Part:BBa_K4004006

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 connect vp1 with LTB through a linker (Figure 2). This part is inserted into plasmid pGEX. Its sequence map is shown in Figure 3.

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

Figure 3. Schematic map of expression module of VP1-LTB...

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.

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. Then we connected VP1-LTB with plasmid to get recombinant plasmid pGEX-6P-1-VP1-LTB.

Proof of function

SDS-PAGE and Coomassie Brilliant Blue staining for whole bacteria, supernate, and precipitation

We transformed pGEX-6P-1-VP1-LTB into E.coli BL21 respectively and incubated them. Firstly, we ran a PAGE gel of the whole bacteria, supernate, and precipitation of E.coli BL21 and then stained the gel through Coomassie Brilliant Blue Staining.

Figure 6. PAGE gel of GST, GST-VP1 and GST-VP1-LTB after staining(W: whole bacteria; S: supernatant; P: precipitation)...

Conclusion: After Coomassie Brilliant Blue Staining, we found that the extent of the brightness of the band in the P group was comparable to that in the W group, while the band in the S group was nearly invisible. In other words, GST, GST-VP1 and GST-VP1-LTB had all been successfully expressed by E.coli BL21, and they mainly existed in the precipitation in the form of inclusion body.

Due to the relatively low rate of growth and efficiency of electroporation of L. casei, our team first transformed E. coli BL21, which is commonly used in plasmids transformation, to verify the expression and antigencity of VP1-LTB protein.

Figure 7. SDS-PAGE and Western Blot for expression of VP1 and VP1-LTB proteins...

Expression optimization

In order to find the optimum condition under which the proteins were expressed the most, we selected bacteria solution of different concentration (OD600=0.5/0.6/0.8/1), and inducted them with IPTG solution of different concentration (IPTG=1mM/10mM). Then we ran a PAGE gel of them and then marked the proteins with Coomassie Brilliant Blue Staining Solution. To visualize and compare the expression of proteins under different conditions, we used the software ImageJ to quantify specific bands on the gel, collected and arranged the data, and constructed a broken line graph with OD600 the x- axis and the gray value as the y-axis.

Figure 8. PAGE gel of GST, GST-VP1 and GST-VP1-LTB under different expression conditions...

References

6. Orlando, A.; Refolo, M. G.; Messa, C.; Amati, L.; Lavermicocca, P.; Guerra, V.; Russo, F. (October 2012). "Antiproliferative and Proapoptotic Effects of Viable or Heat-Killed IMPC2.1 and GG in HGC-27 Gastric and DLD-1 Colon Cell Lines". Nutrition and Cancer. 64 (7): 1103–1111. doi:10.1080/01635581.2012.717676

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 838
  Illegal AgeI site found at 110
  Illegal AgeI site found at 859
  
• 1000
  COMPATIBLE WITH RFC[1000]