Identifying and cloning protein targets
Next generation sequencing-based analysis of TCR and BCR repertoires allows system-wide deciphering of specific receptor configurations and their contribution to adaptive immunity in infections. Once viral genome sequence and host BCR and TCR immune profile data are obtained, further analysis using bioinformatics is performed to reveal the viral antigens and vaccine targets (viral surface receptors) that can be used to effectively select vaccine candidates. Sophisticated bioinformatics pipelines offer a reduction of the complexity of large datasets resulting from such trials to discover reactive clusters of TCR and target-specific BCR-antibody sequences. Virus-specific TCR and BCR (or neutralizing antibody sequences) can be deduced by comparing immune repertoires of sick patients with corresponding control groups (healthy donors) (Schultheiß et al. 2020).
Takara Bio's Cogent NGS Immune Profiler Software can be used along with the SMARTer Human BCR IgG IgM H/K/L Profiling Kit to parse out information about clonotype numbers and V(D)J sequence information. This software is designed to analyze sequence data stored in FASTQ files generated by Illumina sequencers from libraries prepared using the SMARTer Human BCR IgG IgM H/K/L Profiling Kit or SMARTer Human TCR a/b Profiling Kit v2.
Benefits
- Quick results post sequencing—one software covers V(D)J transcripts and clonotypes analysis for both T and B cells
- More confidence in your data—accurate and reliable clonotype calling and quantification, without PCR duplications and errors
- Flexible sequencing options—obtain full-length V(D)J analysis on the MiSeq® or CDR3 analysis on all Illumina platforms
Figure 1. Biological variation in the number of clonotypes provides confidence that subtle differences between healthy or diseased samples can be detected. Clonotype counts from PBMC RNA obtained from different donors. IgG, IgM, IgK, and IgL clonotypes from 10 ng each of PBMC RNA from eight donors (represented by different colors) were determined using the SMARTer Human BCR IgG IgM H/K/L Profiling Kit. Libraries were normalized to 100,000 reads for analysis. The data was analyzed using Cogent NGS immune profiler and then plotted using R.
The vaccine targets identified using bioinformatic analysis of the genomics and immune profiling data are cloned into appropriate vectors for further studies. The use of traditional ligation-based cloning methods to generate target vaccine constructs can be time-consuming, laborious, and expensive. Our In-Fusion Cloning technology can help researchers to accelerate vaccine development workflows by speeding up the generation of target vaccine constructs. In-Fusion Cloning is fast, highly accurate (>95%), sequence-independent (any PCR or synthetic insert can be cloned into any vector at any locus), seamless, directional, and high-throughput ready. The In-Fusion enzyme fuses PCR-generated insert sequences and linearized vectors efficiently and precisely, utilizing a 15-bp overlap at their ends. Use of the 15-bp overlaps eliminates the dependency on restriction site availability, a significant drawback of traditional ligation-based cloning. A simple 15-minute In-Fusion reaction results in seamless and precisely engineered constructs, where no extra bases of vector or restriction-site-derived DNA are added. In-Fusion cloning's speed and accuracy are crucial advantages when generating target vaccine expression constructs, as it allows researchers to avoid adding extra bases/amino acids to their target vaccine, which could result in altered function.
Watch a video outlining applications of In-Fusion Cloning technology.
Benefits
- Subcloning is unnecessary—clone any insert, into any locus, in any vector, with just one reaction
- Highly efficient—over 95% efficiency demonstrated with a broad range of fragment sizes, from 0.5 kb to 15 kb
- Seamless construction—final constructs have no extra base pairs left over (as is often the case with restriction digest or TA cloning)
- Flexibility to clone single or multiple fragments—clone single or multiple DNA fragments simultaneously, in a single reaction
- Can be used to perform site-directed mutagenesis—click here to learn how
There are numerous examples of published work that study and develop vaccines against various pathogens, including coronaviruses, that have utilized In-Fusion Cloning technology within their workflows.
| Title | Link | Product |
|---|---|---|
| Cloning | ||
| SARS-CoV-2 | ||
| Functional assessment of cell entry and receptor usage for SARS-CoV-2 and other lineage B betacoronaviruses |  |
In-Fusion Snap Assembly |
| Cross-reactive Antibody Response between SARS-CoV-2 and SARS-CoV Infections | |
|
| Stability of RNA sequences derived from the coronavirus genome in human cells | |
|
| Development of CRISPR as an Antiviral Strategy to Combat SARS-CoV-2 and Influenza | |
In-Fusion Snap Assembly and Stellar chemically competent cells |
| Other viruses | ||
| Development of a recombinant replication-deficient rabies virus-based bivalent-vaccine against MERS-CoV and rabies virus and its humoral immunogenicity in mice | |
In-Fusion Snap Assembly |
| MERS coronavirus nsp1 participates in an efficient propagation through a specific interaction with viral RNA | |
|
| Genetic manipulation of porcine deltacoronavirus reveals insights into NS6 and NS7 functions: a novel strategy for vaccine design | |
|
| Potency of an inactivated influenza vaccine prepared from a non-pathogenic H5N1 virus against a challenge with antigenically drifted highly pathogenic avian influenza viruses in chickens | |
|
| A rapid strategy for constructing novel simian adenovirus vectors with high viral titer and expressing highly antigenic proteins applicable for vaccine development | |
|
| Cloning of ACE2 isoforms | ||
| The heterogeneous nature of the Coronavirus receptor, angiotensin-converting enzyme 2 (ACE2) in differentiating airway epithelia | |
In-Fusion SMARTer Directional cDNA Library Construction kit |
References
Schultheiß, C., et al. Next-Generation Sequencing of T and B Cell Receptor Repertoires from COVID-19 Patients Showed Signatures Associated with Severity of Disease. Immunity 53, 442–455 (2020).
Featured products
Automated In-Fusion Cloning
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In-Fusion Cloning: general information
Find information about In-Fusion technology, product guides, and performance vs. the competition.
Primer design and other tools
Design your In-Fusion primers with our step-by-step design tool, or access the molar ratio calculator and construct simulator.
In‑Fusion Cloning tips and FAQs
Learn more about In‑Fusion Cloning, including applications, tips, primer design, and vector and insert requirements.
Applications and technical notes
View application data on how In-Fusion technology performs for all of your cloning needs.
Traditional molecular cloning
Choose from a variety of high-quality reagents and kits for every step of your molecular cloning workflow.
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