Part:BBa_K1955005

pSB1C3-5'HYG-HA-3'UTR

The complete coding sequence of Hemagglutinin (HA) acquired from GenBank (Accession# AH002466.2) . HA has been commonly used as the antigen for testing the efficiency of antibody response and T cell activation in previous immunology experiments, thus is a easy to use antigen.



(1) The construction of pSB1C3-5’HYG-HA-3’UTR and pSB1C3-5’HYG-OVA-3’UTR:

The pSB1C3-HA-3’UTR and pSB1C3-OVA-3’UTR were digested with EcoRI and XbaI, while the pSB1C3-5’UTR was digested with EcoRI and SpeI. The pSB1C3-HA-3’UTR, pSB1C3-OVA-3’UTR and 5’UTR were purified by gel extraction, and ligated together. After the transformation step, we used colony PCR to check the correctness of the plasmid. The results showed that the approximately 4100 bp long 5’HYG-HA-3’UTR (1446 bp +1700 bp + 774 bp) and 4500 bp 5’HYG-HA-3’UTR (1446 bp + 2098 bp+ 774 bp) could be amplified from the plasmid, meaning that the pSB1C3-HA-3’UTR, pSB1C3-OVA-3’UTR were finished in the step. In order to transfect the plasmid into leishmania by electroporation, we amplified the plasmid in 200 ml LB broth, and purified the DNA by midiprep.



pSB1C3-HA-3’UTR, pSB1C3-OVA-3’UTR checked by colony PCR

The pSB1C3-5’HYG-HA-3’UTR and pSB1C3-5’HYG-OVA-3’UTR were transformed and the colonies were picked to perform colony PCR. The PCR reaction was performed with Taq polymerase, and screened in 0.8％ agarose gel by electrophoresis. The 4100 bp 5’HYG-HA-3’UTR and 4500 bp 5’HYG-OVA-3’UTR were amplified from pSB1C3-5’HYG-HA-3’UTR and pSB1C3-5’HYG-OVA-3’UTR.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1458
  Illegal BamHI site found at 3164
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 3094