pST1-MITD抗原RNA体外转录载体 BioVector NTCC质粒载体菌种细胞基因保藏中心

pST1-MITD抗原RNA体外转录载体 BioVector NTCC质粒载体菌种细胞基因保藏中心
Plasmid templates for in vitro
transcription of naked antigen-encoding RNAs were based on pSTI-A120 and
pST1-MITD vectors34. pST1-MITD features a signal sequence for routeing to the
endoplasmic reticulum and the major histocompatibility complex (MHC) class
I transmembrane and cytoplasmic domains for improved presentation of MHC
class I and II epitopes. pST1-eGFP-A120 (eGFP), pST1-OVA-MITD (OVA), pST1-
Influenza-HA-MITD (HA) and pST1-Luciferase-A120 (Luc) vectors were
described previously34–36. The OVA construct encodes the H-2Kb
-restricted, immunodominant
epitope OVA257-264, and the HA construct contains a codon-optimized
partial sequence of influenza HA (aa 60–285 fused to aa 517–527; influenza strain
A/PR/8/34) designed to combine all major immunodominant MHC epitopes.
pST1-Thy1.1 encodes the murine Thy1.1 protein. pST1-gp70-MITD encodes the
H-2Ld
-restricted peptide antigen AH1423-431 derived from Moloney murine leukaemia
virus envelope glycoprotein 70 (gp70) with an amino acid substitution at position
five (V/A; AH1-A5)37. pST1-E6-MITD and pST1-E7-MITD encode human
papillomavirus (HPV) 16 full-length E6 and E738, respectively, and the sequence
encoding the point-mutated 27-meric peptide CT26-M90 of ALDH18a1 was cloned
into the pST1-MITD vector. pST1-TRP-1-MITD encodes murine tyrosinaserelated
protein 1 (TRP-1). pST1-empty-MITD does not encode a protein and was
used as an irrelevant RNA control. RNA was generated by in vitro transcription as
described previously39. For some experiments, Luc and Thy1.1 RNA was synthesized
using 1-methyl-pseudouridine instead of uridine. Labelling of RNA with Cy3
or Cy5 was performed according to the manufacturer’s instructions (Amersham
Biosciences), and of total uridine triphosphate (UTP), 15% were replaced with
labelled UTP during in vitro transcription of the HA construct
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