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| Home -> Products & Services -> Custom Plasmid Vector Construction Services -> Custom Construction Service of Adenoviral Shuttle Plasmid Vector | ||
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Custom Construction Service of Adenoviral Shuttle Plasmid Vector Adenovirus is a non-enveloped, double-stranded DNA virus widely found in nature. Due to its high infection efficiency and broad host range, adenovirus has become an important tool in gene therapy, vaccine development, and basic research. Adenovirus vectors can infect both dividing and non-dividing cells and do not integrate into the host genome, making them a safe option for various applications. RGBiotech offers custom construction service of adenoviral shuttle plasmid vector. If you have any needs or questions regarding adenovirus shuttle plasmid vector construction, please feel free to contact us. Our professional team will provide you with the highest quality service and technical support. We look forward to collaborating with you! Why Choose RGBiotech? 1) Rich Backbone Vectors: We have a variety of adenovirus backbone vectors which can meet the diverse needs of customers. Whether for basic research or pre - clinical research, we can provide appropriate vector options.2) Diverse Promoters: We provide a rich resource of promoters, including constitutive promoters (such as CMV, EF1α, etc.), tissue - specific promoters (such as liver - specific Alb promoter, neuron - specific Synapsin promoter, etc.), and inducible promoters (such as Tet - on/Tet - off promoter system). Customers can choose the most suitable promoter to regulate the expression of the target gene according to the experimental purpose and research needs. 3) Diverse Reporter Genes: We are equipped with a variety of commonly used reporter genes, such as green fluorescent protein (GFP), red fluorescent protein (RFP), firefly luciferase (Luciferase), β - galactosidase (β - Gal), etc. These reporter genes can facilitate customers to monitor and evaluate the transduction efficiency of adenovirus vectors and the gene expression situation in real - time. 4) Strict Quality Inspection: We have established a complete quality control system and conduct strict quality inspections on every adenovirus vector construction. We ensure to provide customers with high - quality and reliable adenovirus products. Before delivering the products, we will provide a detailed quality inspection report, allowing customers to clearly understand the product quality. Read More Adenovirus is a group of non - enveloped double - stranded DNA viruses, and its viral particles show an icosahedral symmetric structure. Since its first discovery in 1953, due to its unique biological characteristics, adenovirus has been widely used in fields such as gene therapy, vaccine development, and basic scientific research. 1. Applications of Adenovirus Adenovirus vectors are widely used in multiple fields, including but not limited to: 1) Basic Scientific Research: In basic research fields such as cell biology and molecular biology, adenovirus can be used for gene function research, protein expression regulation, RNA interference, etc. By constructing an adenovirus vector carrying a specific gene and infecting cells, observing the impact of changes in gene expression on cell function provides a powerful tool for in - depth understanding of the molecular mechanisms of life processes.2) Gene Therapy: As a gene therapy vector, adenovirus can efficiently deliver therapeutic genes to target cells, showing great potential in cancer treatment, single - gene genetic disease treatment, etc. For example, in cancer treatment, an adenovirus vector carrying a tumor - suppressor gene can be used to inhibit the growth of tumor cells and induce apoptosis of tumor cells. 3) Vaccine Development: Adenovirus - vector vaccines have good immunogenicity and can stimulate the body to produce strong cellular and humoral immune responses. Vaccines against Ebola virus and some of the COVID - 19 vaccines widely used during the COVID - 19 pandemic use adenovirus as a vector and have achieved remarkable preventive effects. 2. Characteristics of Adenovirus 1) Broad Host Range: It can infect a variety of mammalian cells, including dividing and non - dividing cells, which makes it widely applicable in gene delivery.2) Efficient Transduction: Adenovirus can efficiently infect various cell types, including dividing and non-dividing cells, and the transduction efficiency is usually high, which can meet the needs of most experiments and applications. 3) High Capacity: Adenovirus vectors can accommodate large foreign gene fragments (up to ~8 kb). 4) High Safety: Adenovirus does not integrate into the host genome, reducing the risk of insertional mutagenesis. 5) Easy to Operate and Prepare: The packaging and amplification process of adenovirus is relatively simple and easy to operate in the laboratory, and a large number of viral vectors can be obtained quickly. 6) Regulable Immunogenicity: By modifying the adenovirus vector, its immunogenicity can be regulated, reducing the body's immune response to the vector and improving the safety and effectiveness of the vector in the body. 7) Relatively Stable Viral Particles: The non - enveloped structure makes adenovirus particles relatively stable in the external environment, facilitating storage and transportation. 3. Elements of Adenoviral Shuttle Plasmid Vector 1) Inverted Terminal Repeat (ITR): Located at both ends of the adenovirus genome, it is a crucial site for the initiation of viral DNA replication. In the adenovirus shuttle plasmid, ITR is essential for initiating the replication of the recombinant adenovirus genome. It can provide binding sites for replication - related proteins such as DNA polymerase, guiding the bidirectional replication process of the viral genome.2) Packaging Signal (Ψ): This element determines whether the adenovirus genome can be correctly packaged into the viral capsid. During the construction of the shuttle plasmid, the packaging signal ensures that the recombinant adenovirus genome can be recognized and packaged into complete viral particles. Without this signal, even if the recombinant adenovirus genome is successfully constructed, infectious virus particles cannot be formed. 