Team:Nanjing NFLS/Experiment
“Trojan Horse” antigen selection
Based on the information provided on the NCBI website, we selected a section of HBsAg (containing 92 amino acids) as an artificial antigen nascent antigen. Its sequence is as follows:
YQGMLPVCPLIPGSSTTSTGPCRTCTTPAQGTSMYPSCCCTKPSDGNCTCIPIPSSWAFGKFLWEWASARFSWLSLLVPFVQWFVGLSPTVW
The immunogenicity of this fragment was predicted by the IEDB online prediction website and the results are as follows:
peptide |
Percentile Rank |
HLA |
AA number |
LLVPFVQWFV |
0.13 |
HLA-A02 |
10 |
WLSLLVPFV |
0.2 |
HLA-A02 |
9 |
WLSLLVPFVQWFV |
0.49 |
HLA-A02 |
13 |
LSLLVPFVQWFV |
0.5 |
HLA-A02 |
12 |
SWLSLLVPFVQWFV |
0.54 |
HLA-A02 |
14 |
It can be seen from the predicted results that LLVPFVQWFV in this fragment has a better binding ability to MHC class I molecules of HLA-A02 subtype, indicating that the fragment may have better immunogenicity in vivo.
Establishment of specific regulation system
To ensure that the artificial tumor nascent antigen is only expressed in tumor cells, we use the AND gate to construct a miRNA regulatory gene，which targets the mRNA portion of the artificial tumor neoantigen. On the other hand, a CeRNA designed to competitively bind to the miRNA is constructed on another plasmid. The miRNA is reduced by binding to the ceRNA, thereby reducing the regulation of the mRNA by the miRNA and promoting the expression of the mRNA. To ensure safety, the CeRNA is also regulated by a tumor-specific promoter to ensure its specific initiation in tumor cells.
ThermoFisher's BLOCK-iTTM RNAi Designer software was used to predict miRNA binding sites for selected HBsAg fragments. By scoring, it is proposed to first select the three that have the best score.
miR-HBsAg-70: 5’TGCTGTTGAGCAGTAGTCATGCAGGTGTTTTGGCCACTGACTGACACCTGCATCTACTGCTCAA -3'
miR-HBsAg-125:
5'TGCTGTGCAATTTCCGTCCGAAGGTTGTTTTGGCCACTGACTGACAACCTTCGCGGAAATTGCA -3'
miR-HBsAg-95:
5'TGCTGAACAGGAGGGATACATAGAGGGTTTTGGCCACTGACTGACCCTCTATGTCCCTCCTGTT -3'
For the three sites selected above, combined with the design method of applying TUD[1][2], and with reference to the method of calculation through http://rna.tbi.univie.ac.at/ website and digital model, we finally select the following ceRNA fragments.
CeR-HBsAg-70:
Bulge:GACT
gacggcgctaggatcatcaacACCTGCATGACTGACTACTGCTCAAcaagtattctggtcacagaatacaacACCTGCATGACTGACTACTGCTCAAcaagatgatcctagcgccgtctt
target:TTGAGCAGTAGTCATGCAGGT
Bulge:ACCT
gacggcgctaggatcatcaacACCTGCATGACTACCTACTGCTCAAcaagtattctggtcacagaatacaacACCTGCATGACTACCTACTGCTCAAcaagatgatcctagcgccgtctt
target:TTGAGCAGTAGTCATGCAGGT
CeR-HBsAg-95:
Bulge:TCTC
gacggcgctaggatcatcaacCCTCTATGTATCTCTCCCTCCTGTTcaagtattctggtcacagaatacaacCCTCTATGTATCTCTCCCTCCTGTTcaagatgatcctagcgccgtctt
target:AACAGGAGGGATACATAGAGG
Bulge:AAAC
gacggcgctaggatcatcaacCCTCTATGTATCAAACCCTCCTGTTcaagtattctggtcacagaatacaacCCTCTATGTATCAAACCCTCCTGTTcaagatgatcctagcgccgtctt
target:AACAGGAGGGATACATAGAGG
CeR-HBsAg-125:
Bulge:GAAA
gacggcgctaggatcatcaacAACCTTCGGACGGAAAGAAATTGCAcaagtattctggtcacagaatacaacAACCTTCGGACGGAAAGAAATTGCAcaagatgatcctagcgccgtctt
target:TGCAATTTCCGTCCGAAGGTT
Bulge:GAAT
gacggcgctaggatcatcaacAACCTTCGGACGGAATGAAATTGCAcaagtattctggtcacagaatacaacAACCTTCGGACGGAATGAAATTGCAcaagatgatcctagcgccgtctt
target:TGCAATTTCCGTCCGAAGGTT
Construction of plasmid
Plasmid 1: containing tumor-specific promoter hTERT (454bp), membrane localization sequence SCP1 (75bp), artificial tumor neonatal antigen HBsAg (276bp), flexible linkerGGGGS (15bp), EmGFP (717bp), miRNA inhibition (132bp)
Figure 1. pCDNA6.2-hTERT-HBsAg-EmGFP-miR-HBsAg
Plasmid 2: containing tumor-specific promoter hulc (138 bp), CeRNA competitive binding portion (120 bp)
Figure 2. pCDNA3.1(+)-HULC-ceR-HBsAg
References
[1] HOLLENSEN A K, BAK R O, HASLUND D, et al. Suppression of microRNAs by dual-targeting and clustered Tough Decoy inhibitors [J]. RNA Biol, 2013, 10(3): 406-14.
[2] HOOYKAAS M J G, SOPPE J A, DE BUHR H M, et al. RNA accessibility impacts potency of Tough Decoy microRNA inhibitors [J]. RNA Biol, 2018, 15(11): 1410-9.