQ ID NO.3) gRNA2 AAACATCCTGTCCCTAGTGGCCCC(SEQ ID NO.4)

[0050] 2) pX330 vector (commercially available) was digested with bBSI (Bpil) to recover the 8488 bp vector fragment;

[0051] 3) The double-stranded product was ligated with the vector fragment using T4 ligase, and the pX330-AAVSI plasmid was obtained after transformation ( Figure 1 B).

[0052] 2. Construction and identification of cell lines

[0053] 1. HEK293 cells were plated in 6-well plates. After 24 hours, pAS1-5ADP and pX330-AAVSI plasmids were co-transfected into cells. After 16 hours, the medium was changed to DMEM containing 10% FBS. After 24 hours, the medium was changed to puromycin-containing medium to pressure screen the cells.

[0054] 2. When almost all HEK293 cells in the control wells died, the cells in the transfected wells were passaged and continuously pressure-screened with DMEM medium containing puromycin. Monoclonal cells were picked by limiting dilution method, and after 2 rounds of monoclonal selection and functional verification, the cells were named 293-ADP (the deposit number of this cell line is GDMCC No.65235). Other selected monoclonal cell lines were not deposited and were named 293-ADP 2#, 293-ADP 3#, and 293-ADP 4#.

[0055] 3. Identification of ADP in the genome of 293-ADP cell line. The whole genome of 293-ADP cells was extracted, and the specific ADP target gene in 293-ADP cells was obtained by PCR with primers Ad5ADPF+Ad5ADPR2 ( Figure 2 A), sent for sequencing, proving that the insertion of ADP into the genome was completely correct.

[0056] 4. Identification of ADP transcriptional expression in 293-ADP cell lines. Monoclonal 293-ADP cells were serially passaged, and total cell mRNA was extracted and reverse transcribed into cDNA. PCR was used to identify the ADP target gene band in cDNA ( Figure 2 B), QPCR detection proved that there was a high level of ADP mRNA transcription in 293-ADP ( Figure 2 C), and the cells could be stably passaged for 25 generations, while no ADP transcription was detected in HEK293 control cells, demonstrating a high level of ADP transcription in 293-ADP cells.

[0057] 5. Identification of 293-ADP cell proliferation ability. HEK293 and 293-ADP cell lines were plated in 96-well plates at 2000 cells / well, and cells were collected at 24, 48, 96, 144, 192, and 240 hours, and the number of viable cells was calculated ( Figure 2 D), and CCK8 kit was used to detect cell activity ( Figure 2 E), the results showed that the proliferation ability of HEK293 cell line overexpressing ADP was no different from that of HEK293.

[0058] Example 2 Plaque formation experiment of cell lines infected with replication-defective Ad5 vector reporter virus

[0059] HEK293 and 293-ADP cells were plated in 6-well plates, and after culturing for 24 hours, Ad5-EGFP reporter virus was diluted and infected with multiple doses of cells. After infection, the medium was changed and DMEN without phenol red containing 0.7% agarose gel was plated. The formation of Ad5-EGFP fluorescent plaques was observed at 72 hours and 120 hours, and the number and diameter of plaques were measured by taking pictures with an inverted fluorescence microscope.

[0060] Plaque formation results Figure 3 As shown, the number of plaques formed by Ad5-EGFP in HEK293 cells is small and smaller than that in 293-ADP cells ( Figure 3 A), by measuring the diameter of all plaques in the fixed area, the results showed that the diameter of plaques formed in 293-ADP was significantly larger than that in HEK293 cells ( Figure 3 B) confirmed that the propagation and proliferation rate of a single replication-deficient Ad5 using the 293-ADP cell line was significantly faster than that of HEK293 cells.

[0061] Example 3 Variations in the production of replication-deficient Ad5 vector vaccines in 293-ADP cell lines

[0062] HEK293 and multiple monoclonal cells overexpressing ADP (293-ADP in the present application, and undeposited 293-ADP 2#, 293-ADP 3#, and 293-ADP 4#) were plated separately. After culturing for 24 hours, the replication-deficient Ad5 COVID-19 vaccine was inoculated with 10 6 vp / well infected cells, total cell DNA was extracted 24h and 48h after infection, and the copy number of replication-defective Ad5 genome in the cells was determined by QPCR.

[0063] The results are as follows Figure 4 As shown in Figure 2, the copy number of replication-deficient Ad5 genome in multiple monoclonal cell lines overexpressing ADP for 48 h was significantly higher than that in HEK293 cells, and the copy number of 293-ADP cells was 7.3 times higher than that of HEK293 cells ( Figure 4A), and using the antibody staining method, it was determined that the titers of the replication-deficient Ad5 vaccine produced in multiple monoclonal cell lines overexpressing ADP were higher than those of HEK293 cells, among which 293-ADP had the highest titer, which was 5.2 times that of HEK293 ( Figure 4 B).

[0064] It can be seen from the above that the HEK293-derived cell line (293-ADP) stably expressing adenovirus ADP deposited in the present application has produced unexpected technical effects.

[0065] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

[0066] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.

[0067] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0068] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A HEK293-derived cell line stably expressing adenovirus ADP, characterized in that: The deposit number of the cell line is GDMCC No.65235.

2. A method for constructing a HEK293-derived cell line stably expressing adenovirus ADP according to claim 1, characterized in that: The method comprises: The ADP gene fragment was obtained, digested with HindIII+NotI, and then ligated with the pEGFP-N1 vector digested with the same enzymes to obtain the pAS1-5ADP plasmid; The sgRNA targeting AAVS1 was annealed to synthesize a double-stranded fragment and connected to the pX330 vector after being digested by bBSI to obtain the pX330-AAVSI plasmid; The pAS1-5ADP plasmid and the pX330-AAVSI plasmid were co-transfected into HEK293 cells, and the HEK293-derived cell line stably expressing adenovirus ADP was obtained through pressure screening and selection and identification in a medium containing puromycin.

3. The construction method according to claim 2, characterized in that: The ADP fragment is obtained by PCR using the Ad5 genome as a template and the primers shown in the sequences SEQ ID NO.1 and SEQ ID NO.

2.

4. The construction method according to claim 2, characterized in that: The nucleotide sequence of the ADP gene fragment is shown as SEQ ID NO.

5.

5. The construction method according to claim 2, characterized in that: The nucleotide sequence of the sgRNA is shown in SEQ ID NO.3-SEQ ID NO.

4.

6. The construction method according to claim 2, characterized in that: The pX330 vector fragment is obtained by digesting the pX330 vector with bBSI.

7. The construction method according to claim 2, characterized in that: The pressure screening of the puromycin-containing culture medium comprises: The pAS1-5ADP plasmid and the pX330-AAVSI plasmid are co-transfected into HEK293 cells, and after 16-18 hours, the cells are replaced with DMEM containing 10% FBS, and after 24-25 hours, the cells are pressure screened with a culture medium containing puromycin, and a control well is set at the same time, and the HEK293 cells in the control well are not transfected; When almost all the HEK293 cells in the control wells died, the co-transfected HEK293 cells were passaged and continuously pressure-screened with DMEM medium containing puromycin, and single clones were picked by limiting dilution method.

8. Use of the HEK293-derived cell line stably expressing adenovirus ADP according to claim 1 in preparing a replication-deficient Ad5 vector vaccine.

9. Use of the HEK293-derived cell line stably expressing adenovirus ADP according to claim 1 in the preparation of gene therapy products.

10. A replication-deficient Ad5 vector vaccine, characterized in that: The replication-deficient Ad5 vector vaccine is produced by using the HEK293-derived cell line stably expressing adenovirus ADP as described in claim 1.

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