Difference between revisions of "Part:BBa K3992000"

 
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VP7
 
VP7
  
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=== Profile ===
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==== Name: Vp7 ====
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====Base Pairs:898 bp====
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====Origin: E. coli , synthetic====
 +
 
 +
====Properties: Preparation of rotavirus oral vaccine====
 +
 
 +
 
 +
 
 
===Usage and Biology===
 
===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.
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==== Experimental approach ====
 +
====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.
 +
 +
[[File:T--Shanghai high school--BBa K3992000-Figure1.png|500px|thumb|center|Figure 1. Figure 1 PCR verification of VP7 and VP7-LTB.]]
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=== Proof of function ===
 +
====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.
 +
[[File:T--Shanghai high school--BBa K3992001-Figure2.png|500px|thumb|center|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 right figure with the other one, we could find that the adding of LTB stimulates the expression of VP7
 +
[[File:T--Shanghai high school--BBa K3992001-Figure3.png|500px|thumb|center|Figure 3 SDS-PAGE of WB800n protein expression.]]
 +
VP7 and VP7-LTB were successfully expression in E. coli predominantly as inclusion bodies. The protein expression in B. subtilis was not successful as indicated by SDS-PAGE and Western blot
 +
These graphs show the relationship between time and protein expression
 +
[[File:T--Shanghai high school--BBa K3992001-Figure4.png|500px|thumb|center|Figure 4 Figure 4 VP7 expression with 1mM IPTG.]]
 +
[[File:T--Shanghai high school--BBa K3992001-Figure5.png|500px|thumb|center|Figure 5 Figure 5 VP7 expression with 2 mM IPTG.]]
 +
Conclusion: VP7 was present in both the supernatant and sediment, but predominantly as inclusion bodies in the sediment.
 +
=== 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).====
  
 
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Revision as of 04:24, 20 October 2021


VP7

VP7

Profile

Name: Vp7

Base Pairs:898 bp

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.

Experimental approach

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. Figure 1 PCR verification of VP7 and VP7-LTB.

Proof of function

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.

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 right figure with the other one, we could find that the adding of LTB stimulates the expression of VP7

Figure 3 SDS-PAGE of WB800n protein expression.

VP7 and VP7-LTB were successfully expression in E. coli predominantly as inclusion bodies. The protein expression in B. subtilis was not successful as indicated by SDS-PAGE and Western blot These graphs show the relationship between time and protein expression

File:T--Shanghai high school--BBa K3992001-Figure4.png
Figure 4 Figure 4 VP7 expression with 1mM IPTG.
File:T--Shanghai high school--BBa K3992001-Figure5.png
Figure 5 Figure 5 VP7 expression with 2 mM IPTG.

Conclusion: VP7 was present in both the supernatant and sediment, but predominantly as inclusion bodies in the sediment.

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
    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]