Difference between revisions of "Part:BBa K4004004"

Revision as of 03:45, 20 October 2021

pGEX-vp1

promoter

Profile

Name: pGEX-vp1

Base Pairs: 5860bp

Origin: E. coli, synthetic

Properties: Hand-foot-mouth disease Drinkable EV71 Vaccine

Usage and Biology

Hand-foot-mouth disease (HFMD) is an infectious disease caused by enterovirus 71 (EV71). The virus is an important pathogenic factor of hand, foot and mouth disease. Vp1 protein is the viral capsid protein and promotes the infection of host cells by virus particles. Vp1 is also the main antigen gene of the EV71 virus. Generally, the vaccinated population, especially infants and young children, are more compliant with oral vaccines, so we are trying to develop oral HFMD vaccines. Probiotics bacteria Bifidobacteria, as the natural host of the intestinal tract, can adhere to intestinal epithelial cells and are ideal oral live vaccine expression vectors, and related studies have found that their preventive effects on gastrointestinal pathogens are more significant. Therefore, we can use the bifidobacterium in lactic acid bacteria as an expression system to express EV71 vp1.

Figure 1. Concept map of the EV71 oral vaccine...

Construct design

Figure 2. The expression system of EV71 vp1 in plasmid pGEX...

Figure 3. Schematic map of expression system of pGEX-VP1 plasmids...

The profiles of every basic part are as follows:

BBa_K4004001

Name: vp1

Base Pairs: 891bp

Origin: E. coli

Properties: Vp1 is also the main antigen gene of the EV71 virus

Usage and Biology

BBa_K4004001 is a coding sequence of from E. coli . Vp1 protein is the viral capsid protein and promotes the infection of host cells by virus particles. Vp1 is also the main antigen gene of the EV71 virus.

BBa_K4004003

Name: pGEX vector

Base Pairs: 4969bp

Origin: Addgene

Properties: A plasmid that allows cloning gene.

Usage and Biology

BBa_K4004003 is a plasmid backbone. pGEX plasmid allows cloning of gene of interest into bacterial expression vector with PreScission Protease cleavable N-terminal GST tag.

Experimental approach

Firstly, to amplify VP1 fragments from pUC57-VP1, we added VP1-FP and VP1-RP into two tubes to amplify VP1 fragments.

To confirm whether we successfully amplified the fragment, we ran the electrophoresis of the fragment. We then scanned the gel, compared the strong bands with the markers, and identified VP1 fragment on the gel.

Figure 4. Gel electrophoresis of VP1 fragments after PCRs...

Conclusion: Theoretically, VP1 fragment is 891bp in length. Compared with the markers, the strong bands all fit in the right range, so it proved that our PCR of fragment was successful, and we could continue our experiments.

·Clonexpress Ligation reaction for pGEX-VP1

We first needed to use the same restriction enzymes, SalⅠ and BamHⅠ, to digest pGEX-6P-1 and make the plasmids available for ligation. We then run the gel electrophoresis of digested pGEX-6P-1, identified the fragments we wanted, and extracted them from the gel. After that, we conducted ClonExpress ligation reaction to ligate VP1 fragments with pGEX-6P-1.

Figure 5. gel electrophoresis of pGEX-6P-1 after double digestion...

Conclusion: Theoretically, pGEX-6P-1 after double digestion of SalI and BamHI is 4975bp in length. Compared with the markers, the strong band fit in the right range, so we can continue to conduct ClonExpress ligation reaction for pGEX-6P-1-VP1.

Plasmid transformation

Sequence and Features