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* These authors contributed equally
In this study, an infectious clone of human adenovirus type 7 (HAdV-7) was constructed, and an E3-deleted HAdV-7 vector system was established by modifying the infectious clone. This strategy used here can be generalized to make gene transfer vectors from other wild-type adenoviruses.
Adenoviral vectors have been used as a gene transfer tool in gene therapy for more than three decades. Here, we introduce a protocol to construct an adenoviral vector by manipulating the genomic DNA of wild-type HAdV-7 by using a DNA assembly method. First, an infectious clone of HAdV-7, pKan-Ad7, was generated by fusing the viral genomic DNA with a PCR product from plasmid backbone, comprising of the kanamycin-resistant gene and the origin of replication (Kan-Ori), through DNA assembly. This was done by designing a pair of PCR primers, that contained ~25 nucleotides of the terminal sequence of HAdV-7 inverted terminal repeat (ITR) at the 5' end, a non-cutter restriction enzyme site for HAdV-7 genome in the middle, and a template-specific sequence for PCR priming at the 3' end. Second, an intermediate plasmid-based strategy was employed to replace the E3 region with transgene-expressing elements in the infectious clone to generate an adenoviral vector. Briefly, pKan-Ad7 was digested with dual-cutter restriction enzyme Hpa I, and the fragment containing the E3 region was ligated to another PCR product of plasmid backbone by Gibson assembly to construct an intermediate plasmid pKan-Ad7HpaI. For convenience, restriction-assembly was used to designate the plasmid cloning method of combined restriction digestion and assembly. Using restriction-assembly, the E3 genes in pKan-Ad7HpaI was replaced with a green fluorescent protein (GFP) expression cassette, and the modified E3 region was released from the intermediate plasmid and restored to the infectious clone to generate an adenoviral plasmid pKAd7-E3GFP. Finally, pKAd7-E3GFP was linearized by Pme I digestion and used to transfect HEK293 packaging cells to rescue recombinant HAdV-7 virus. To conclude, a DNA assembly-based strategy was introduced here for constructing adenoviral vectors in general laboratories of molecular biology without the need of specialized materials and instruments.
Over the past three decades, recombinant adenoviral vectors have been widely used in vaccine development and gene therapy1,2,3,4 as well as in basic research due to their outstanding biological properties, such as high gene transduction efficiency, non-integration to the host genome, the manipulative viral genome, and the ease of large-scale production.
Currently, the most commonly used adenoviral vectors are constructed based on human adenovirus 5 (HAdV-5)5,6. Although HAdV-5 vector-mediated transduction provides encouraging results, preclinical and clinical applications have revealed several disadvantages, (e.g., high pre-existing anti-vector immunity within the human population and low transduction efficiency in cells lacking the coxsackievirus and adenovirus receptor (CAR)). To circumvent these problems, there has been a great interest to construct vectors based on other human or mammalian adenovirus types3,7,8.
Until now, the most popular method to construct an adenoviral vector is homologous recombination in bacteria5. Such bacterial strains must express recombinases, which can affect the stability or amplification of the plasmids they bear. Some strains are even commercially unavailable. Recently, methods based on other principles, including bacterial artificial chromosomes, direct cloning, or direct DNA assembly, have been employed to generate infectious clones of adenovirus or recombinant adenoviral vectors9,10,11,12. However, these methods are somewhat unfriendly to researchers with little experience in this field.
In 2018, the process of constructing an adenovirus infectious clone is simplified in the laboratory by directly ligating the virus genome with a PCR product carrying plasmid backbone through Gibson assembly13. After that, the methods of restriction digestion and Gibson assembly are combined together to load transgenes to existing adenoviral plasmids14,15,16,17,18. For the sake of convenience, restriction-assembly is used hereafter to refer to the method of combined restriction enzyme digestion and Gibson assembly. Strategies were further developed to construct adenoviral vectors from infectious clones by using restriction-assembly19. The essence of restriction-assembly is to include fragments excised from plasmids as much as possible in a DNA assembly reaction, while short PCR products serve as linkers or patches for plasmid modification. At the same time, the number of fragments included is kept as low as possible. Such efforts reflect the payoff; the possibility of unwanted mutations caused by PCR or DNA assembly can be minimized, and the success rate can be improved. Conclusively, a pipeline from a wild-type adenovirus to an adenoviral vector has been set up in the laboratory13,14,15,16,17,18,19.
