{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,16]],"date-time":"2026-05-16T05:45:14Z","timestamp":1778910314689,"version":"3.51.4"},"reference-count":51,"publisher":"MDPI AG","issue":"3","license":[{"start":{"date-parts":[[2022,3,6]],"date-time":"2022-03-06T00:00:00Z","timestamp":1646524800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["82161138001"],"award-info":[{"award-number":["82161138001"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>Gene therapy and vaccine development need more novel adenovirus vectors. Here, we attempt to provide strategies to construct adenovirus vectors based on restriction-assembly for researchers with little experience in this field. Restriction-assembly is a combined method of restriction digestion and Gibson assembly, by which the major part of the obtained plasmid comes from digested DNA fragments instead of PCR products. We demonstrated the capability of restriction-assembly in manipulating the genome of simian adenovirus 1 (SAdV-1) in this study. A PCR product of the plasmid backbone was combined with SAdV-1 genomic DNA to construct an infectious clone, plasmid pKSAV1, by Gibson assembly. Restriction-assembly was performed repeatedly in the steps of intermediate plasmid isolation, modification, and restoration. The generated adenoviral plasmid was linearized by restriction enzyme digestion and transfected into packaging 293 cells to rescue E3-deleted replication-competent SAdV1XE3-CGA virus. Interestingly, SAdV1XE3-CGA could propagate in human chronic myelogenous leukemia K562 cells. The E1 region was similarly modified to generate E1\/E3-deleted replication-defective virus SAdV1-EG. SAdV1-EG had a moderate gene transfer ability to adherent mammalian cells, and it could efficiently transduce suspension cells when compared with the human adenovirus 5 control vector. Restriction-assembly is easy to use and can be performed without special experimental materials and instruments. It is highly effective with verifiable outcomes at each step. More importantly, restriction-assembly makes the established vector system modifiable, upgradable and under sustainable development, and it can serve as the instructive method or strategy for the synthetic biology of adenoviruses.<\/jats:p>","DOI":"10.3390\/v14030546","type":"journal-article","created":{"date-parts":[[2022,3,6]],"date-time":"2022-03-06T20:40:38Z","timestamp":1646599238000},"page":"546","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":13,"title":["Restriction-Assembly: A Solution to Construct Novel Adenovirus Vector"],"prefix":"10.3390","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-1960-183X","authenticated-orcid":false,"given":"Xiaojuan","family":"Guo","sequence":"first","affiliation":[{"name":"NHC Key Laboratory of Medical Virology and Viral Diseases, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 100052, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yangyang","family":"Sun","sequence":"additional","affiliation":[{"name":"NHC Key Laboratory of Medical Virology and Viral Diseases, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 100052, China"},{"name":"School of Laboratory Medicine, Weifang Medical University, Weifang 261053, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Juan","family":"Chen","sequence":"additional","affiliation":[{"name":"NHC Key Laboratory of Medical Virology and Viral Diseases, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 100052, China"},{"name":"School of Public Health, Baotou Medical College, Inner Mongolia University of Science and Technology, Baotou 014040, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6834-1341","authenticated-orcid":false,"given":"Xiaohui","family":"Zou","sequence":"additional","affiliation":[{"name":"NHC Key Laboratory of Medical Virology and Viral Diseases, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 100052, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Wenzhe","family":"Hou","sequence":"additional","affiliation":[{"name":"NHC Key Laboratory of Medical Virology and Viral Diseases, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 100052, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Wenjie","family":"Tan","sequence":"additional","affiliation":[{"name":"NHC Key Laboratory of Medical Virology and Viral Diseases, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 100052, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Tao","family":"Hung","sequence":"additional","affiliation":[{"name":"NHC Key Laboratory of Medical Virology and Viral Diseases, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 100052, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5767-401X","authenticated-orcid":false,"given":"Zhuozhuang","family":"Lu","sequence":"additional","affiliation":[{"name":"NHC Key Laboratory of Medical Virology and Viral Diseases, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 100052, China"},{"name":"Chinese Center for Disease Control and Prevention\u2013Wuhan Institute of Virology, Chinese Academy of Sciences Joint Research Center for Emerging Infectious Diseases and Biosafety, Wuhan 430071, China"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2022,3,6]]},"reference":[{"key":"ref_1","unstructured":"King, A.M.Q., Adams, M.J., Carstens, E.B., and Lefkowitz, E.J. (2011). Virus Taxonomy, Elsevier Academic Press. Classification and Nomenclature of Viruses: Ninth Report of the International Committee on Taxonomy of Viruses."