Selected article for: "eukaryotic expression vector and final extension"
Author: Meng, Fandan; Ren, Yudong; Suo, Siqingaowa; Sun, Xuejiao; Li, Xunliang; Li, Pengchong; Yang, Wei; Li, Guangxing; Li, Lu; Schwegmann-Wessels, Christel; Herrler, Georg; Ren, Xiaofeng
Title: Evaluation on the Efficacy and Immunogenicity of Recombinant DNA Plasmids Expressing Spike Genes from Porcine Transmissible Gastroenteritis Virus and Porcine Epidemic Diarrhea Virus
  • Document date: 2013_3_19
    • ID: 081o2zmd_8
    Snippet: The amplification profile for the TGEV S1 gene (primers P1/ P2) was as follows: 95uC for 5 min followed by 30 cycles of 94uC for 1 min, 54.4uC for 1 min, and 72uC for 3 min. A final extension of 72uC for 10 min was performed at the end of the cycling period. The amplification profile for PEDV-S1 gene (primers P3/P4) and PEDV-S gene (primers P3/P5) were the same as for the TGEV-S1 gene except that the annealing temperature was raised to 56.8uC and.....
    Document: The amplification profile for the TGEV S1 gene (primers P1/ P2) was as follows: 95uC for 5 min followed by 30 cycles of 94uC for 1 min, 54.4uC for 1 min, and 72uC for 3 min. A final extension of 72uC for 10 min was performed at the end of the cycling period. The amplification profile for PEDV-S1 gene (primers P3/P4) and PEDV-S gene (primers P3/P5) were the same as for the TGEV-S1 gene except that the annealing temperature was raised to 56.8uC and 56.4uC, respectively. All PCR products were purified prior to cloning into the eukaryotic expression vector pIRES (TaKaRa, Japan). Briefly, TGEV-S1 was digested with NheI and EcoRI and then inserted into the multiple clone site (MCS) A of the pIRES vector, resulting in a recombinant plasmid pIRES-TGEV-S1; PEDV-S1 and PEDV-S were digested with SalI and NotI and then inserted into MCS B of pIRES-TGEV-S1, respectively. The resulting recombinant plasmids were designated as pIRES-(TGEV-S1-PEDV-S1) and pIRES-(TGEV-S1-PEDV-S), respectively. The cloning steps are shown in Figure 1 . After transformation, clones were picked and validated by DNA sequencing.

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