WO2021139548A1

WO2021139548A1 - Expression vector based on chimpanzee chad 63-type adenovirus and construction method thereof

Info

Publication number: WO2021139548A1
Application number: PCT/CN2020/139819
Authority: WO
Inventors: 张超; 刘海涛; 侯永凡; 张玲莉; 周晨亮; 周东明; 刘革; 史力; 莫呈钧; 张智
Original assignee: 怡道生物科技(苏州)有限公司
Priority date: 2020-01-08
Filing date: 2020-12-28
Publication date: 2021-07-15
Also published as: ZA202208391B; CN113088530A

Abstract

A method of constructing an expression vector based on chimpanzee ChAd 63-type adenovirus. The method uses wild-type chimpanzee ChAd 63-type adenovirus to construct a vector capable of expressing exogenous genes. A chimpanzee ChAd 63-type adenovirus vector and a novel method for constructing an adenovirus vector from scratch are thus provided for the research and development of novel adenovirus vector-related products as well as for biomedical basic research thereof. The method is simple and precise, and allows for controllable quality and cost, shorter processing time, and lower sequence mutation risk. In addition, advance acquisition of wild-type strains is not required, improving the general applicability of the method.

Description

An expression vector based on chimpanzee ChAd63 adenovirus and its construction method

Technical field

The present invention belongs to the fields of biotechnology and virology; more specifically, the present invention relates to an expression vector based on a chimpanzee ChAd63 adenovirus and a construction method thereof.

Background technique

Adenovirus belongs to the family of Adenovirus and is a medium-sized (70-90nm in diameter) non-enveloped double-stranded DNA virus. In the 1950s, adenovirus was first isolated from human glands (Rowe et al. 1953). In the following forty years, adenovirus vectors have been widely used in gene therapy, oncolytic viruses, and vaccine development (Chen et al. 2016). As of 2019, four adenovirus-related products have been approved for the market worldwide, including an attenuated oral vaccine for adenovirus type 4 and type 7 infection (Adenovirus Type 4 and Type 7 Vaccine, Live, Oral, U.S.). Recombinant human type 5 adenovirus injection (Ankerui), recombinant human p53 adenovirus injection (now born again), and recombinant Ebola virus adenovirus vector vaccine (Pearson et al. 2004, Wu et al. 2017). In addition, there are more than 200 clinical trials related to adenovirus vectors in the world. Adenovirus-based medical products have played an increasingly important role in the field of gene therapy, tumor therapy and vaccine research and development, and it is of great significance to develop products based on adenovirus vectors.

The genome of adenovirus is about 30-40kb in size and encodes 30-40 genes, divided into early genes and late genes. At present, more than one hundred adenovirus serotypes have been isolated and identified (Alonso-Padilla et al. 2016). Viral vectors developed based on adenoviruses have the following advantages: 1) After adenoviruses infect cells, their genomes can enter the nucleus, so the foreign antigens of adenovirus vectors can stimulate a stronger CD8+T cell response through the endogenous protein processing pathway Reacts with antibodies; 2) the modified replication-deficient adenovirus has good safety; 3) the adenovirus genome does not integrate into the host genome; 4) the adenovirus is invasive to many types of cells; 4) Adenovirus vectors can load foreign genes up to 8kb; 5) Adenovirus vectors have longer timeliness for expressing foreign genes in vivo; 6) Adenovirus amplification and purification technology is mature, and higher titer virus particles can be obtained ( Vannucci et al. 2013).

The most commonly used adenovirus vector type in clinical trials is human adenovirus type 5 (HuAd5), including the three adenovirus-related products approved for marketing in China, all of which use HuAd5 as the carrier. However, because 40-60% of people have been naturally infected by HuAd5, there are already neutralizing antibodies against viral vectors in their bodies, and the pre-existing antibodies will greatly inhibit the expression efficiency of foreign genes and increase the vector-mediated cytotoxicity (Xiang et al. 2002, Peruzzi et al. 2009). At present, it is commonly used to modify rare human adenovirus serotypes (26, 35, etc.) or use non-human primate-derived adenovirus serotypes as vaccine carriers to overcome the impact of pre-existing immunity in the population on the effect of vaccine products. For example, since the human body does not contain neutralizing antibodies against chimpanzee adenovirus, products developed based on chimpanzee adenovirus as an expression vector may have significantly better effects than human serotype adenovirus vectors. Moreover, more and more products developed based on chimpanzee adenovirus vectors also show excellent clinical safety.