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"veab018","DOI":"10.1093\/ve\/veab018","article-title":"Emergence and characterization of a putative novel human adenovirus recombinant HAdV-C104 causing pneumonia in Southern China","volume":"7","author":"Ji","year":"2021","journal-title":"Virus Evol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"3","DOI":"10.1089\/hum.2013.2527","article-title":"Adenovirus: The first effective in vivo gene delivery vector","volume":"25","author":"Crystal","year":"2014","journal-title":"Hum. Gene Ther."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"831","DOI":"10.1111\/cas.13228","article-title":"Recent advances in genetic modification of adenovirus vectors for cancer treatment","volume":"108","author":"Yamamoto","year":"2017","journal-title":"Cancer Sci."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1881","DOI":"10.1016\/S0140-6736(08)61591-3","article-title":"Efficacy assessment of a cell-mediated immunity HIV-1 vaccine (the Step Study): A double-blind, randomised, placebo-controlled, test-of-concept trial","volume":"372","author":"Buchbinder","year":"2008","journal-title":"Lancet"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"2875","DOI":"10.4161\/hv.29594","article-title":"Pre-existing immunity against Ad vectors: Humoral, cellular, and innate response, what\u2019s important?","volume":"10","author":"Kobinger","year":"2014","journal-title":"Hum. Vaccin. Immunother."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"5579","DOI":"10.1016\/j.vaccine.2016.09.043","article-title":"Prevalence of serum neutralizing antibodies to adenovirus type 5 (Ad5) and 41 (Ad41) in children is associated with age and sanitary conditions","volume":"34","author":"Yang","year":"2016","journal-title":"Vaccine"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"597","DOI":"10.1080\/14760584.2019.1588113","article-title":"A review of 65 years of human adenovirus seroprevalence","volume":"18","author":"Mennechet","year":"2019","journal-title":"Expert Rev. Vaccines"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"285","DOI":"10.1089\/hum.2013.228","article-title":"Circumventing antivector immunity: Potential use of nonhuman adenoviral vectors","volume":"25","author":"Podgorski","year":"2014","journal-title":"Hum. Gene Ther."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1241","DOI":"10.1080\/14760584.2017.1394842","article-title":"Chimpanzee adenoviral vectors as vaccines\u2014challenges to move the technology into the fast lane","volume":"16","author":"Vitelli","year":"2017","journal-title":"Expert Rev. Vaccines"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"e01924-17","DOI":"10.1128\/JVI.01924-17","article-title":"Rapid Cloning of Novel Rhesus Adenoviral Vaccine Vectors","volume":"92","author":"Abbink","year":"2018","journal-title":"J. Virol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"101430","DOI":"10.1016\/j.smim.2020.101430","article-title":"New viral vectors for infectious diseases and cancer","volume":"50","author":"Sasso","year":"2020","journal-title":"Semin. Immunol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"e6","DOI":"10.4110\/in.2021.21.e6","article-title":"Adenovirus Vectors: Excellent Tools for Vaccine Development","volume":"21","author":"Chang","year":"2021","journal-title":"Immune Netw."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"3609","DOI":"10.1002\/1873-3468.13691","article-title":"State-of-the-art human adenovirus vectorology for therapeutic approaches","volume":"593","author":"Gao","year":"2019","journal-title":"FEBS Lett."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"475","DOI":"10.1002\/ijc.20129","article-title":"Modified adenoviruses for cancer gene therapy","volume":"110","author":"Kanerva","year":"2004","journal-title":"Int. J. Cancer"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"e50","DOI":"10.1093\/nar\/gkv031","article-title":"Ad 2.0: A novel recombineering platform for high-throughput generation of tailored adenoviruses","volume":"43","author":"Solanki","year":"2015","journal-title":"Nucleic Acids Res."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"4805","DOI":"10.1128\/jvi.70.7.4805-4810.1996","article-title":"Efficient generation of recombinant adenovirus vectors by homologous recombination in Escherichia coli","volume":"70","author":"Chartier","year":"1996","journal-title":"J. Virol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1679","DOI":"10.1080\/21645515.2017.1419108","article-title":"Development of novel vaccine vectors: Chimpanzee adenoviral vectors","volume":"14","author":"Guo","year":"2018","journal-title":"Hum. Vaccin. Immunother."