Part:BBa_K4276005
GLBP-vp7-vp1-IL12-amilGFP
GLBP-vp7-vp1-IL12-amilGFP
Characterization by Shanghai_HS
BBa_K4276005
Name: GLBP-vp7-vp1-IL12-amilGFP
Base Pairs: 5459 bp
Origin: enterovirus 71, rotaviruses, genome
Properties: tool for monitor the protein expression
Usage and biology
We intend to develop a bivalent vaccine containing the two viruses’ antigens. Specifically, VP1 of enterovirus 71 and VP7 of rotaviruses are the viral proteins constitutes the capsid, respectively. Both proteins are exposed to the surface and are accessible for vaccine targeted VP1 and VP7. In addition, the proinflammatory cytokines interleukin-12 (IL-12) was used as mucosal adjuvant to further enhance the immunome response. And, we used alacto-N-biose/lacto-N-biose I-binding protein (GL-BP) to assist the antigen and IL-12 in expressing on the surface of Bifidobacterium membrane. The antigens and IL12 expression is monitored by the GFP intensity. The plasmid of GLBP-vp7-vp1-IL12-amilGFP is shown as 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
BBa_K4276001
Name: IL12
Base Pairs: 1593 bp
Origin: synthesis
Properties: pro-inflammatory type-1 cytokine
Usage and biology
Interleukin-12 (IL-12) is a pro-inflammatory type-1 cytokine which bridges the innate resistance and adaptor immunity. Also, IL-12 has long been studied as a potential vaccine adjuvant to stimulate the immune. In a study reported that oral administration of IL-12 could induce immune responses.
Experiment approach
1.1PCR cloning vp1, vp7, GLBP, GFP and IL-12 DNA fragments
The vp1, vp7, GLBP, GFP and IL-12 DNA fragments amplified by PCR, the result was shown in Figure 2. Compared to DNA maker, the PCR products exhibits the correct bands.
1.2 Overlap extension PCR cloning GLBP-VP7-VP1-IL12-GFP
The electrophoresis result showed the PCR-amplified GLBP-VP7-VP1-IL12-GFP fragment.
1.3 colony PCR of GLBP-VP7-VP1-IL12-GFP plasmid
We picked up 4 colonies to verify whether the colony containing the recombinant plasmid pET28a-GLBP-RV-EV-IL12-GFP. The result shown as figure 17 and the transformants 1, 3, and 4 have right size. Thus, we picked up positive colony 3 to sequencing.
1.4 Sanger sequencing
The correct colony 3 containing pET28a-GLBP-vp7-vp1-IL12-GFP plasmid was sent to sequencing in Genwiz. The results showed as Figure 5 and the sequence well matched with the template. Thus, we used this plasmid to perform the subsequent experiment.
Proof of function
In Figure 6, we found the GLBP-vp7-vp1-IL12-GFP protein in lane S, P and E, it indicated that the GLBP-vp7-vp1-IL12-GFP protein successfully expressed in E.coli BL21 (DE3).
This new composite part is a technical innovation of the project as a new attempt.
Reference
1. Rotavirus Overview[J] David I. Bernstein. The Pediatric Infectious Disease Journal . 2009 (3 Su).
2. Isanaka Sheila,Djibo Ali,Grais Rebecca F. Heat-Stable Oral Rotavirus Vaccine.[J]. The New England journal of medicine,2017,377(3).
3. Kanchan Vibhu, Zaman Khalequ, Aziz Asma Binte,Zaman Sheikh Farzana,Zaman Farzana,Haque Warda,Khanam Mahbuba,Karim Mohammad Mahbubul, Kale Sachin, Ali Syed Khalid,Goveia Michelle G.,Kaplan Susan S.,Gill Davinder,Khan Wasif Ali,Yunus Mohammad,Singh Ajitpal,Clemens John D.A randomized Phase I/II study to evaluate safety and reactogenicity of a heat-stable rotavirus vaccine in healthy adults followed by evaluation of the safety, reactogenicity, and immunogenicity in infants[J]. Human Vaccines & Immunotherapeutics,2020,16(3).
4. Yamamoto S, Wada J, Katayama T, Jikimoto T, Nakamura M, Kinoshita S, et al. Genetically modified Bifidobacterium displaying Salmonella-antigen protects mice from lethal challenge of Salmonella Typhimurium in a murine typhoid fever model[J].Vaccine,2010,28: 6684–6691.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 3359
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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