There are currently two methods for constructing recombinant adenovirus vectors: homologous recombination and direct cloning. The homologous recombination method usually requires at least two plasmids, one shuttle plasmid carries the target gene, and the other backbone plasmid contains the genome sequence required for virus assembly. The two plasmids are homologously recombined in eukaryotic cells or E. coli through inverted repeat sequences or specific recombinase systems to obtain recombinant viruses or recombinant adenovirus vectors, such as AdMax system or AdEasy system. The disadvantage of this method is low recombination efficiency, easy to be contaminated by parental virus, and complicated steps. Although it is relatively convenient to use a commercial vector system based on human HuAd5, on the one hand, many product developments cannot be carried out due to patent restrictions, and because many new adenoviruses have not yet established commercial backbone vectors, it is very time-consuming and labor-intensive (Miravet et al. .2014). The direct cloning method is to transform and clone the wild-type adenovirus genome into a plasmid vector through molecular cloning, and then linearize it into cell lines to rescue the virus. The disadvantage of this method is that due to the large adenovirus genome (about 36kb), molecular cloning is quite difficult (Zhou et al. 2010, Yang et al. 2016). Moreover, since the acquisition of wild-type adenovirus usually has considerable difficulties (for example, unable to purchase, difficult to isolate, etc.), it also greatly limits the use of this method.

On the contrary, the method described in the present invention solves the above problems faced by the current construction of recombinant adenovirus vectors. The method has the advantages of simpler, accurate, controllable quality, controllable cost, shorter time, no risk of sequence mutation, no need to obtain wild-type strains first, and more universal applicability.

Summary of the invention

The purpose of the present invention is to provide an expression vector based on chimpanzee ChAd63 adenovirus and its construction method.

In the first aspect of the present invention, there is provided a method for constructing an expression vector based on a chimpanzee ChAd63 adenovirus, the method comprising the complete genome sequence of a wild-type chimpanzee ChAd63 adenovirus (GenBank, #MS912777.1), namely SEQ ID NO: The E1, E3, and E4 regions in the sequence shown in 1 are functionally deleted.

In a specific embodiment, the nucleotides in the E1 region corresponding to positions 576-3128 of the sequence shown in SEQ ID NO: 1, that is, the sequence shown in SEQ ID NO: 3, are deleted; the E3 region corresponding to SEQ ID NO : The nucleotides at positions 27917-31786 in the sequence shown in 1, that is, the sequence shown in SEQ ID NO: 4 is deleted; and the nucleotides in the E4 region corresponding to the nucleotides at positions 33825-36215 in the sequence shown in SEQ ID NO: 1, namely The sequence shown in SEQ ID NO: 5 is deleted and replaced with the E4 ORF6 expression box sequence of the full genome sequence of the chimpanzee adenovirus ChAd68 type shown in SEQ ID NO: 2. Preferably, the E4 ORF6 expression box sequence is the sequence shown in SEQ ID NO: 8.

In a further embodiment, the sequence containing the restriction site is inserted into the deleted E1 region. Preferably, the sequence containing restriction sites is a sequence containing one or more restriction sites selected from AsiSI, I-CeuI, MluI, EcoRI, and PI-SceI. More preferably, the sequence containing the restriction site is the sequence shown in SEQ ID NO:6.

Alternatively or additionally, the sequence containing the restriction site is inserted into the deleted E3 region. Preferably, the sequence containing the restriction site is a sequence containing the restriction site of HpaI and/or PacI. More preferably, the sequence containing the restriction site is the sequence shown in SEQ ID NO:7.

In a further embodiment, the foreign gene is inserted into one or more regions of the deleted E1, E3, and E4 regions. Preferably, the foreign gene is EGFP or mCherry. Preferably, EGFP is inserted into the deleted E1 region; or EGFP and mCherry are inserted into the deleted E1 and E3 regions, respectively.

In a further embodiment, the method further includes adding a linearization restriction site and inserting it into a prokaryotic replication plasmid. Preferably, the linearization restriction site is a SwaI restriction site. Preferably, the sequence of the prokaryotic replication plasmid is shown in SEQ ID NO: 9.

In the second aspect of the present invention, an expression vector based on a chimpanzee ChAd63 adenovirus is provided, which is constructed according to the method described in any one of the foregoing embodiments.

