Difference between revisions of "Part:BBa K4004001"

Latest revision as of 14:08, 12 October 2022

vp1

Profile

Name: vp1

Base Pairs: 891bp

Origin: Enterovirus 71,genome

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

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. 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.

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 1. Gel electrophoresis of VP1 fragments after PCR...

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. Then we put vp1 fragment into plasmid pGEX to get a recombinant plasmid pGEX-vp1 and we performed functional test on this recombinant plasmid.

Proof of function

SDS-PAGE and Coomassie Brilliant Blue staining for whole bacteria, supernate, and precipitation

We transformed pGEX-6P-1-VP1 into E.coli BL21 respectively and incubated them. Firstly, we ran a PAGE gel of the whole bacteria, supernate, and precipitation of E.coli BL21 and then stained the gel through Coomassie Brilliant Blue Staining.

Figure 2. PAGE gel of GST, GST-VP1 and GST-VP1-LTB after staining(W: whole bacteria; S: supernatant; P: precipitation)...

Conclusion: After Coomassie Brilliant Blue Staining, we found that the extent of the brightness of the band in the P group was comparable to that in the W group, while the band in the S group was nearly invisible. In other words, GST, GST-VP1 had all been successfully expressed by E.coli BL21, and they mainly existed in the precipitation in the form of inclusion body.

Due to the relatively low rate of growth and efficiency of electroporation of L. casei, our team first transformed E. coli BL21, which is commonly used in plasmids transformation, to verify the expression and antigencity of VP1 protein.

Figure 3. SDS-PAGE and Western Blot for expression of VP1 and VP1-LTB proteins...

Expression optimization

In order to find the optimum condition under which the proteins were expressed the most, we selected bacteria solution of different concentration (OD600=0.5/0.6/0.8/1), and inducted them with IPTG solution of different concentration (IPTG=1mM/10mM). Then we ran a PAGE gel of them and then marked the proteins with Coomassie Brilliant Blue Staining Solution. To visualize and compare the expression of proteins under different conditions, we used the software ImageJ to quantify specific bands on the gel, collected and arranged the data, and constructed a broken line graph with OD600 the x- axis and the gray value as the y-axis.

Figure 4. PAGE gel of GST, GST-VP1 and GST-VP1-LTB under different expression conditions...

Improvement by 2022 Shanghai_HS

There is a summary of the background of vp1 and vp7 protein-related parts. To the production of the combined vaccine, we searched the iGEM Biological Parts library for similar fragments of EV71 and RV, and selected BBa_K4004001, vp-1. This is a basic part built by iGEM21_Shanghai_Metropolis team in 2021, they tried to fuse vp-1 with vaccine adjuvant LTB, protein expression. We further screened for part BBa_K3992000, vp7, submitted by the iGEM21_Shanghai_high_school team to add some basic sequence information.

Compared to the 2021 oral vaccine project o, we designed the plasmid pET28a-GLBP-vp7-vp1-GFP which combines vp1 with vp7 antigens. In addition, the antigen also fused a membrane-bound protein GLBP to allow the antigens to display on the bacterial surface. Meanwhile, to facilitate the detection of vp7-vp1 localization in bacteria, a green fluorescent protein (GFP) was added. Eventually, we detected the GFP fluorescence signal, indicating that IPTG-induced protein GLBP-vp7-vp1-GFP successfully expressed. The results showed that the main antigens vp1 of EV71 and vp7 of RV were co-expressed fused with GLBP and expressed on the surface of bacteria to obtain a combined bivalent vaccine. This innovative idea is feasible. In the future, we will let the protein GLBP-vp7-vp1-GFP into Lactobacilli Bifidobacteria, made of an oral bivalent vaccine. It not only can improve the vaccination rate but also does not need to be vaccinated professionals, which the consumption of social resources is lower.