Here, we attempt to introduce these methods by providing examples of constructing an HAdV-7 infectious clone and an E3-deleted replication-competent HAdV-7 vector.
NOTE: Adenoviruses are classified as Biosafety Level 2 (BSL-2); all the steps to use the viruses were carried out in a biosafety level 2 laboratory. The wild-type HAdV-7 was isolated in 2017 from the nasopharyngeal aspirate specimen of a 10-month-old infant who was hospitalized with acute respiratory tract infection in Beijing Children's Hospital20. The virus stocks were stored at -80 °C.
1. Extraction of HAdV-7 genomic DNA
2. Construction of infectious clone of HAdV-7 by DNA assembly
3. In silico construction and modification of an intermediate plasmid
NOTE: Generally, an intermediate plasmid should be constructed, and how to construct an ideal intermediate plasmid is the key step. DNA analysis software with a restriction enzyme module, such as pDRAW32 (a free software, available at www.acaclone.com), is needed for the design of an intermediate plasmid.
4. Rescue of infectious recombinant adenovirus in HEK293 cells
NOTE: The three adenoviruses generated in this article are all rescued according to the following protocol.
5. Construction of E3-deleted HAdV-7 genome plasmid
NOTE: The genome of HAdV-7 is 35,239 bp in length, and the E3 region is located between 27,354 bp and 31,057 bp of the genome. To construct an intermediate plasmid, find an endonuclease with two cutting sites on the infectious clone plasmid, or two endonucleases with a single cutting site on the plasmid. There are Hpa I, Sal I, or Avr II and Mlu I available. The sequence between BstZ17 I (28,312 bp) and Mlu I (30,757 bp) is to be deleted and replaced with the CMV-GFP-PA cassette or CMV-mCherry-PA cassette.
6. Amplification and purification of the recombinant adenovirus in HEK293 cells
7. Identification of recombinant adenovirus genome by restriction enzyme digestion
The strategy for the construction of an infectious clone of HAdV-7 is shown in Figure 1 and Figure 2. Two infectious clone plasmids were randomly selected and identified by BstZ17 I, BamH I, and EcoR V, respectively. The results showed that the fragments were consistent with the expected size (Figure 2A), indicating that the plasmids were constructed correctly. Comet-foci could be seen in the cells 12 days after the Pme I-linearized...
Different adenoviruses have various tissue tropisms, and the prevalence of host pre-existing immunity against different adenoviruses can fluctuate intensively in human beings24, which attracts the interest in constructing novel adenoviral vectors for gene therapy or vaccine development. However, the establishment of a new adenoviral vector system remains cumbersome for generic laboratories of molecular biology.
Here, we introduced a protocol for generating vectors from ...
The authors declare no conflicts of interest.
This research was funded by Beijing Natural Science foundation (7204258), National Natural Science Foundation of China (82161138001, 82072266), CAMS Innovation Fund for Medical Sciences (2019-I2M-5-026), and the research and application on molecular tracing of essential respiratory pathogens in Beijing, by the Capital Health Development and Research of Special (2021-1G-3012).