In one embodiment, the expression vector contains a modified sequence shown in SEQ ID NO: 1, in which the E1, E3, and E4 regions are functionally deleted.

In a specific embodiment, the nucleotides at positions 576-3128 in the sequence shown in SEQ ID NO: 1 in the E1 region, that is, the sequence shown in SEQ ID NO: 3 is deleted; the E3 region corresponds to SEQ ID NO: The nucleotides at positions 27917-31786 in the sequence shown in 1, that is, the sequence shown in SEQ ID NO: 4 is deleted; and the nucleotides in the E4 region corresponding to the nucleotides at positions 33825-36215 in the sequence shown in SEQ ID NO:1, that is, SEQ The sequence shown in ID NO: 5 was deleted and replaced with the E4ORF6 expression box sequence of the chimpanzee adenovirus ChAd68 type genome sequence shown in SEQ ID NO: 2. Preferably, the E4ORF6 expression box sequence is the sequence shown in SEQ ID NO: 8.

In a further embodiment, the expression vector further includes a sequence containing a restriction site.

In a specific embodiment, the sequence containing the restriction site is inserted into the deleted E1 region. Preferably, the sequence containing restriction sites is a sequence containing one or more restriction sites selected from AsiSI, I-CeuI, MluI, EcoRI, and PI-SceI. More preferably, the sequence containing the restriction site is the sequence shown in SEQ ID NO:6.

In a further embodiment, the vector further comprises a linearization restriction site and a prokaryotic replication plasmid. Preferably, the linearization restriction site is a SwaI restriction site. Preferably, the sequence of the prokaryotic replication plasmid is shown in SEQ ID NO: 9.

In one embodiment, the expression vector includes the sequence shown in SEQ ID NO: 12. In another embodiment, the sequence of the expression vector is shown in SEQ ID NO: 12.

In a further embodiment, the expression vector may also contain foreign genes.

In one embodiment, foreign genes can be inserted into one or more regions of the deleted E1, E3, and E4 regions. The exogenous gene can be any gene, and can be the same or different. Preferably, the foreign gene is EGFP or mCherry. Preferably, EGFP is inserted into the deleted E1 region; or EGFP and mCherry are inserted into the deleted E1 and E3 regions, respectively. Preferably, the sequence of the expression vector containing the foreign gene is shown in SEQ ID NO: 13 or SEQ ID NO: 14.

In the third aspect of the present invention, a host cell is provided, which comprises the expression vector according to any one of the foregoing embodiments.

In one embodiment, the host cell is a eukaryotic cell. Preferably, the eukaryotic cell is a HEK293 cell.

The fourth aspect of the present invention provides a method for packaging and amplifying recombinant adenovirus using host cells. Optionally, the harvested virus stock is purified. In one embodiment, the host cell is a eukaryotic cell. Preferably, the eukaryotic cell is a HEK293 cell.

Due to the disclosure herein, other aspects of the present invention will be apparent to those skilled in the art.

Description of the drawings

Figure 1: The genome map of the wild-type ChAd63 adenovirus and the plasmid map of the recombinant chimpanzee adenovirus vector carrying the reporter gene. A. Genome map of wild-type adenovirus ChAd63. ΔE1, ΔE3, ΔE4 respectively represent three deleted regions in the genome. B. Sequence map of recombinant chimpanzee adenovirus vector plasmid pChAd63-EGFP carrying a single reporter gene EGFP. C. Sequence map of recombinant chimpanzee adenovirus vector plasmid pChAd63-EGFP-mCherry carrying two reporter genes EGFP and mCherry at the same time.

Figure 2: Enzyme digestion map to verify the complete sequence of the recombinant chimpanzee adenovirus vector plasmid carrying the reporter gene.

Figure 3: The typical cytopathic phenomenon after the recombinant chimpanzee adenovirus carrying the reporter gene infects HEK293 cells.

Figure 4: The expression detection of the fluorescent reporter gene inserted after the recombinant chimpanzee adenovirus carrying the reporter gene was infected with HEK293 cells.

Figure 5: Enzyme digestion map to verify the complete genome sequence of the purified recombinant adenovirus ChAd63-EGFP.

