Part:BBa_K3992004

Vp7-GS linker-LTB

Vp7-GS linker-LTB

Profile

Origin: E. coli , synthetic

====Properties: Preparation of rotavirus oral vaccine

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 plasmid (Figure 1). The sequence of pHT43-VP7-LBT and pET28a-VP7-LBT are shown in Figure 2.

Figure 1. Genetic design of the plasmid.

Figure 2. Figure 2. Schematic map of plasmids.

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_K3992002

Profile

Usage and Biology

BBa_K3992002 is a part of GS linker. It can link different proteins to make them become a fusion protein. In our group, we use this part to link VP7 protein and LTB protein.

Plasmid Construction

Polymerase Chain Reaction

A PCR verification was performed to confirm whether the sequence of synthesized VP7-LTB is correct. 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 two empty plasmid vectors 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.

pET28a

Once digestion completed, we set up gel electrophoresis again to assess the result. The first three lanes were DNA marker ladder, pET28a after digestion, and the control, respectively. The second lane was our linear pET28a with 5639 nt. The place of band in the gel was consistent with its real size.

Figure 4 The result of enzyme digestion of pET28a.

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 5 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.

pET28a-VP7-LTB

According to the agarose gel electrophoresis image, we were able to identify that the target genes of the correct sizes have been successfully amplified. Sequence VP7 had 846 nt, and same was for VP7-LTB that had 1242 nt.

Figure 6 PCR verification of E. coli BL21 containing pET28a-VP7 and pET28a-VP7-LTB.

pHT43-His-VP7-LTB

We expected these two plasmids to be inserted into WB800N at the end of the project. 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. As a result, the gel image clearly demonstrates that our target genes with correct size have been amplified from the cell’s DNA.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 1
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1
  Illegal BamHI site found at 799
  Illegal XhoI site found at 893
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 1
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 1
  
• 1000
  COMPATIBLE WITH RFC[1000]