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− | <!--图片--> | + | <!--图片--> |
− | <div class="page-header"> | + | <div class="page-header"> |
− | <img src="https://static.igem.org/mediawiki/2021/d/d2/T--Shanghai_Metropolis--bg-1.png" alt="">
| + | <img src="https://static.igem.org/mediawiki/2021/d/d2/T--Shanghai_Metropolis--bg-1.png" alt=""> |
− | <h1>Description</h1>
| + | <h1>Description</h1> |
− | </div> | + | </div> |
| | | |
− | <!--内容--> | + | <!--内容--> |
− | <div class="content-small"> | + | <div class="content-small"> |
− | <section class="article p-t-54 p-b-54">
| + | <section class="article p-t-54 p-b-54"> |
− | <section>
| + | <section> |
− | <h1 class="title">I.Background</h1>
| + | <h1 class="title">I.Background</h1> |
− | <img src="https://static.igem.org/mediawiki/2021/b/b6/T--Shanghai_Metropolis--img_desc_1.jpg" alt="">
| + | <img src="https://static.igem.org/mediawiki/2021/b/b6/T--Shanghai_Metropolis--img_desc_1.jpg" alt=""> |
| | | |
− | <h2 class="title2 blue">HFMD</h2>
| + | <h2 class="title2 blue">HFMD</h2> |
− | <p>Hand, foot, and mouth disease (HFMD) is an infectious disease caused by enteroviruses. HFMD occurs in
| + | <p>Hand, foot, and mouth disease (HFMD) is an infectious disease caused by enteroviruses. HFMD occurs in |
− | children under 5 years of age. It can cause herpes in the mouth, hands, and feet, or complications such as
| + | children under 5 years of age. It can cause herpes in the mouth, hands, and feet, or complications such as |
− | myocarditis and meningitis.</p>
| + | myocarditis and meningitis.</p> |
− | <p>From July to December in 1997, 11 previously healthy children in Peninsular Malaysia succumbed to an acute
| + | <p>From July to December in 1997, 11 previously healthy children in Peninsular Malaysia succumbed to an acute |
− | severe refractory leftventricular heart failure, following a brief prodromal illness characterized by fever
| + | severe refractory leftventricular heart failure, following a brief prodromal illness characterized by fever |
− | (axillary temperature > 38 degrees C) (100%), oral ulcers (72%), extremity rashes (45%) and significant
| + | (axillary temperature > 38 degrees C) (100%), oral ulcers (72%), extremity rashes (45%) and significant |
− | vomiting (55%); they were in the midst of an outbreak of hand, foot and mouth disease (HFMD). China has the
| + | vomiting (55%); they were in the midst of an outbreak of hand, foot and mouth disease (HFMD). China has the |
− | largest number of patients of it (8:87% of the total number of nine foot diseases in 2014), with enterovirus
| + | largest number of patients of it (8:87% of the total number of nine foot diseases in 2014), with enterovirus |
− | 71(EV71) as its main pathogenic agent. Hence, it is then highly urgent for people to think up a solution to
| + | 71(EV71) as its main pathogenic agent. Hence, it is then highly urgent for people to think up a solution to |
− | control and forestall the infection of EV71.</p>
| + | control and forestall the infection of EV71.</p> |
| | | |
− | <h2 class="title2 blue">Vaccine</h2>
| + | <h2 class="title2 blue">Vaccine</h2> |
− | <p>The most effective way to deal with the high prevalence of HFMD caused by EV71 is vaccination. The mechanism
| + | <p>The most effective way to deal with the high prevalence of HFMD caused by EV71 is vaccination. The mechanism |
− | of the vaccine is to introduce viruses or parts of them to human bodies and to build up specific memory
| + | of the vaccine is to introduce viruses or parts of them to human bodies and to build up specific memory |
− | cells, in a word, immunity, for future virus infection.</p>
| + | cells, in a word, immunity, for future virus infection.</p> |
− | <p>There are already various types of EV71 vaccines, including live attenuated viruses, inactivated whole
| + | <p>There are already various types of EV71 vaccines, including live attenuated viruses, inactivated whole |
− | viruses, VLPs, and recombinant protein subunits. Nonetheless, these injection-based vaccines are never
| + | viruses, VLPs, and recombinant protein subunits. Nonetheless, these injection-based vaccines are never |
− | coming without their limitations--- relatively low coverage rate due to children’s fear of needles, high
| + | coming without their limitations--- relatively low coverage rate due to children’s fear of needles, high |
− | consumption of time and labor due to requirements of injection, huge production of biohazardous waste, and
| + | consumption of time and labor due to requirements of injection, huge production of biohazardous waste, and |
− | high incidence of side effects. There isneed to design safer, more convenient and more effective vaccine
| + | high incidence of side effects. There isneed to design safer, more convenient and more effective vaccine |
− | delivery methods to protect people, especially children, from unaddressed and emerging diseases.</p>
| + | delivery methods to protect people, especially children, from unaddressed and emerging diseases.</p> |
− | </section>
| + | </section> |
| | | |
− | <section>
| + | <section> |
− | <h1 class="title">II.General Concept</h1>
| + | <h1 class="title">II.General Concept</h1> |
− | <p>Vaccinations include injections (hepatitis B, BCG, and influenza, etc.) and oral vaccines (polio, cholera,
| + | <p>Vaccinations include injections (hepatitis B, BCG, and influenza, etc.) and oral vaccines (polio, cholera, |
− | and rotavirus). Given the prevalent needle fears of children and a series of problems caused by injections,
| + | and rotavirus). Given the prevalent needle fears of children and a series of problems caused by injections, |
− | we select oral vaccine as the form of our EV71 vaccine.</p>
| + | we select oral vaccine as the form of our EV71 vaccine.</p> |
− | <p>Oral vaccine is self-administered, more acceptable to children, and biohazardous-waste-free; additionally,
| + | <p>Oral vaccine is self-administered, more acceptable to children, and biohazardous-waste-free; additionally, |
− | the oral route enables stimulation of humoral and cellular immune responses at both systemic and mucosal
| + | the oral route enables stimulation of humoral and cellular immune responses at both systemic and mucosal |
− | sites. It seems that oral EV71 vaccine can compensate for the limitations of traditional injection-based
| + | sites. It seems that oral EV71 vaccine can compensate for the limitations of traditional injection-based |
− | vaccine and provide people with a broader and long-lasting protection from HFMD.</p>
| + | vaccine and provide people with a broader and long-lasting protection from HFMD.</p> |
| | | |
− | <h2 class="title2 blue">Lactobacillus casei</h2>
| + | <h2 class="title2 blue">Lactobacillus casei</h2> |
− | <p><i>Lactobacillus casei</i> is an important probiotic to maintain the stability of human intestinal flora. L.