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"16","DOI":"10.1002\/0471142727.mb1626s113","article-title":"Rapid, Efficient, and Modular Generation of Adenoviral Vectors via Isothermal Assembly","volume":"113","author":"Yang","year":"2016","journal-title":"Curr. Protoc. Mol. Biol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.virusres.2019.05.008","article-title":"A rapid strategy for constructing novel simian adenovirus vectors with high viral titer and expressing highly antigenic proteins applicable for vaccine development","volume":"268","author":"Luo","year":"2019","journal-title":"Virus Res."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"193","DOI":"10.1016\/j.jbiotec.2019.11.009","article-title":"Site-directed modification of adenoviral vector with combined DNA assembly and restriction-ligation cloning","volume":"307","author":"Guo","year":"2020","journal-title":"J. Biotechnol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"188","DOI":"10.1016\/j.jviromet.2011.05.010","article-title":"An improved HAdV-41 E1B55K-expressing 293 cell line for packaging fastidious adenovirus","volume":"175","author":"Zou","year":"2011","journal-title":"J. Virol. Methods"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1681","DOI":"10.1099\/vir.0.80757-0","article-title":"Complete genome sequence of simian adenovirus 1: An Old World monkey adenovirus with two fiber genes","volume":"86","author":"Kovacs","year":"2005","journal-title":"J. Gen. Virol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"760","DOI":"10.2144\/98245bm14","article-title":"Modified Hirt procedure for rapid purification of extrachromosomal DNA from mammalian cells","volume":"24","author":"Arad","year":"1998","journal-title":"BioTechniques"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"128","DOI":"10.1002\/jgm.1284","article-title":"Novel recombinant adenovirus type 41 vector and its biological properties","volume":"11","author":"Lu","year":"2009","journal-title":"J. Gene Med."},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Lv, Y., Xiao, F.J., Wang, Y., Zou, X.H., Wang, H., Wang, H.Y., Wang, L.S., and Lu, Z.Z. (2019). Efficient gene transfer into T lymphocytes by fiber-modified human adenovirus 5. BMC Biotechnol., 19.","DOI":"10.1186\/s12896-019-0514-x"},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Yan, B., Zou, X., Liu, X., Zhao, J., Zhang, W., Guo, X., Wang, M., Lv, Y., and Lu, Z. (2020). User-Friendly Reverse Genetics System for Modification of the Right End of Fowl Adenovirus 4 Genome. Viruses, 12.","DOI":"10.3390\/v12030301"},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Zou, X., Rong, Y., Guo, X., Hou, W., Yan, B., Hung, T., and Lu, Z. (2021). Fiber1, but not fiber2, is the essential fiber gene for fowl adenovirus 4 (FAdV-4). J. Gen. Virol., 102.","DOI":"10.1099\/jgv.0.001559"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"85","DOI":"10.1016\/j.jviromet.2018.04.001","article-title":"DNA assembly technique simplifies the construction of infectious clone of fowl adenovirus","volume":"257","author":"Zou","year":"2018","journal-title":"J. Virol. Methods"},{"key":"ref_30","first-page":"515","article-title":"Construction of adenoviral vectors","volume":"135","author":"Davis","year":"2000","journal-title":"Methods Mol. Biol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"1313","DOI":"10.1007\/s00705-012-1293-z","article-title":"Construction of an infectious clone of human adenovirus type 41","volume":"157","author":"Chen","year":"2012","journal-title":"Arch. Virol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"63","DOI":"10.1023\/A:1009838416621","article-title":"Preparation of pure cell cultures by cloning","volume":"22","author":"McFarland","year":"2000","journal-title":"Methods Cell Sci."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"8263","DOI":"10.1128\/JVI.77.15.8263-8271.2003","article-title":"Replication-deficient human adenovirus type 35 vectors for gene transfer and vaccination: Efficient human cell infection and bypass of preexisting adenovirus immunity","volume":"77","author":"Vogels","year":"2003","journal-title":"J. Virol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"13207","DOI":"10.1128\/JVI.78.23.13207-13215.2004","article-title":"Novel replication-incompetent vector derived from adenovirus type 11 (Ad11) for vaccination and gene therapy: Low seroprevalence and non-cross-reactivity with Ad5","volume":"78","author":"Holterman","year":"2004","journal-title":"J. Virol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"1171","DOI":"10.1016\/j.exphem.2006.05.005","article-title":"Efficient gene transfer into hematopoietic cells by a retargeting adenoviral vector system with a chimeric fiber of adenovirus serotype 5 and 11p","volume":"34","author":"Lu","year":"2006","journal-title":"Exp. Hematol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"442","DOI":"10.1016\/j.tips.2012.04.005","article-title":"Adenovirus receptors: Implications for targeting of viral vectors","volume":"33","author":"Arnberg","year":"2012","journal-title":"Trends Pharmacol. Sci."