Coding

Part:BBa_K3992001

Designed by: Ke ZHU   Group: iGEM21_Shanghai_high-school   (2021-10-12)
Revision as of 10:04, 21 October 2021 by Kimmy2020 (Talk | contribs) (→‎Proof of function)

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LTB

LTB

Profile

Name: LTB

Base Pairs: 604bp

Origin: E. coli

Properties: The B subunit in the heat-labile enterotoxin (LT)

Usage and Biology

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).

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 1 PCR verification of VP7 and VP7-LTB.

Proof of function

Inducible expression

SDS PAGE

Figure 2 shows the protein expression of E. coli with SDS-PAGE. Our purpose was to identify the presence of new proteins and confirm whether they are our proteins of interest --- VP7 and VP7-LTB.

Figure 2 SDS-PAGE of BL21 protein expression.

There are supernatants and 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. 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.

Figure 3 SDS-PAGE of WB800n protein expression.
Western Blot

These graphs show the relationship between time and protein expression

Figure 4 VP7-LTB expression with1mM IPTG.

Conclusion: VP7 LTB was successfully induced to express and was present as inclusion bodies, which increased gradually with increasing induction time.

Figure 5 VP7-LTB expression with 2mM IPTG.
Conclusion:

VP7 LTB was successfully induced to express and was present as inclusion bodies, which gradually increased in expression with increasing induction time, but the heteroprotein appeared so the optimal induction conditions were: 1 mM IPTG for 6 h induction. In order to determine the optimum induction duration of our engineered BL21 strains for the antigen protein expression which is designed to induce neutralizing antibody immune response inside the human body, we conducted several Western Blot and collected the gray value which was calculated by ImageJ to quantify the protein expression against hours.

T--Shanghai high school--BBa K3992001-table.png

When IPTG concentration was given 1mM, VP7-LTB expresses the most fast during the third hour of the induction and it starts to flatten out after 4 hours. So we could imply that our engineered strain PET28a-VP7-LTB would start its maximum response efficiency after 2 hours and its optimum induction duration will be 4 hours around. When IPTG concentration was given 2 mM, the strain expresses the fast in the second hour of the induction but it is also earlier to get the steady phase which the peak of gray value is 120 around. In this case, the optimum induction duration of VP7-LTB shall be 2 hours.

References

1.Liya Hu,Sue E Crawford,Joseph M Hyser,Mary K Estes,BV Venkataram Prasad. Rotavirus non-structural proteins: structure and function[J]. Current Opinion in Virology,2012,2(4).

2.Isanaka Sheila,Djibo Ali,Grais Rebecca F. Heat-Stable Oral Rotavirus Vaccine.[J]. The New England journal of medicine,2017,377(3).

3.Bernstein David I. Rotavirus Vaccines-Going Strong After 15 Years.[J].

4.Carl D. Kirkwood,Lyou-Fu Ma,Megan E. Carey,A. Duncan Steele. The rotavirus vaccine development pipeline[J]. Vaccine,2019,37(50).

5.C.A. Perez,C. Eichwald,O. Burrone,D. Mendoza. Rotavirus vp7 antigen produced by Lactococcus lactis induces neutralizing antibodies in mice[J]. Journal of Applied Microbiology,2005,99(5).

6.Alexander Falkenhagen,Corinna Patzina-Mehling,Ashish K. Gadicherla,Amy Strydom,Hester G. O’Neill,Reimar Johne. Generation of Simian Rotavirus Reassortants with VP4- and VP7-Encoding Genome Segments from Human Strains Circulating in Africa Using Reverse Genetics[J]. Viruses,2020,12(2).

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

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
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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