Part:BBa_K3992005

PHT43- VP7-LTB

PHT43-VP7-LTB

Usage and Biology

Otavirus (RV) is the main viral pathogen that causes severe acute diarrhea in infants and young children. Almost all children under five weeks of age have been infected with the virus, causing nearly 130,000 deaths worldwide each year. Social conditions in developing countries have led to reduced effectiveness of oral rehydration solutions and vaccines, as well as a lack of approved antiviral drugs, making rotavirus infection a global health problem. RV structural protein vp7, on the outermost layer of virus particles, is the first choice for the development of genetic engineering vaccines. We are trying to develop a new oral vaccine for hand, foot and mouth disease due to its advertisement for infants and young children. The B subunit LTB in the heat-labile enterotoxin (LT) of Escherichia coli heat-labile enterotoxin (LT) has strong immunogenicity and adjuvant activity, and will not cause harm to the human body. LTB and a variety of non-related proteins and their non-protein antigens can increase the mucosal IgA and humoral immune IgG response levels of the antigen through different immunization pathways. Currently, there are three main types of vaccines, including inactivated vaccines/attenuated vaccines, mRNA vaccines/DNA vaccines, and neutralizing antibody/non-neutralizing antibody vaccines. Human vaccination methods include injection (hepatitis B vaccine, BCG vaccine, flu vaccine, etc.) and oral administration (poliomyelitis, cholera vaccine, and rotavirus vaccine).

Construct design

VP7-LTB had been linked by P2A linker. VP7-LTB is inserted into pHT43 plasmid (Figure 1). The sequence of pHT43-VP7-LBT is shown in Figure 2.

Figure 1. The expression system of vp7.

Figure 2. Schematic map of expression system of pHT43-vp7-LTB plasmid.

The profiles of every basic part are as follows:

=BBa_K3992000

Usage and Biology

BBa_K3992000 is a coding sequence of from E. coli. RV structural protein vp7 is on the outermost layer of virus particles.

BBa_K3992001

=Usage and Biology

BBa_K3992001 is a coding sequence of E. coli, which has strong immunogenicity and adjuvant activity, and will not cause harm to the human body.

BBa_K3992003

Usage and Biology

BBa_K3992003 is a part that can express proteins. In our team, we hope that we can express Vp7 protein in B. subtilis.

Experimental approach

Plasmid Construction

Polymerase Chain Reaction

A PCR verification was performed to confirm whether the sequence of synthesized VP7-LTB is cerrect. Agarose gel electrophoresis was used to assess the PCR’s result. According to the 15000 bp DNA marker, the PCR amplified DNA fragments possess the desired right size.

Figure 3 PCR verification of VP7 and VP7-LTB.

Restriction enzyme digestion

At this point we had an empty plasmid vector pHT43-his and two DNA fragments (vp7 and Ltb) awaiting to be inserted. To that end, we used BamHI to digest and linearize the plasmid, making a specific site for fragment insertion.

pHT43-His

We did the same digestion of pHT43-His. As shown in Figure 5, lanes 2 to 7 were the linearized plasmids and the last was the original supercoiled and nicked DNA. It can be identified that the DNA was around 8000 bp, which was consistent to pHT43-His’s standard size-8101 bp.

Figure 4 The result of enzyme digestion of pHT43-His.

Conclusion

The two gel images demonstrated that we had successfully digested the plasmid vector and pHT43-his. According to the standard ladder, linearized DNAs are consistent with their size and distinctively different to the controls.

Colony PCR

After completing restriction enzyme digestion and ligation, we had transformed the new plasmids into different cultures of E. coli BL21 for cloning. Additionally, we also transformed the empty plasmid vector pHT43-HIS into WB800N through electroporation instead of normal processes. We then performed a colony PCR to verify if the two types of bacteria contain the target plasmids.

pHT43-His-VP7 & pHT43-His-VP7-LTB

We expected these two plasmids to be inserted into WB800N at the end of the project. However, considering that WB800N is Gram-positive, we suspect whether they are able to be entered at all. For this reason, we first transformed the plasmids into E. coli BL21, a Gram-negative bacterium with substantially thinner cell wall, to test the feasibility, and performed a colony PCR using 1 μL of the solution containing the cell as DNA template. As a result, the gel image clearly demonstrates that our target genes with correct size have been amplified from the cell’s DNA.

