Part:BBa_K4276008
GLBP-vp7-vp1-amilGFP
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.
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
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
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
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
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
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.
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.
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.
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.
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.
Improvement
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.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 539
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 260
Illegal NgoMIV site found at 700
Illegal NgoMIV site found at 1222
Illegal NgoMIV site found at 3100
Illegal AgeI site found at 2372
Illegal AgeI site found at 3121 - 1000COMPATIBLE WITH RFC[1000]
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