Part:BBa_K4004007

pGEX-vp1-LTB

promoter

Usage and Biology

Hand-foot-mouth disease (HFMD) is an infectious disease caused by enterovirus 71 (EV71). The virus is an important pathogenic factor of hand, foot and mouth disease. Vp1 protein is the viral capsid protein and promotes the infection of host cells by virus particles. Vp1 is also the main antigen gene of the EV71 virus. Generally, the vaccinated population, especially infants and young children, are more compliant with oral vaccines, so we are trying to develop oral HFMD vaccines. Probiotics bacteria Bifidobacteria, as the natural host of the intestinal tract, can adhere to intestinal epithelial cells and are ideal oral live vaccine expression vectors, and related studies have found that their preventive effects on gastrointestinal pathogens are more significant. Therefore, we can use the bifidobacterium in lactic acid bacteria as an expression system to express EV71 vp1.

Figure 1. Concept map of the EV71 oral vaccine...

Construct design

We link vp1 and LTB with a linker. The vp1-LTB is inserted into plasmid pGEX, respectively (Figure 2 and 3).

Figure 2. The expression system of EV71 vp1...

Figure 3. Schematic map of expression system of pGEX-vp1-LTB plasmid...

The profiles of every basic part are as follows:

Usage and Biology

BBa_K4004001 is a coding sequence of from E. coli . Vp1 protein is the viral capsid protein and promotes the infection of host cells by virus particles. Vp1 is also the main antigen gene of the EV71 virus.

Usage and Biology

BBa_K4004005 is a coding sequence of E. coli, which has strong immunogenicity and adjuvant activity, and will not cause harm to the human body.

BBa_K4004003

Name: pGEX vector

==== Base Pairs: 4969bp ==== Origin: Addgene

Usage and Biology

BBa_K4004003 is a plasmid backbone. pGEX plasmid allows cloning of gene of interest into bacterial expression vector with PreScission Protease cleavable N-terminal GST tag.

Experimental approach

PCR for VP1, VP1-linker and LTB fragments

Firstly, to amplify VP1 fragments and VP1-linker fragments from pUC57-VP1 and LTB fragments from pUC57-LTB, we added VP1-FP and VP1-RP into two tubes to amplify VP1 fragments, VP1-FP and VP1-linker-RP into another two tubes to amplify VP1-linker fragments, and LTB-FP and LTB-RP into another two tubes to amplify LTB fragments.

To confirm whether we successfully amplified the fragments we wanted from the plasmids, we ran the electrophoresis of the fragments in the six tubes. We then scanned the gel, compared the strong bands with the markers, and identified VP1, VP1-linker and LTB fragments on the gel. If we got the expected results, we can extract the three types of fragments from the gel and continue our experiments: digestion of VP1 fragments and OE PCR of VP1-LTB fragments.

Figure 4. Gel electrophoresis of VP1, VP1-linker and LTB fragments after PCR...

Conclusion: Theoretically, VP1 fragment is 891bp in length; VP1-linker fragment is 948bp in length; LTB fragment is 604bp in length. Compared with the markers, the strong bands in the six tubes all fit in the right range, so it proved that our PCR for the three types of fragments was successful, and we could continue our experiments.

·OE PCR for VP1-LTB fragments

After obtaining VP1-linker and LTB fragments from PCR, we overlapped them through OE PCR. We added the two types of fragments, VP1-FP, and LTB-RP into one tube and waited for them to overlap. Then we conducted double digestion on the newly ligated fragments.

To confirm whether we successfully overlapped the two fragments, we ran the electrophoresis of the fragments in the tubes. We then scanned the gel, compared the strong bands with the markers and identified VP1-LTB fragments on the gel. If we got the expected results, we can extract the fragments from the gel and insert them into the vectors.

Figure 5. Gel electrophoresis of VP1-LTB fragments after OE PCR and enzyme digestion...

Conclusion: Theoretically, digested VP1-LTB fragment is 1552bp in length. Compared with the markers, the strong band fit in the right range, so it proved that our OE PCR for VP1-LTB was successful, and we could continue our experiments.

·Clonexpress Ligation reaction for pGEX-VP1-LTB

We had already obtained digested VP1-LTB fragment after PCR and OE PCR. In order to insert them into the vectors pGEX-6P-1 respectively, we first needed to use the same restriction enzymes, SalⅠ and BamHⅠ, to digest pGEX-6P-1 and make the plasmids available for ligation. We then run the gel electrophoresis of digested pGEX-6P-1, identified the fragments we wanted, and extracted them from the gel. After that, we conducted ClonExpress ligation reaction to ligate VP1-LTB fragment with pGEX-6P-1.

Figure 6. gel electrophoresis of pGEX-6P-1 after double digestion...

Conclusion: Theoretically, pGEX-6P-1 after double digestion of SalI and BamHI is 4975bp in length. Compared with the markers, the strong band fit in the right range, so we can continue to conduct ClonExpress ligation reaction for pGEX-6P-1-VP1-LTB.

Simultaneously, we used Snapgene to construct the profiles of the plasmids we constructed, pGEX-6P-1-VP1-LTB. We demonstrated and understood the parts of the plasmids: restriction gene, target gene, and multiple cloning site.

Plasmid transformation

In this part, we transformed the plasmids we constructed into E.coli to replicate them, then extracted and verified the plasmids, and transformed them into E.coli and L.casei again. Finally, we verified the expression of VP1-LTB proteins in the two types of bacteria.

Verification of transformed plasmids

·Electrophoresis of pGEX-VP1-LTB

After transforming the plasmids into E.coli DH5α to replicate them, we picked 6 individual colonies from each petri dish and extracted the plasmids from them. To confirm whether the extracted plasmids were the ones we required, we ran a DNA gel electrophoresis of the transformed plasmids; we also run gel electrophoresis of pGEX-6P-1 after single digestion on the same gel as a negative group. We then scanned the gel, compared the brightest DNA bands with the markers, and identifies pGEX-6P-1-VP1-LTB on the gel.

Figure 7. gel electrophoresis of pGEX-6P-1-VP1 and pGEX-6P-1-VP1-LTB...

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 930
  Illegal XhoI site found at 954
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 5807
  Illegal AgeI site found at 5079
  Illegal AgeI site found at 5828
  
• 1000
  COMPATIBLE WITH RFC[1000]