Name | Company | Catalog Number | Comments |
1.5 mL polypropylene microcentrifuge Tube | Axygen | MCT-150-C | Storage of virus |
15 mL polypropylene centrifuge tubes | Corning | 430790 | Storage of virus |
150 mm TC-treated culture dishes | Corning | 430599 | Growth of HEK29E cells |
20 K MWCO dialysis cassette | ThermoFisher Scientific | 66005 | Dialysis of virus |
Acetic acid | Amresco | 714 | Extraction of DNA |
Afl II | NEB | R0520 | Digestion |
Agarose | Takara | 5260 | Electrophoresis |
Age I | NEB | R0552 | Digestion |
Asc I | NEB | R0558 | Digestion |
BamH I | NEB | R0136 | Digestion |
Benzonase Nuclease | Sigma | E8263-25KU | Purification of virus |
BsrG I | NEB | R0575 | Digestion |
Cell lifter | Corning | 3008 | Scrape off the cells |
CsCl | Sigma | C3032 | Purification of virus |
DNA gel recovery kit | Zymo | D4045 | Recovery of DNA |
Dulbecco’s modified Eagle’s medium (DMEM) | Cytiva | SH30022.01 | HEK293 cells medium |
E.coli TOP10 competent cells | TIANGEN BIOTECH (BEIJING) CO.,LTD. | CB-104 | Transformation of assembly product |
EcoR V | NEB | R3195 | Digestion |
EDTA | Thermo Fisher Scientific | R3104 | Extraction of DNA |
Fetal bovine serum (FBS) | Cytiva | SV30208.02 | HEK293 cells culture |
Genomic DNA Clean and concentrator kit | Zymo | D4065 | Purification of DNA |
Glycerol | Shanghai Macklin Biochemical Co., Ltd | G810575 | Dialysis of virus |
HEK293 cells | ATCC | CRL-1573 | Amplification of virus |
High-Fidelity DNA Polymerase | NEB | M0491 | PCR |
Hind III | NEB | R3104 | Digestion |
Kanamycin sulfate | Amresco | 408 | Selection of plasmid |
Kpn I | NEB | R3142 | Digestion |
Lambda/HindIII DNA marker | Takara | 3403 | Electrophoresis |
LB broth | BD | 240230 | LB plate for bacteria |
LB medium | Solarbio Life Science | L1010 | Medium for bacteria |
MgCl2 | Sigma | 63068 | Dialysis of virus |
Microcentrifuge | Thermo Fisher Scientific | Sorvall Legend Micro 21R | Extraction of DNA |
NaCl | Sigma | S5886 | Dialysis of virus |
Nde I | NEB | R0111 | Digestion |
NEBuilder HiFi DNA Assembly Master Mix | NEB | E2621 | DNA assembly |
Nhe I | NEB | R0131 | Digestion |
Phosphate Buffered Saline | Cytiva | SH30256.01 | Washing of cells |
Pipette | Thermo Fisher Scientific | Matrix | Aspirate the medium |
Plasmid Maxprep Kit | Vigorous Biotechnology Beijing Co., Ltd. | N001 | Extraction of DNA |
Plasmid Miniprep Kit | TIANGEN BIOTECH (BEIJING) CO.,LTD. | DP103 | Extraction of DNA |
Pme I | NEB | R0560 | Digestion |
Potassium acetate | Amresco | 698 | Extraction of DNA |
Protease K | Thermo Fisher Scientific | AM2542 | Extraction of DNA |
pShuttle-CMV | Stratagene | 240007 | PCR template |
RNase | Beyotime | D7089 | Extraction of DNA |
Sal I | NEB | R0138 | Digestion |
Sbf I | NEB | R3642 | Digestion |
SDS | Amresco | 227 | Extraction of DNA |
Swinging-bucket rotor | HITACHI | S52ST | Purification of virus |
T-25 cell flask | Corning | 430639 | Growth of HEK29E cells |
T-75 cell flask | Corning | 430641 | Growth of HEK29E cells |
Transfection reagent | Polyplus-transfection | 114-15 | Transfection |
Transmission electron microscope | FEI | TECNAI 12 | Obsevation of virus |
Tris-HCl | Amresco | 234 | Dialysis of virus |
Ultracentrifuge | HITACHI | Himac CS120GXII | Purification of virus |
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