Adenovirus vector and production method for same

Abstract

The present invention relates to a new adenovirus vector and its production method. The production of the vector includes two main steps, one is cloning the foreign gene into the shuttle vector, and the other is the recombination of the shuttle vector and the adenovirus backbone vector. The shuttle vector used in the present invention is improved on the basis of pShuttle-CMV vector—pCMV-FH; the adenovirus backbone vector used is a yeast gene expressing ISceI cloned into the existing adenovirus backbone vector; the recombinant strain used is SW102. The cloning method of the shuttle vector and the recombination method of the shuttle vector and the adenovirus skeleton vector of the present invention all adopt a high-throughput production method. Using the high-throughput shuttle vector cloning method and the high-throughput recombination method to repeat the operation until the fifth round, 99% of the shuttle vector clones can be recombined into the adenovirus vector. The recombination efficiency of the high-throughput adenovirus vector production method of the present invention is as high as 85-90%, the experimental steps are greatly simplified, the time is also greatly shortened, and the experimental cost is significantly reduced.

Description

technical field

[0001] The invention belongs to the field of bioengineering and relates to a new adenovirus vector and a production method thereof, in particular to a high-throughput adenovirus vector and a production method thereof. Background technique

[0002] Adenoviral vectors are the most efficient and versatile system for transfecting human cells, and they are widely used in biomedical research and gene therapy. Adenoviral vectors can effectively transfect dividing or non-dividing cells, and the transfection efficiency can reach 100%. The adenoviral vector does not integrate into the human genome after entering the cell, so it is relatively safe.

[0003] The production of adenovirus is relatively easy to achieve high titers, and can accommodate up to 30kb of foreign genes. The expression of adenovirus recombinant protein can reach 20-30% of the cell weight. Although the adenoviral vector has many advantages, its production process is cumbersome and the production ...

Examples

Embodiment 1

[0040] Example 1 .shuttle vector pCMV-FH

[0041] The shuttle carrier pCMV-FH of the present invention is transformed on the basis of pCMV-Shuttle by conventional molecular biology techniques, and the specific steps are:

[0042] (1). Shorten the left and right homology arms. The recombinant sequences on the left and right sides of the vector were shortened to 800 bases. The right homology arm of 1243-2043 bases and the left homology arm of 3545-4428 bases of the original plasmid were retained. Primers were designed using general molecular biology primer design methods. A PmeI restriction site was added at both ends of the PCR primers. After PCR amplification, the PCR products were self-ligated, then transformed into competent cells, extracted and sequenced.

[0043] (2). Add a Puromycin gene at the PmeI restriction site. The Puromycin gene, SV40 promoter and BGH PolyA signal were used for gene synthesis in vitro. After the synthetic product was digested with PmeI, it ...

Embodiment 2

[0046] Example 2. Construction of high-throughput shuttle clones

[0047] It is transformed by conventional molecular biology techniques, and the specific steps are:

[0048] (1). The full-length DNA template for PCR (the full-length human gene comes from the RefSeq database at http: / / www.ncbi.nlm.nih.gov / RefSeq / ) is first arranged on a 96-well PCR plate. 30,000 ORF primers (purchased from Eurofins / MWG / Operon Biological Company in the United States, see the attached Figure 5 ) are also designed in the order of the DNA template.

[0049] (2). 15,000 existing clones (12,000 from the National Health Service of the United States and 3,000 from Qingdao Macleay Biotechnology Co., Ltd.) were amplified in a 96-well plate format. To reduce mutations during PCR, each gene was amplified for only 15 rounds, and a high-fidelity polymerase was used. (Example 3 takes the NM_000632 gene as an example to describe in detail the human whole genome gene PCR)

Embodiment 3

[0049] (2). 15,000 existing clones (12,000 from the National Health Service of the United States and 3,000 from Qingdao Macleay Biotechnology Co., Ltd.) were amplified in a 96-well plate format. To reduce mutations during PCR, each gene was amplified for only 15 rounds, and a high-fidelity polymerase was used. (Example 3 takes the NM_000632 gene as an example to describe in detail the human whole genome gene PCR)

[0050] (3). According to the size of the ORF, take 2 microliters of each PCR product, combine 50 genes of similar size into a test tube, and then purify.