Sequence description

SEQ ID NO: 1: Full sequence of wild-type chimpanzee ChAd63 adenovirus (wt-ChAd63);

SEQ ID NO: 2: Full sequence of wild-type chimpanzee adenovirus ChAd68;

SEQ ID NO: 3: Deleted E1 region sequence;

SEQ ID NO: 4: Deleted E3 region sequence;

SEQ ID NO: 5: deleted E4 region sequence;

SEQ ID NO: 6: The sequence containing the restriction site inserted into the deleted E1 region;

SEQ ID NO: 7: The sequence containing the restriction site inserted into the deleted E3 region;

SEQ ID NO: 8: ChAd68E4ORF6 sequence inserted into the deleted E4 region;

SEQ ID NO: 9: modified prokaryotic replication plasmid empty vector sequence;

SEQ ID NO: 10: The reading frame sequence of the foreign reporter gene containing EGFP inserted between the restriction sites MluI and EcoRI of the deleted E1 region;

SEQ ID NO: 11: The foreign reporter gene encoding expression reading frame sequence containing mCherry inserted between the restriction sites HpaI and PacI of the deleted E3 region;

SEQ ID NO: 12: Recombinant chimpanzee ChAd63 adenovirus empty vector plasmid sequence;

SEQ ID NO: 13: Recombinant chimpanzee ChAd63 adenovirus vector plasmid sequence expressing EGFP (pChAd63-EGFP);

SEQ ID NO: 14: Chimpanzee ChAd63 adenovirus vector plasmid sequence for recombinant expression of EGFP and mCherry (pChAd63-EGFP-mCherry).

Detailed ways

After a series of in-depth studies, the inventor designed a concise and effective strategy, and successfully constructed a new expression vector based on the existing published chimpanzee ChAd63 adenovirus wild-type sequence. The adenovirus expression vector can be used to prepare preventive vaccines, therapeutic vaccines, oncolytic viruses or gene therapy products.

The invention not only solves the problem of not being able to obtain wild-type virus strains, but also has a simpler and more direct design scheme, can have complete quality control, and greatly avoid sequence mutations in the cloning construction process, and the obtained viral vector carrying the reporter gene The plasmid can also verify the packaging of the virus and the expression of foreign genes in the shortest time to verify the success of the vector construction.

The term "functional deletion" refers to a modification that has the effect of rendering a specific gene product non-functional, thereby producing a replication-deficient recombinant chimpanzee ChAd63 adenovirus expression vector. More specifically, it is preferable to delete most of the E1 area, most of the E3 area, and replace most of the E4 area.

In a specific embodiment, the nucleotides in the E1 region corresponding to positions 576-3128 of the sequence shown in SEQ ID NO: 1, that is, the sequence shown in SEQ ID NO: 3, are deleted; the E3 region corresponding to SEQ ID NO : The nucleotides at positions 27917-31786 in the sequence shown in 1, that is, the sequence shown in SEQ ID NO: 4 is deleted; and the nucleotides in the E4 region corresponding to the nucleotides at positions 33825-36215 in the sequence shown in SEQ ID NO: 1, namely The sequence shown in SEQ ID NO: 5 is deleted and replaced with the E4 ORF6 expression box sequence of the full genome sequence of the chimpanzee adenovirus ChAd68 type shown in SEQ ID NO: 2. Preferably, the E4ORF6 expression box sequence is the sequence shown in SEQ ID NO: 8.

Aiming at the full sequence of the wild-type chimpanzee ChAd63 adenovirus, the inventors carefully studied the published literature to make the modified replication-deficient recombinant chimpanzee ChAd63 adenovirus expression vector obtain a larger insert length space. Most of the E4 region is deleted, only the ORF6 reading expression frame necessary for virus replication is retained, and the coding frame is replaced with the chimpanzee ChAd68 adenovirus E4 ORF6 expression frame sequence, which is conducive to obtaining a vector adenovirus with stronger replication ability.

Aiming at the full sequence of the wild-type chimpanzee ChAd63 adenovirus, the inventors finally determined the application of the restriction sites AsiSI, I-CeuI, MluI, EcoRI, and PI-SceI as the foreign genes in the E1 deletion region after careful sequence alignment. The insertion site not only does not cause erroneous cutting in other positions of the adenovirus expression vector, but a variety of site selections are also conducive to subsequent more convenient selection. At the same time, HpaI and PacI, as the insertion sites of the foreign genes in the E3 deletion region, will not cause erroneous cutting, and also provide convenient insertion sites for the insertion of the foreign genes in the E3 region.