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"849","DOI":"10.1089\/hum.2007.067","article-title":"Promoter choice for retroviral vectors: Transcriptional strength versus trans-activation potential","volume":"18","author":"Weber","year":"2007","journal-title":"Hum. Gene Ther."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"49","DOI":"10.1016\/j.coviro.2021.03.006","article-title":"Adenovirus\u2014A blueprint for gene delivery","volume":"48","author":"Greber","year":"2021","journal-title":"Curr. Opin Virol."},{"key":"ref_39","doi-asserted-by":"crossref","unstructured":"Gao, J., Zhang, W., and Ehrhardt, A. (2020). Expanding the Spectrum of Adenoviral Vectors for Cancer Therapy. Cancers, 12.","DOI":"10.3390\/cancers12051139"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"2883","DOI":"10.1182\/bloodadvances.2019000706","article-title":"High-level protein production in erythroid cells derived from in vivo transduced hematopoietic stem cells","volume":"3","author":"Wang","year":"2019","journal-title":"Blood Adv."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"363","DOI":"10.2174\/156652311797415890","article-title":"Development of adenovirus hybrid vectors for Sleeping Beauty transposition in large mammals","volume":"11","author":"Hausl","year":"2011","journal-title":"Curr. Gene Ther."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"909","DOI":"10.3389\/fimmu.2020.00909","article-title":"Factors Which Contribute to the Immunogenicity of Non-replicating Adenoviral Vectored Vaccines","volume":"11","author":"Coughlan","year":"2020","journal-title":"Front. Immunol."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"165","DOI":"10.1002\/rmv.612","article-title":"Adenovirus receptors: Implications for tropism, treatment and targeting","volume":"19","author":"Arnberg","year":"2009","journal-title":"Rev. Med. Virol."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"5288","DOI":"10.1128\/JVI.75.11.5288-5301.2001","article-title":"Construction of avian adenovirus CELO recombinants in cosmids","volume":"75","author":"Francois","year":"2001","journal-title":"J. Virol."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"631","DOI":"10.1002\/jgm.543","article-title":"Development of an adenoviral vector system with adenovirus serotype 35 tropism; efficient transient gene transfer into primary malignant hematopoietic cells","volume":"6","author":"Nilsson","year":"2004","journal-title":"J. Gene Med."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"e98","DOI":"10.1093\/nar\/gnh094","article-title":"A simple, rapid, high-fidelity and cost-effective PCR-based two-step DNA synthesis method for long gene sequences","volume":"32","author":"Xiong","year":"2004","journal-title":"Nucleic Acids Res."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"777","DOI":"10.1089\/hum.2018.258","article-title":"Single Plasmid-Based, Upgradable, and Backward-Compatible Adenoviral Vector Systems","volume":"30","author":"Liu","year":"2019","journal-title":"Hum. Gene Ther."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"2509","DOI":"10.1073\/pnas.95.5.2509","article-title":"A simplified system for generating recombinant adenoviruses","volume":"95","author":"He","year":"1998","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"343","DOI":"10.1038\/nmeth.1318","article-title":"Enzymatic assembly of DNA molecules up to several hundred kilobases","volume":"6","author":"Gibson","year":"2009","journal-title":"Nat. Methods"},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"13970","DOI":"10.1021\/acsnano.1c04556","article-title":"Synthetic Biology Approaches for Engineering Next-Generation Adenoviral Gene Therapies","volume":"15","author":"Collins","year":"2021","journal-title":"ACS Nano"},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"231","DOI":"10.1016\/0003-2697(92)90358-E","article-title":"Quantification of gene expression over a wide range by the polymerase chain reaction","volume":"206","author":"Kinoshita","year":"1992","journal-title":"Anal. Biochem."}],"container-title":["Viruses"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4915\/14\/3\/546\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T22:32:55Z","timestamp":1760135575000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4915\/14\/3\/546"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,3,6]]},"references-count":51,"journal-issue":{"issue":"3","published-online":{"date-parts":[[2022,3]]}},"alternative-id":["v14030546"],"URL":"https:\/\/doi.org\/10.3390\/v14030546","relation":{},"ISSN":["1999-4915"],"issn-type":[{"value":"1999-4915","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,3,6]]}}}