MRNA Vector

Constructs for in vitro transcription. pIVT vector was generated by modifying the pGEM-3Z Vector (Promega, Madison, WI) and used for in vitro transcription of antigen-encoding RNAs. First, a 100 bp poly A tail was inserted into the pGEM-3Z Vector using HindIII restriction sites. The 3′UTR of the β-globin flanked by PstI and SphI restriction enzyme sites was amplified from activated PBMCs using β-globin 3′ UTR-Forward: 5′-TAC CTGCAG AGC TCG CTT TCT TGC TGT CCA ATT TCT-3′) and β-globin 3′ UTR-Reverse: 5′-TAC GCATGC GCA GCA ATG AAA ATA AAT GTT TTT TAT TAG GCA-3′. The amplified β-globin 3′ UTR was then inserted before the poly A tail. To direct secretion of the cloned antigens, the MHC-I secretion signal peptide (78 bp) was amplified from activated PBMCs and inserted into the pGEM-3Z Vector using sec-EcoRI-Forward, 5′-TAC GAA TTC ATG GCC GTC ACG GCG CCC CGA ACC-3′ and sec-KpnI-Reverse, 5′-TAC GGT ACC GGA GCC CGC CCA GGT CTG GGT CAG-3′. Two blunt end restriction sites, EcoRV and BpiI, were introduced before the T7 promoter and after the poly(A) tail using site-directed mutagenesis in order to linearize the construct.