In a further embodiment, the foreign gene is inserted into one or more regions of the deleted E1, E3, and E4 regions. The exogenous gene can be any gene, and can be the same or different. Preferably, the foreign gene is EGFP or mCherry. Preferably, EGFP is inserted into the deleted E1 region; or EGFP and mCherry are inserted into the deleted E1 and E3 regions, respectively.

In a further embodiment, the method further includes adding a linearization restriction site and inserting it into a prokaryotic replication plasmid. Preferably, the linearization restriction site is a SwaI restriction site, and the recognition sequence is: ATTTAAAT. This site not only does not cause wrong cutting of the sequence, but also has a blunt end after cutting, which is more conducive to the stability of the entire adenovirus sequence after linearization.

Preferably, the sequence of the prokaryotic replication plasmid is shown in SEQ ID NO: 9. More specifically, the prokaryotic replication plasmid is modified from the pcDNA3.1 plasmid, and contains a basic replication origin site, ampicillin resistance screening gene and a single enzyme cut site SwaI to facilitate the insertion of adenovirus vector sequences, such as SEQ ID NO: Sequence shown in 9. After careful sequence comparison, the inventors deleted most of the pcDNA3.1 sequence, such as a large number of restriction site sequences, to avoid erroneous cutting by the endonuclease. Only the core replication initiation site sequence and resistance screening gene expression box sequence of the plasmid are retained for subsequent plasmid amplification and screening.

In a specific embodiment, the method includes inserting a sequence containing a specific restriction site into the deleted E1 region (such as AsiSI, I-CeuI, MluI, EcoRI, PI-SceI), E3 region (such as HpaI and Pact). ); Replace the deleted E4 region with the chimpanzee adenovirus ChAd68 type (also known as Paniscus Adenovirus 6 or Simian adenovirus 25, GenBank, #AC_000011.1) (SEQ ID NO: 2) E4 ORF6 expression box sequence, preferably SEQ ID NO ：Sequence shown in 8; Finally, add a linearization site (such as Swat) at the beginning and end of the entire sequence, and insert this site as a boundary into the prokaryotic replication plasmid to obtain the replication-deficient recombinant chimpanzee ChAd63 gland Virus expression vector, its sequence is shown in SEQ ID NO: 12. The obtained full sequence was entrusted to a professional gene synthesis company for full sequence synthesis, and finally a plasmid with accurate sequence information and sufficient transfection grade purification was obtained.

In a specific embodiment, the nucleotides at positions 576-3128 in the sequence shown in SEQ ID NO: 1 in the E1 region, that is, the sequence shown in SEQ ID NO: 3 is deleted; the E3 region corresponds to SEQ ID NO: The nucleotides at positions 27917-31786 in the sequence shown in 1, that is, the sequence shown in SEQ ID NO: 4 is deleted; and the nucleotides in the E4 region corresponding to the nucleotides at positions 33825-36215 in the sequence shown in SEQ ID NO:1, that is, SEQ The sequence shown in ID NO: 5 was deleted and replaced with the E4 ORF6 expression box sequence of the chimpanzee adenovirus ChAd68 type genome sequence shown in SEQ ID NO: 2. Preferably, the E4 ORF6 expression box sequence is the sequence shown in SEQ ID NO: 8.

In a specific embodiment, the sequence containing the restriction site is inserted into the deleted E1 region. Preferably, the sequence containing the restriction site is a sequence containing one or more restriction sites selected from AsiSI, I-CeuI, MluI, EcoRI, and PI-SceII. More preferably, the sequence containing the restriction site is the sequence shown in SEQ ID NO:6.

In a further embodiment, the expression vector may also include one or more foreign genes.

In one embodiment, foreign genes can be inserted into one or more regions of the deleted E1, E3, and E4 regions. The exogenous gene can be any gene, and can be the same or different. Preferably, the foreign gene is EGFP or mCherry. Preferably, EGFP is inserted into the deleted E1 region; or EGFP and mCherry are inserted into the deleted E1 and E3 regions, respectively.

In a specific embodiment, an exogenous reporter gene encoding and expression reading frame containing EGFP is inserted between the insertion sites MluI and EcoRI of the deleted E1 region to obtain a recombinant adenovirus vector that can express an exogenous gene. Its sequence is shown in SEQ ID NO: 13.

