Part:BBa_K4004004

pGEX-vp1

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

Figure 2. The expression system of EV71 vp1 in plasmid pGEX...

Figure 3. Schematic map of expression system of pGEX-VP1 plasmids...

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_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

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

To confirm whether we successfully amplified the fragment, we ran the electrophoresis of the fragment. We then scanned the gel, compared the strong bands with the markers, and identified VP1 fragment on the gel.

Figure 4. Gel electrophoresis of VP1 fragments after PCRs...

Conclusion: Theoretically, VP1 fragment is 891bp in length. Compared with the markers, the strong bands all fit in the right range, so it proved that our PCR of fragment was successful, and we could continue our experiments.

·Clonexpress Ligation reaction for pGEX-VP1

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 fragments with pGEX-6P-1.

Figure 5. 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.

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.

Verification of transformed plasmids

·Electrophoresis of pGEX-VP1

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 on the gel.

Figure 6. gel electrophoresis of pGEX-6P-1-VP1...

Conclusion: Theoretically, pGEX-6P-1 is 4984 bp long in linear shape; pGEX-6P-1-VP1 is 5853 bp long in linear shape. Accordingly, pGEX-6P-1, which became linear-shaped after single digestion, should be around 5000bp in length, while pGEX-6P-1-VP1 was in the form of cccDNA, should be around 2500bp in length. Compared with the markers, the plasmids that we extracted from E.coli DH5α all fit in the right range. Therefore, we reached a preliminary conclusion that we succeeded in the experiments of plasmid transformation and obtained the plasmids we wanted.

·Restriction enzyme double digestion of pGEX-VP1 and

We had tested the plasmids we extracted from E.coli DH5α in terms of their length. To further confirm whether the plasmids carried the fragments we wanted, we used SalⅠ and BamHⅠ to cut off the fragments between their cutting sites from pGEX-6P-1-VP1; simultaneously, we conducted single digestion on each group as the negative control group. Then we run the gel electrophoresis of the digested plasmids to separate and identify the fragments cut off from the plasmids.

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

Conclusion: Theoretically, VP1 fragment is 891bp in length; pGEX-6P-1 after double digestion by SalⅠ and BamHⅠis 4975 in length. Compared with the markers, samples 3 and 4 of pGEX-6P-1-VP1 were both correct. We discarded this sample and sent the samples that were proved right after enzyme digestion to a company for sequencing.

Sequencing for VP1 and VP1-LTB fragments on transformed plasmids

After enzyme digestion and gel electrophoresis of pGEX-6P-1-VP1, we tentatively confirmed that the experiment of plasmid transformation into E.coli DH5α was successful, and we obtained the plasmids we wanted. To further verify whether there was any mutation in VP1 fragments on the plasmids, we needed to obtain and compare the upstream and downstream sequences of the two fragments.

Proof of function

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

We transformed pGEX-6P-1-VP1 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 8. 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 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 protein.

Figure 9. 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 10. 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
  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]