GLBP-vp7-vp1-amilGFP

BBa_K4276008

Name: GLBP-vp7-vp1-amilGFP

Base Pairs: 3858 bp

Origin: enterovirus 71, rotaviruses, genome

Properties: tool for monitor the antigens expression

Usage and biology

The researches indicate that the major capsid protein vp1 is the main component of the polyomavirus capsid and the main antigens of EV71. vp1 monomers are typically about 350 amino acids in length and can self-assemble into icosahedral structures consisting of 72 pentamers of 360 vp1 molecules. A capsid protein, vp7, is the main antigens of RV, early studies using sera from hyperimmunized animals in cross-neutralization assays described some different serotypes that were found to infect humans and animals based on the vp7 protein. GLBP is a membrane protein belonging to the family of ATP-binding cassette transport proteins. It is widely expressed in the cell wall of wild-type Bifidobacteria and actively transports specific substances across the cell membrane of all organisms using the energy of adenosine triphosphate (ATP). Antigens expressed in fusion with GLBP can induce mucosal immunity and immune memory. Green fluorescent protein (GFP) is a fluorescent tag that facilitates the detection of vp7-vp1 localization on Bifidobacteria.

Our goal is to produce vp1 and vp7 combined vaccine to protect infants from these two viruses. In addition, the oral method is much more convenient, which can not only improve the vaccination rate, but also does not require professionals to vaccinate, and the consumption of social resources is lower. The plasmid of GLBP-vp7-vp1-amilGFP showed in figure 1.

Figure 1. map of GLBP-vp7-vp1-amilGFP.

The components of the plasmid are described as follows:

BBa_K4276000

Name: GLBP

Base Pairs: 1314 bp

Origin: Bifidobacterium, genome

Properties: a protein anchored in the membrane of gram-positive bacteria

Usage and biology

Galacto-N-biose/lacto-N-biose I-binding protein (GL-BP) belongs to the family of ATP-binding cassette-type transporter from Bifidobacterium. The GL-BP are anchored in the membrane of gram-positive bacteria by lipid.

BBa_K4004001

Name: vp1

Base Pairs: 891 bp

Origin: Enterovirus 71 (EV71), genome

Properties: capsid protein and used to be antigen

Usage and biology

The EV71 is a single-stranded RNA virus coated capsid which consists of structural proteins (VP1-VP4). Among the four structural proteins, VP1 is an immunodomaint protein due to distributing in the capsid surface. The VP1 mutations allow to escape the host immune response. Thus, the vaccines were designed based on the VP1 region

BBa_K4276009

Name: vp7

Base Pairs: 883 bp

Origin: Rotaviruses, genome

Properties: a protein anchored in the membrane of gram-positive bacteria

Usage and biology

Rotaviruses are the most common pathogen of gastroenteritis among infants and young children. Rotavirus are members of the Reoviridae family and transmitted through the fecal-oral route. These viruses consist of multilayered, non-enveloped particles with double-stranded RNA. VP4 and VP7 are exposed on the surface of capsid. Thus, these proteins are considered as the antigen to induce the neutralizing antibodies against the Rotavirus. VP7 is a calcium-binding glycoprotein.

Experiment approach

1.1 DNA fragments amplified by PCR

Figure 2. Gel electrophoresis result showed vp1, vp7, GLBP, and GFP fragments M: DNA marker.

The vp1, vp7, GLBP, and GFP DNA fragments PCR result shown in Figure 2. Compared to DNA maker, the PCR products exhibits the correct bands.

1.2 overlap extension PCR

Figure 3. Gel electrophoresis result showed overlap extension PCR. M: DNA marker.

Due to large GLBP-VP7-VP1-GFP DNA fragment, we used overlap extension PCR in two steps. Firstly, we splice GLBP and VP7, VP1 and GFP, respectively (shown as left panel). Then, combine two fragments GLBP-VP7 with VP1-GFP to the final DNA fragment (shown as middle panel).

1.3 Restriction Enzyme Digestion

Figure 4. Gel electrophoresis result exhibited difference between undigested pET28a and digested pET28a. Line 1 is undigested supercoiled pET28a. Line 2-4 are digested linear pET28a.