Preferably, the foreign reporter gene encoding expression reading frame containing EGFP refers to the EGFP expression frame sequence initiated by the CMV promoter, as shown in SEQ ID NO: 10.

In addition, preferably, the recombinant adenovirus vector plasmid pChAd63-EGFP that can express an exogenous gene is directly obtained by the method of complete gene combination after the said sequence is completely constructed in the sequence analysis software.

In another specific embodiment, an exogenous reporter gene encoding expression reading frame containing EGFP is inserted between the insertion sites MluI and EcoRI of the deleted E1 region, and at the same time the insertion sites HpaI and HpaI of the deleted E3 region are inserted. Insert an exogenous reporter gene encoding and expression reading frame containing mCherry between PacI to obtain a recombinant adenovirus vector that can express two exogenous genes at the same time. The sequence is shown in SEQ ID NO: 14.

Preferably, the exogenous reporter gene encoding expression reading frame containing mCherry refers to the EGFP expression frame sequence initiated by the CB promoter, as shown in SEQ ID NO: 11.

In addition, preferably, the recombinant adenovirus vector plasmid pChAd63-EGFP-mCherry, which can express two foreign genes, is directly obtained by the method of complete gene combination after all the said sequences are constructed in the sequence analysis software.

Specifically, in order to verify the effectiveness of the constructed adenovirus expression vector for expressing one or two foreign genes, the present inventors inserted the fluorescent reporter gene EGFP into the deleted E1 region; and inserted EGFP and mCherry respectively into the deleted E1 region; The E1 area and E3 area. The results show that the constructed chimpanzee ChAd63 adenovirus carrying the reporter gene can efficiently express foreign genes, which proves that the inventors have successfully obtained a new adenovirus expression vector, which can be used in the research and development of biological products or basic biomedical research.

The fourth aspect of the present invention provides a method for packaging and amplifying recombinant adenovirus using host cells. Preferably, the harvested virus can repeatedly infect the production cells and continue to be passaged. Optionally, the harvested virus stock can be purified. The determination of virus titer can be carried out according to conventional methods known in the art.

The adenovirus expression vector of the present invention, as a vector platform that can express foreign genes, is suitable for expressing one, two, or even multiple foreign genes, thereby being applied to the preparation of preventive vaccines, therapeutic vaccines, oncolytic viruses or genes Treatment and other products.

The method of the present invention has the following outstanding advantages:

A. After obtaining the complete sequence through software sequence analysis, the present invention successfully circumvents the traditional method of constructing adenovirus vector through homologous recombination by directly synthesizing the entire sequence of the adenovirus vector plasmid, and greatly reduces the adenovirus vector sequence. The possibility of mutation.

B. Compared with the method of direct cloning through wild-type adenovirus sequences, the method of the present invention has simpler and more direct operation, without the disadvantages of the direct cloning method. The direct cloning method is often due to the large adenovirus genome sequence (about 36kb), and the failure rate of molecular cloning experiments is high. In addition, molecular cloning usually relies on specific restriction sites and PCR amplified sequences for splicing, and the restriction sites are difficult to find; multiple and long fragment PCR amplifications increase the possibility of sequence mutations; for molecular cloning of sequences with high GC content The failure rate is higher.

C. Because the method of the present invention adopts more expensive industrialized and standardized gene synthesis, it can ensure 100% sequence accuracy, quality control of purified plasmids, cost control of plasmid preparation, and time control of vector construction. For ordinary laboratory operators, they only need to master simple sequence analysis and alignment.

D. The method of the present invention does not rely on the acquisition of wild-type adenovirus strains, and can be carried out only by using the published complete sequence of the wild-type adenovirus genome, which ensures a wider and more batch application.

Therefore, the present invention not only provides a new type of adenovirus expression vector, but also provides a new type of adenovirus vector construction method, which is more conducive to promoting the adenovirus vector to exert its excellence in the field of biomedical basic research and clinical application development. The characteristics of this lay a certain foundation for human health.

Example

The present invention will be further explained below in conjunction with specific embodiments. It should be understood that the embodiments of the present invention are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without conditions indicated in the following examples are usually carried out in accordance with conventional experimental conditions, molecular cloning experimental guidelines, or conditions recommended by manufacturers of related reagents (kits).