3) Target Gene Expression Cassette: The expression cassette includes a promoter, multiple cloning site (MCS) and a terminator. 4) Reporter Gene: A reporter gene can be used to monitor virus packaging efficiency and infection effectiveness. Common reporter genes include fluorescent proteins such as GFP (Green Fluorescent Protein) and RFP (Red Fluorescent Protein), enzymatic reporter genes such as luciferase and LacZ (β-galactosidase). Reporter genes are often co-expressed or separately expressed with the target gene. 4. Different Generations of Adenoviruses 1) First - Generation Adenovirus 2) Second - Generation Adenovirus 3) Third - Generation Adenovirus 5. Chimeric Adenovirus 1) Ad5/F35 Adenovirus 2) Ad5/RGD Adenovirus 6. Oncolytic Adenovirus Oncolytic adenovirus is a type of genetically engineered adenovirus. It can specifically replicate and lyse tumor cells in tumor cells, while it cannot effectively replicate in normal cells. By modifying the gene regulatory elements of adenovirus, it can respond to unique environmental signals in tumor cells (such as hypoxia, high expression of certain oncogenes, etc.) and initiate the virus replication process. Oncolytic adenovirus is mainly applied in the field of tumor treatment, it achieves the purpose of treating tumors by directly lysing tumor cells and triggering the body's anti - tumor immune response. Currently, a variety of oncolytic adenovirus products have entered the clinical trial stage and achieved certain therapeutic effects. Oncolytic adenovirus has tumor - specific killing ability, can act locally in tumors, and reduce damage to normal tissues. At the same time, after oncolytic adenovirus lyses tumor cells, it can also release tumor - associated antigens, activate the body's immune system, and generate a systemic anti - tumor immune response. 7. Different Adenovirus Packaging Systems 1) AdEasy System The AdEasy system is an adenovirus packaging system based on homologous recombination in bacteria. First, the target gene is cloned into a shuttle plasmid, which contains part of the adenovirus sequence and screening markers. Then, the shuttle plasmid and bacteria containing the adenovirus backbone are electroporated. Inside the bacteria, the target gene is integrated into the adenovirus backbone through homologous recombination to form a recombinant adenovirus plasmid. Finally, the recombinant adenovirus plasmid is transfected into an adenovirus packaging cell line (such as HEK293 cells) to package recombinant adenovirus particles. 2) AdMax System The AdMax system uses the Cre/loxP recombinase system to construct adenovirus vectors. First, a shuttle plasmid and an adenovirus backbone plasmid containing loxP sites are constructed, and the target gene is cloned into the shuttle plasmid. Then, the shuttle plasmid and the adenovirus backbone plasmid are co - transfected into a cell line expressing Cre recombinase (such as 293Cre cells). Under the action of Cre recombinase, the shuttle plasmid and the backbone plasmid recombine to generate a recombinant adenovirus plasmid and package adenovirus particles. 8. Adenovirus Packaging Process 1) Vector Construction: First, an adenovirus shuttle plasmid vector is constructed, and the target gene is inserted into the shuttle plasmid. This shuttle plasmid contains part of the adenovirus sequence and marker genes for screening and identification.2) Generation of Recombinant Adenovirus Plasmid: The shuttle plasmid and the adenovirus backbone plasmid are co - transfected into specific competent bacteria, and a recombinant adenovirus plasmid is generated through homologous recombination. Inside the bacteria, the shuttle plasmid and the backbone plasmid recombine to form a complete recombinant adenovirus genome. Please note that: This step varies depending on the packaging system you use. If you are using the AdMax system, the recombination should be directly achieved within the packaging HEK293 cell line. 3) Transfection of Recombinant Adenovirus Plasmid: The recombinant adenovirus plasmid is transfected into an adenovirus packaging cell line (such as HEK293 cells). The transfected cells use their own transcription and translation systems to express various structural and non - structural proteins of adenovirus, and then assemble into complete adenovirus particles. 4) Harvest and Preliminary Purification of Adenovirus: After culturing the transfected cells for a period of time, when obvious cytopathic effects (CPE) occur, the cell culture supernatant is collected. Cell debris is removed by methods such as low - speed centrifugation to obtain a preliminarily purified adenovirus crude extract. 9. Adenovirus Amplification Process 1) Cell Seeding: Adenovirus - sensitive cells (such as HEK293 cells) are seeded into cell culture flasks or plates. When the cells grow to the logarithmic growth phase, they are ready for adenovirus infection.2) Adenovirus Infection: The harvested adenovirus crude extract is used to infect the seeded cells according to a certain multiplicity of infection (MOI). During the infection process, adenovirus particles adsorb and enter the cells, and use the cell's metabolic system to replicate their own genomes and synthesize viral proteins. 3) Culture and Observation: The infected cells are continuously cultured under appropriate culture conditions, and the cytopathic conditions are observed regularly. As the virus continues to replicate and assemble, the cells gradually show cytopathic changes, such as cell rounding and detachment. 4) Repeated Infection and Amplification: When the cytopathic effect reaches a certain level, the cell culture supernatant is collected and used as the virus source for a new round of infection. It is then used to infect new cells again. This process of repeated infection is carried out multiple times to achieve large - scale amplification of adenovirus. 5) Final Harvest and Purification: After multiple rounds of amplification, a large amount of cell culture supernatant containing adenovirus is collected. Through a series of purification steps, such as ultra - centrifugation and column chromatography, impurities are removed to obtain a high - purity and high - titer adenovirus product. 6) Titer Determination: Measuring virus titer using methods such as PFU, TCID50 or qPCR to ensure quantity and quality. |
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