To obtain pET28a-vp7-vp1 plasmid, we used restriction enzymes EcoRI and XhoI to digest the gene fragments and plasmid to make double digestion samples, then used DNA ligase to join the vp7-vp1 DNA fragment and pET28a vector. After the pET28a plasmids were digested, we used electrophoresis to confirm whether the cleavage was thorough. In vivo, linear (cleaved) plasmids travel a shorter distance compared to circular plasmids due to the more friction. Thus, the digested plasmids in wells 2-4 ran slower than the supercoiled pET28a plasmid in the first well. This result showed that the plasmids were successfully cleaved by restriction enzymes.

1.4 Colony PCR

Figure 5. Gel electrophoresis result demonstrated the length of pET28a-GLBP-RV-EV-GFP in DH10B. Line 1-10 colony PCR of pET28a-GLBP-RV-EV-GFP colonies.

We picked up 10 colonies to verify whether the colony containing the recombinant plasmid pET28a-GLBP-RV-EV-GFP. the result shown as figure 15, and the transformants 2, 4, 7, and 9 has right size. Thus, we picked up positive colony 2 to sequence.

1.5 Sequencing

Figure 6. Sanger sequencing of pET28a-GLBP-vp7-vp1-GFP plasmid.

The correct colony 2 containing pET28a-GLBP-vp7-vp1-GFP plasmid was sent to sequencing. The results showed as Figure and the sequence well matched with the template. Thus, we used this plasmid to perform the subsequent experiment.

Proof of function

2.1 Protein expression and purification

We added IPTG to induce protein expression when the OD600 reached 0.3-0.5. After overnight induction and culture, we collected the cells and ultrasonic fragmentation of cells to release the intracellular proteins pET28a-GLBP-vp7-vp1-GFP. Next, we used nickel column purification to purify the protein pET28a-GLBP-vp7-vp1-GFP we wanted.

Figure 7. SDS-PAGE gel demonstrated the size of the protein samples. P: precipitant S: supernatant E: elution.

The negative control is pET28a plasmid without exogenous gene. The expression of GLBP-VP7-VP1-GFP were confirmed by SDS-PAGE, as shown in Figure 11.

2.2 Fluorimetric determination

The GLBP-vp7-vp1-GFP contains a green fluorescent protein (GFP) tag. Under blue light, the green fluorescence can be detected, and the GFP can locate the protein GLBP-vp7-vp1-GFP. Figure 8 showed that GLBP-vp7-vp1-GFP protein was successfully expressed.

Figure 8. The comparison of supernatant of GLBP-VP7-VP1 fused GFP and without GFP.

The left tube is bacteria expressing pET28a-VP7-VP1 without GFP as the negative control. The right tube is bacteria expressing pET28a-GLBP-VP7-VP1-GFP. The lift panel showed the supernatant is under visible light, and the right panel showed the supernatant is under blue light.

Figure 9. Fluorescence microscopy of GLBP-VP7-VP1-GFP.

It’s obvious that the supernatant contains lots of protein tagged GFP after ultrasonic crushing bacteria expressing pET28a-GLBP-VP7-VP1-GFP, which indicating the VP7 and VP1 overexpressed in the bacteria and bond to the cell surface.

Figure 10. The GFP intensity indicated protein expression.

At the same time, we monitored the bacteria expressed the VP1 and VP7 for 0 to 8 hours using GFP intensity. as shown in figure 10, the result exhibited the relationship of protein expression with time and the effect of IPTG on fluorescence intensity. The longer induction time is beneficial for protein expression, but according to the result, 3 hours is enough for VP1 and VP7 expression. In addition, the IPTG concentration has a significant influence on the GFP intensity during the cultivation. Concentration of IPTG in 0.25 to 10 mM has a little effect on the protein expression. However, higher concentration of IPTG (10 mM) increased the GFP intensity.

Sequence and Features