1. Experimental materials

1.1 Cell

The HEK293 cell line was purchased from the American ATCC cell bank (Cat. No.: CRL-1573), and the medium was DMEM (GIBCO, #11995-040) containing 10% fetal bovine serum (GIBCO, #10091-148).

1.2 Restriction endonucleases

Restriction endonucleases were purchased from New England Biolabs.

2. Experimental method

2.1 Construction of chimpanzee ChAd63 adenovirus vector plasmid carrying reporter gene.

In sequence analysis software (such as SnapGene, Vector NTI, etc.), delete the nucleotides at positions 576-3128 in the sequence shown in SEQ ID NO:1 in the E1 region, that is, the sequence shown in SEQ ID NO:3; delete E3 The nucleotides in the region corresponding to the 27917-31786 of the sequence shown in SEQ ID NO: 1, that is, the sequence shown in SEQ ID NO: 4, are deleted; and the sequence 33825-36215 in the E4 region corresponding to the sequence shown in SEQ ID NO: 1 The nucleotide at the position, that is, the sequence shown in SEQ ID NO: 5 is deleted and replaced with the sequence shown in SEQ ID NO: 8. In addition, insert two restriction sites MluI and EcoRI in the deleted E1 region; insert restriction sites HpaI and PacI in the deleted E3 region. Then, insert the fluorescent reporter gene EGFP into the deleted E1 region; or insert the gene EGFP into the deleted E1 region and insert the gene mCherry into the deleted E3 region. Finally, add the linearization restriction site SwaI at the beginning and end of the entire sequence, and insert this site as a boundary into the prokaryotic replication plasmid (the sequence is shown in SEQ ID NO: 9) to obtain recombinant expression of EGFP and simultaneous expression The replication-deficient recombinant chimpanzee ChAd63 adenovirus expression vector of EGFP and mCherry, namely plasmid pChAd63-EGFP and pChAd63-EGFP-mCherry complete sequence. The obtained full sequence was entrusted to Suzhou Jinweizhi Biotechnology Co., Ltd. to complete the sequence synthesis, and finally the transfection-grade purified plasmids pChAd63-EGFP and pChAd63-EGFP-mCherry were obtained, as shown in Figure 1 plasmid map.

2.2 Identification of the plasmid sequence of the chimpanzee ChAd63 adenovirus vector carrying the reporter gene

The plasmids obtained by gene synthesis were subjected to preliminary restriction map verification, and standardized restriction digestion with restriction enzymes respectively, followed by DNA analysis and identification, including HindIII&XbaI double restriction digestion, XhoI single restriction restriction digestion, NotI&XbaI double restriction restriction digestion, NdeI&XbaI Double digestion and PvuI single digestion. Compare the actual restriction map with the restriction map predicted by the software according to the expected sequence to verify the correctness of the sequence. The results show that the obtained two plasmid sequences are fully in line with expectations, as shown in Figure 2.

2.3 Packaging of Chimpanzee ChAd63 Adenovirus Carrying Reporter Gene

The plasmids pChAd63-EGFP and pChAd63-EGFP-mCherry were linearized and digested with restriction enzyme SwaI. Spread a 6-well plate with HEK293 cells. When the cell density reaches 80%-90%, transfect approximately 6 μg of linearized pChAd63-EGFP and pChAd63-EGFP-mCherry with the transfection reagent Lipofectamine 2000 (Invitrogen, #11668019) Product, observe the cell cytopathy (see Figure 3) and fluorescent reporter gene expression (see Figure 4) every day. Obvious fluorescence expression and cytopathic changes were observed after about 4-6 days, proving that the virus packaging was successful. After harvesting the transfected cell product, freezing and thawing three times and centrifuging the supernatant, it can be regarded as the initial virus stock.

2.4 Amplification of chimpanzee ChAd63 adenovirus carrying reporter gene

HEK293 cells were cultured in DMEM medium containing 10% fetal bovine serum to a cell density close to 100%, the initial virus stock solution was added to the cell culture medium, and the cells were continued to be cultured. When the cells become cytopathic, almost all cells change from adherent to floating state, then all infected cells can be collected and centrifuged to collect cells. Resuspend the cells with an appropriate amount of DMEM, freeze and thaw the cells three times to lyse the cells, and centrifuge to collect the supernatant. The supernatant can be further amplified repeatedly according to the above method to make the virus stock solution reach a certain amount.