[0051] (4). Digest according to the enzyme cut point, separate and purify on agarose gel.

[0052] (5). Linked to the shuttle vector pCMV-FH and transformed into competent cells.

[0053] (6). Pick 96 bacterial cells, culture them in 96-well deep-well plates, extract and sequence the plasmids.

[0054] (7). The sequence at the 5' end is compared with the sequence of the original 50 ORFs.

[0055] (8). The 5'...

technical field

[0001] The invention belongs to the field of bioengineering and relates to a new adenovirus vector and a production method thereof, in particular to a high-throughput adenovirus vector and a production method thereof. Background technique

[0002] Adenoviral vectors are the most efficient and versatile system for transfecting human cells, and they are widely used in biomedical research and gene therapy. Adenoviral vectors can effectively transfect dividing or non-dividing cells, and the transfection efficiency can reach 100%. The adenoviral vector does not integrate into the human genome after entering the cell, so it is relatively safe.

[0003] The production of adenovirus is relatively easy to achieve high titers, and can accommodate up to 30kb of foreign genes. The expression of adenovirus recombinant protein can reach 20-30% of the cell weight. Although the adenoviral vector has many advantages, its production process is cumbersome and the production ...

Examples

Embodiment 1

[0040] Example 1 .shuttle vector pCMV-FH

[0041] The shuttle carrier pCMV-FH of the present invention is transformed on the basis of pCMV-Shuttle by conventional molecular biology techniques, and the specific steps are:

[0042] (1). Shorten the left and right homology arms. The recombinant sequences on the left and right sides of the vector were shortened to 800 bases. The right homology arm of 1243-2043 bases and the left homology arm of 3545-4428 bases of the original plasmid were retained. Primers were designed using general molecular biology primer design methods. A PmeI restriction site was added at both ends of the PCR primers. After PCR amplification, the PCR products were self-ligated, then transformed into competent cells, extracted and sequenced.

[0043] (2). Add a Puromycin gene at the PmeI restriction site. The Puromycin gene, SV40 promoter and BGH PolyA signal were used for gene synthesis in vitro. After the synthetic product was digested with PmeI, it ...

Embodiment 2

[0046] Example 2. Construction of high-throughput shuttle clones

[0047] It is transformed by conventional molecular biology techniques, and the specific steps are:

[0048] (1). The full-length DNA template for PCR (the full-length human gene comes from the RefSeq database at http: / / www.ncbi.nlm.nih.gov / RefSeq / ) is first arranged on a 96-well PCR plate. 30,000 ORF primers (purchased from Eurofins / MWG / Operon Biological Company in the United States, see the attached Figure 5 ) are also designed in the order of the DNA template.

[0049] (2). 15,000 existing clones (12,000 from the National Health Service of the United States and 3,000 from Qingdao Macleay Biotechnology Co., Ltd.) were amplified in a 96-well plate format. To reduce mutations during PCR, each gene was amplified for only 15 rounds, and a high-fidelity polymerase was used. (Example 3 takes the NM_000632 gene as an example to describe in detail the human whole genome gene PCR)

Embodiment 3

[0049] (2). 15,000 existing clones (12,000 from the National Health Service of the United States and 3,000 from Qingdao Macleay Biotechnology Co., Ltd.) were amplified in a 96-well plate format. To reduce mutations during PCR, each gene was amplified for only 15 rounds, and a high-fidelity polymerase was used. (Example 3 takes the NM_000632 gene as an example to describe in detail the human whole genome gene PCR)

[0050] (3). According to the size of the ORF, take 2 microliters of each PCR product, combine 50 genes of similar size into a test tube, and then purify.

[0051] (4). Digest according to the enzyme cut point, separate and purify on agarose gel.

[0052] (5). Linked to the shuttle vector pCMV-FH and transformed into competent cells.

[0053] (6). Pick 96 bacterial cells, culture them in 96-well deep-well plates, extract and sequence the plasmids.

[0054] (7). The sequence at the 5' end is compared with the sequence of the original 50 ORFs.

[0055] (8). The 5'...

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