Peptide-mhc compacts
A technology of HLA-C and HLA-A, applied in the field of HC compounding, can solve the problems of low signal-to-noise ratio of multimer staining, laborious design and verification, and instability, and achieve the effect of obvious commercial potential.
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0222] Example 1: Design and Cloning of the comPACT Minigene Cloned by Restriction Digestion
[0223] Structure of the compPACT minigene used for restriction digestion:
[0224] Basic exemplary components of a compPACT minigene are a signal sequence directing protein secretion, a universal target sequence such as a restriction site or a primer binding site, an antigenic peptide (or neoantigen, NeoE), a second universal target site, a constant β2M, the extracellular domain of the MHC allele and a purification cluster that enables enzymatic modification (eg, biotinylation) and compPACT purification via an affinity tag. Clusters may also contain protease cleavage sites and linker sequences between peptide components. Minigenes can also contain cysteine mutations that act as disulfide bond traps. A map of the comPACT minigene is shown in figure 1 middle. Additional restriction sites upstream and downstream of the MHC heavy chain sequence can be used to insert other MHC allel...
Embodiment 2
[0231] Example 2: Design and cloning of the comPACT minigene via primer annealing
[0232] In a third variation of MHC template-carrier ligation, PCR and restriction digestion are bypassed by annealing two reverse-complementary neoantigen-encoding primers. These primers were designed with 5' and 3' ends starting and ending in complementary sequences that mimic the overhangs from the restriction digest ( Figure 5 ). The sense and antisense primers were incubated with T4 polynucleotide kinase and ATP to phosphorylate the 5' ends ( Figure 22A ). When these primers anneal to each other, they form a double-stranded oligonucleotide sequence with overhanging nucleotides as if digested with restriction enzymes. The phosphorylated neoantigen insert is ligated into a pre-cut MHC template in the vector. The compPACT minigene has the same structure as described in Example 1. The ligation products were then used for PCR amplification of linear compPACT amplicons using bookend univer...
Embodiment 3
[0235] Example 3: Design and cloning of compPACT minigenes assembled via PCR
[0236] Structure of the comPACT minigene used for PCR assembly:
[0237] A fourth method of inserting neoantigens can also be used. In this method, neoantigens are inserted via polymerase chain reaction into an MHC template flanked by an upstream promoter and a downstream polyadenylation signal to form a 2.5 kb minigene. The PCR assembly reaction is shown in Image 6 middle.
[0238] In this example, the comPACT minigene is shown with the following structure: a promoter at the 5' end; a signal sequence with a first universal target sequence; an antigenic peptide; a linker sequence with predominant glycine and serine residues (i.e., the GlySer linker ); the β2M sequence; the second Gly-Ser linker sequence; the MHC heavy chain allele; the third Gly-Ser linker sequence; the purification cluster; and the polyA sequence. In this method the universal target sequences are not identical.
[0239]PCR as...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More