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  • Characterizing the viral genome and host response
  • Identifying and cloning protein targets
  • Expressing and purifying protein targets
  • Immunizing mice and optimizing vaccines

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Home › Applications › mRNA and protein therapeutics › Identifying and cloning protein targets

mRNA and protein therapeutics

  • Characterizing the viral genome and host response
  • Identifying and cloning protein targets
  • Expressing and purifying protein targets
  • Immunizing mice and optimizing vaccines
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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.

Cogent analysis

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

Cat. # Product Size Price License Quantity Details
638948 In-Fusion® Snap Assembly Master Mix 50 Rxns USD $750.00

In-Fusion Snap Assembly Master Mix is designed for fast, directional cloning of one or more fragments of DNA into any vector. This proprietary master mix fuses DNA fragments (e.g., PCR-generated sequences and linearized vectors) efficiently and precisely by recognizing a 15-bp overlap at their ends. This 15-bp overlap can be engineered into the primers designed for PCR amplification of the desired sequences. In Fusion Snap Assembly Master Mix offers high efficiency, even for applications that can be challenging, including the cloning of long fragments, short oligonucleotides, and multiple fragments.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

Documents Components You May Also Like Image Data

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638948: In-Fusion Snap Assembly Master Mix

638948: In-Fusion Snap Assembly Master Mix

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Performance comparison between In-Fusion Snap Assembly and NEBuilder HiFi using inverse PCR.

Performance comparison between In-Fusion Snap Assembly and NEBuilder HiFi using inverse PCR.

Performance comparison between In-Fusion Snap Assembly and NEBuilder HiFi using inverse PCR. A single 3.8-kb insert (Panel A) or a 34.2-kb adenovirus insert (Panel B) was cloned into a 2.7-kb vector which was linearized via inverse PCR. These cloning reactions were performed in triplicate with both In-Fusion Snap Assembly and NEBuilder HiFi. Primers were designed according to the manufacturers' specifications. After transformation and plating, 20 colonies from each replicate were analyzed by Sanger sequencing (for the 3.8-kb insert) or colony PCR (for the adenovirus insert) to determine the cloning accuracy. In-Fusion Snap Assembly yielded 2X more colonies than NEBuilder HiFi.

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The In-Fusion cloning protocol

The In-Fusion cloning protocol

The In-Fusion cloning protocol.

636767 Stellar™ Competent Cells (96-well plate) 96 x 20 uL USD $578.00

Stellar Competent Cells arranged in a 96-well plate for high-throughput transformation. Stellar Cells are an E. coli HST08 strain that provides high transformation efficiency. These cells can be used in a wide variety of applications: from preparation of cDNA and genomic libraries, to construction of longer-length genomic libraries, to subcloning, and even methylated DNA cloning. Stellar Competent Cells lack the gene cluster for cutting foreign methylated DNA (mrr-hsdRMS-mcrBC and mcrA), and are therefore useful for cloning methylated DNA. The cells can also be used for blue/white screening (i.e., α-complementation) when transformed with vectors containing the lacZα gene. A pUC19 vector, SOC Medium, 8-Cap Strips, and a Plate Lid are included. These competent cells are recommended for use with Takara Bio's In-Fusion PCR Cloning Kits.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

Documents Components Image Data

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Stellar Competent Cells provided transformation efficiencies equivalent to or better than those obtained using DH5α or DH10B competent cells

Stellar Competent Cells provided transformation efficiencies equivalent to or better than those obtained using DH5α or DH10B competent cells

The high efficiency of Stellar Competent Cells was especially noticeable in the transformation of 10 kb and 20 kb plasmids. Using DNA plasmids of 2 kb (100 pg), 10 kb (1 ng), and 20 kb (1 ng), each strain of competent cells was transformed and plated on ampicillin-containing LB agar plates. Transformation efficiencies were determined based on the colony counts obtained.

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Stellar Competent Cells provided the highest transformation efficiencies for all the ligation reaction mixtures tested.

