Part:BBa_K3992005

PHT43- VP7-LTB

pSIP403- 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.

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]