2.5 Purification of Chimpanzee ChAd63 Adenovirus Carrying Reporter Gene

The amplified virus stock solution was purified and titer determined according to the cesium chloride density gradient centrifugation method described in the published literature (Zhou et al. 2010), and then added glycerol and stored in a refrigerator at -80°C for long-term storage.

2.6 Identification of the genome sequence of the chimpanzee ChAd63 adenovirus carrying the reporter gene

A purified virus solution with a titer of 0.5-1× ^{10 12} virus particles (vps) was taken, and the virus genome was extracted using the kit DNeasy Blood&Tissue Kit (QIAGEN, #69504). The extracted virus genome was also subjected to standardized digestion with restriction enzymes and then subjected to DNA analysis and identification, including HindIII&XbaI double digestion, XhoI single digestion, NotI&XbaI double digestion, NdeI&XbaI double digestion, and PvuI single digestion. The actual restriction map is compared with the restriction map predicted by the software according to the expected sequence to verify the correctness of the sequence. The results show that the obtained two plasmid sequences are fully in line with expectations, as shown in Figure 5.

3. Conclusion

Through the above method of direct gene synthesis of adenovirus genome, the inventors constructed a wild-type chimpanzee ChAd63 adenovirus into a vector capable of expressing foreign genes. Therefore, the present inventors provided a chimpanzee ChAd63 adenovirus vector for the development of new adenovirus vector-related products and its biomedical basic research, and at the same time provided a new method for constructing an adenovirus vector de novo.

All references mentioned in the present invention are cited as references in this application, as if each document was individually cited as a reference. In addition, it should be understood that after reading this application, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

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Claims

A method for constructing an expression vector based on a chimpanzee ChAd63 adenovirus, the method comprising functionally deleting the E1, E3 and E4 regions in the sequence shown in SEQ ID NO:1.
The method according to claim 1, wherein the nucleotides corresponding to positions 576-3128 in the sequence shown in SEQ ID NO:1 in the E1 region are deleted; and the nucleotides in the E3 region corresponding to the sequence shown in SEQ ID NO:1 are 27917- The nucleotides at position 31786 are deleted; and the nucleotides corresponding to positions 33825-36215 in the sequence shown in SEQ ID NO:1 in the E4 region are deleted and replaced with the whole genome of the chimpanzee adenovirus ChAd68 type shown in SEQ ID NO:2 Sequence of the E4 ORF6 expression box sequence.
The method according to claim 2, wherein the sequence of the E4 ORF6 expression box is the sequence shown in SEQ ID NO: 8.
The method according to any one of claims 1 to 3, wherein a sequence containing one or more restriction sites selected from AsiSI, I-CeuI, MluI, EcoRI, PI-SceI is inserted into the deleted E1 region In; and/or insert the sequence containing the restriction site of HpaI and/or PacI into the deleted E3 region.
The method according to claim 4, wherein the sequence containing the restriction site of MluI and EcoRI is inserted into the deleted E1 region.
The method according to claim 4 or 5, wherein the sequence shown in SEQ ID NO: 6 is inserted into the deleted E1 region.
The method according to claim 4 or 5, wherein the sequence shown in SEQ ID NO: 7 is inserted into the deleted E3 region.
The method according to any one of the preceding claims, wherein the foreign gene is inserted into one or more regions of the deleted E1, E3 and E4 regions.
The method according to claim 8, wherein the exogenous gene is EGFP or mCherry.
The method according to claim 8, wherein EGFP and mCherry are inserted into the deleted E1 and E3 regions, respectively.
The method according to any one of the preceding claims, further comprising adding a linearization restriction site and inserting into a prokaryotic replication plasmid.
The method according to claim 11, wherein the linearization restriction site is a SwaI restriction site.
The method according to claim 11, wherein the sequence of the prokaryotic replication plasmid is shown in SEQ ID NO: 9.
A chimpanzee ChAd63 adenovirus-based expression vector constructed according to the method of any one of the preceding claims.
The expression vector according to claim 14, which comprises the sequence shown in SEQ ID NO: 12.
A host cell comprising the expression vector of claim 14 or 15.
The host cell of claim 16, which is a HEK293 cell.
A method for packaging and amplifying recombinant adenovirus using the host cell of claim 16 or 17.
The method according to claim 18, comprising the step of purifying the harvested virus stock.