Figure 5 PCR verification of E. coli BL21 containing pHT43-His-VP7.

Figure 6 PCR verification of E. coli BL21 containing pHT43-His-VP7-LTB.

pHT43-His

In the previous experiments, pHT43-His had been transferred into WB800N by electroporation, which opened up temporary pores on host’s membrane to facilitate the entry of the plasmid. Again, a colony PCR was used to verify the transformation using 1 μL of DNA template from the host. The gel image with a 2000 bp DNA ladder identified that our pHT43-His sequence had been successfully amplified from the solution. Unlike BL21’s plasmid transformation gel results, WB800N’s showed additional vague gene bands whose bp values deviate from the standard. This was because, during PCR, not all DNA from the template was paired with the primers; the remaining DNA may paired with random gene sequences, which lead to various sizes.

Figure 7 PCR verification of WB800N containing pHT43.

PHT43-His-VP7 & PHT43-His-VP7-LTB (WB800N)

A colony PCR was conducted to verity the plasmid transformation of PHT43-His-VP7 & PHT43-His-VP7-LTB into bacteria WB800N. According to the gel electrophoresis image, we collected 2 samples of WB800N containing PHT43-His-VP7 and 1 sample of WB800N containing PHT43-His-VP7-LTB. Again, the band of VP7 is at 846 bp and that of VP7-LTB presents at 1242 bp. Therefore, the location of each gene bands on the following image is consistent with the values, which demonstrates successful transformations.

Figure 8 PCR verification of WB800N containing PHT43-His-VP7 & PHT43-His-VP7-LTB.

Conclusion

All gel images are the proofs that our target plasmids have been amplified from the DNA template, which indicates that they have been successfully connected to the cell’s DNA. Furthermore, It can be identified that some lanes have gene bands substantially thicker than the others. This tells us that the amount of DNA amplified in each sample is different. This is caused by the difference between cell cultures from which the DNA template is extracted from. Whether the template is exactly 1 μL can also influence the result.

Proof of function

Inducible expression

SDS PAGE

Figure 9 shows the protein expression of E. coli with SDS-PAGE. SDS-PAGE is a gel electrophoresis technique used for protein separation based on their molecular weight. Our purpose was to identify the presence of new proteins and confirm whether they are our proteins of interest --- VP7 and VP7-LTB.

Figure 9 SDS-PAGE of BL21 protein expression

There are supernatants and precipitates. The precipitates are usually debris of the cell such as organelle. The supernatants are usually protein, carbohydrates, nucleic acids of the bacteria. Usually, the soluble protein should appear in the supernatant. However, it appeared in the precipitates. We suggest that the virus-induced proteins existed in the form of insoluble inclusion body.

Compared the samples of the precipitates and the supernatants at 0 h and other times of the figure on the left, we could see an extra band that located below 50 kD. If there are extra bands, it is highly possible that they are our target proteins. Due to the presence of IPTG, the amount of proteins could be limited. We should see thinner bands in the samples, which are collected after adding IPTG. However, the extra bands are thicker than the parallel bands which are at the 0 h. This phenomenon demonstrates that the thicker bands could be our target proteins. The masses of the bands match pretty well with the theoretical molecular weight of VP7-LTB. Compared the left figure and the middle one, we could find that VP7-LTB induced by 1nM of IPTG expressed better than that induced by 2nM of IPTG. Because IPTG may make the bacteria less active, 1nM is a suitable concentration.

Figure 10 Figure 10 SDS-PAGE of WB800n protein expression

References

7.Offit Paul A. Challenges to Developing a Rotavirus Vaccine.[J]. Viral immunology,2018,31(2).

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 8102
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 8102
  Illegal NheI site found at 1091
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 8102
  Illegal BglII site found at 3022
  Illegal BglII site found at 6103
  Illegal BamHI site found at 2905
  Illegal BamHI site found at 8900
  Illegal XhoI site found at 6107
  Illegal XhoI site found at 8994
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 8102
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 8102
  Illegal AgeI site found at 2932
  
• 1000
  COMPATIBLE WITH RFC[1000]