Stellar Competent Cells provided the highest transformation efficiencies for all the ligation reaction mixtures tested.

The difference in transformation efficiencies of the competent cells was particularly prominent when cloning 20 kb fragments. Ligation reactions were set up with either 100 ng of a 2 kb, Hind III-cut DNA fragment + 50 ng of Hind III/BAP-cut pUC118 vector, or 75 ng of a 20 kb, Hind III-cut DNA fragment + 25 ng of Hind III/BAP-cut pUC118 vector. All reactions were performed at 16℃ for 6 hours using the Takara DNA Ligation Kit (Cat# 6023 or 6024). Each strain of competent cells was transformed using portions of these reaction mixtures and plated on ampicillin-containing LB agar (+ X-gal). Transformation efficiencies were determined based on the white colony counts obtained.

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Stellar Competent Cell colonies showed a faster growth rate than DH10B colonies

Stellar Competent Cell colonies showed a faster growth rate than DH10B colonies

Stellar colonies transformed with larger plasmid clearly showed a faster growth rate. DNA plasmids of 2 kb (Panel A) and 10 kb (Panel B) were each used to transform Stellar and DH10B competent cells with similar genetic characteristics, including methylation requiring restriction. This experiment used the same method as in Figure 1. Photographs of colonies on agar were taken after 15 hours of incubation.

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Stellar Competent Cells (96-well plate)Catalog No.636767

Stellar Competent Cells (96-well plate)Catalog No.636767
634933 In-Fusion® SMARTer® Directional cDNA Library Construction Kit 10 Rxns USD $1633.00

The In-Fusion SMARTer Directional cDNA Library Construction Kit provides a sensitive and efficient method for producing high-quality, full-length cDNA libraries. The kit utilizes two of our most innovative technologies: SMART (Switching Mechanism At 5' end of RNA Transcript) technology coupled with PCR amplification makes it possible to generate high yields of full-length double-stranded cDNA from nanograms of poly A+ or total RNA. In-Fusion cloning technology makes it easy to clone your SMARTer cDNA library into any location within any vector. You may use the pSMART2IFD Linearized Vector included in the kit, or any vector of your choice, for cloning your library. Isolated clones from finished libraries can be transferred directly to any linearized expression vector for functional analysis-without the need for compatible restriction sites.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

Documents Components Image Data

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SMART(er) cDNA synthesis compared to conventional cDNA synthesis

SMART(er) cDNA synthesis compared to conventional cDNA synthesis
SMART(er) cDNA synthesis compared to conventional cDNA synthesis. Unlike conventional cDNA synthesis methods, which involve a multiple enzyme/multiple step procedure, the SMART(er) cDNA synthesis protocol is performed by one reverse transcription reaction, in a single tube, with no adaptor ligation or intervening purification steps. Following PCR amplification, SMART(er) cDNA is immediately available for a variety of downstream applications.

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The In-Fusion SMARTer Directional cDNA Library Construction Kit includes components for cDNA synthesis, library construction, and library amplification

The In-Fusion SMARTer Directional cDNA Library Construction Kit includes components for cDNA synthesis, library construction, and library amplification
The In-Fusion SMARTer Directional cDNA Library Construction Kit includes components for cDNA synthesis, library construction, and library amplification.

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Insert size screening by PCR

Insert size screening by PCR

Insert size screening by PCR. An In-Fusion SMARTer Directional cDNA library was generated from 10 ng of Control Mouse Liver Total RNA. SMARTer ds cDNA was synthesized, size-fractionated, and then cloned into pSMART2IFD using the In-Fusion Cloning Kit. 15 randomly selected colonies from the unamplified library were size screened by PCR according to the protocol in the User Manual. 3-μl samples of PCR products were electrophoresed on a 1.2% agarose/EtBr gel. Lane M: 1-kb DNA ladder.


Automated In-Fusion Cloning

View application notes demonstrating automated cloning solutions.

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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