| + | <p><i>Lactobacillus casei</i> is an important probiotic to maintain the stability of human intestinal flora. L. |
− | <i>casei</i> can tolerate the body's defense mechanisms, including enzymes in the mouth, low pH in gastric
| + | <i>casei</i> can tolerate the body's defense mechanisms, including enzymes in the mouth, low pH in gastric |
− | juices, and bile acids in the small intestine. Therefore, L. <i>casei</i> can survive in the intestinal
| + | juices, and bile acids in the small intestine. Therefore, L. <i>casei</i> can survive in the intestinal |
− | tract in
| + | tract in |
− | large quantities after entering the human body, regulating the balance of intestinal flora, and promoting
| + | large quantities after entering the human body, regulating the balance of intestinal flora, and promoting |
− | human digestion and absorption; it is an ideal expression vector for an oral live vaccine. Therefore, after
| + | human digestion and absorption; it is an ideal expression vector for an oral live vaccine. Therefore, after |
− | we transform the plasmid into E. coli, we will also transform them into L. casei to express the target
| + | we transform the plasmid into E. coli, we will also transform them into L. casei to express the target |
− | proteins.</p>
| + | proteins.</p> |
| | | |
− | <h2 class="title2 blue">EV71 vaccine</h2>
| + | <h2 class="title2 blue">EV71 vaccine</h2> |
− | <p><i>VP1</i> is the main antigenic gene of the EV71 virus, which can encode capsid protein and promote the
| + | <p><i>VP1</i> is the main antigenic gene of the EV71 virus, which can encode capsid protein and promote the |
− | invasion
| + | invasion |
− | of virus particles into host cells (figure 1). Therefore, we selected VP1 as the target gene for the
| + | of virus particles into host cells (figure 1). Therefore, we selected VP1 as the target gene for the |
− | development of the EV71 vaccine. The B-subunit of heat-labile enterotoxin (LT) in E. coli, called LTB, has
| + | development of the EV71 vaccine. The B-subunit of heat-labile enterotoxin (LT) in E. coli, called LTB, has |
− | strong immunogenicity and adjuvant activity, and will not do harm to the human body. LTB and a variety of
| + | strong immunogenicity and adjuvant activity, and will not do harm to the human body. LTB and a variety of |
− | unrelated proteins and their non-protein antigens can enhance the mucosal IgA and humoral IgG responses of
| + | unrelated proteins and their non-protein antigens can enhance the mucosal IgA and humoral IgG responses of |
− | antigens through different immune pathways. In conclusion, we will combine VP1 and LTB in our vaccine.</p>
| + | antigens through different immune pathways. In conclusion, we will combine VP1 and LTB in our vaccine.</p> |
− | <div class="img-container">
| + | <div class="img-container"> |
− | <img src="https://static.igem.org/mediawiki/2021/4/4d/T--Shanghai_Metropolis--img_desc_2.jpg" alt="">
| + | <img src="https://static.igem.org/mediawiki/2021/4/4d/T--Shanghai_Metropolis--img_desc_2.jpg" alt=""> |
− | <span>Figure-1 VP1 is the main antigen gene of EV71</span>
| + | <span>Figure-1 VP1 is the main antigen gene of EV71</span> |
− | </div>
| + | </div> |
− | <p>We use subunit vaccine and adjuvant, VP1-LTB in this case since it includes the immunological part of EV71
| + | <p>We use subunit vaccine and adjuvant, VP1-LTB in this case since it includes the immunological part of EV71 |
− | but does not confront people with the risk of counteractive effects, such as the virus particles returning
| + | but does not confront people with the risk of counteractive effects, such as the virus particles returning |
− | back to active stages and infecting people.</p>
| + | back to active stages and infecting people.</p> |
| + | |
| + | <h2 class="title2 blue">Design</h2> |
| + | <section> |
| + | <p>First of all, we will manipulate PCR on two types of plasmids: pUC57-VP1 and pUC57-LTB. After each PCR |
| + | process, we will manipulate electrophoresis on the samples that we have got, in order to examine if our |
| + | PCR |
| + | is successful and to extract the DNA fragments we want. Therefore, from the first step, we will obtain |
| + | DNA |
| + | fragments VP1, VP1-Lkr, and LTB. With the linker, we are able to manipulate overlap extension PCR (OE |
| + | PCR) |
| + | to connect VP1-Lkr and LTB together to form VP1-LTB. After this, we will conduct enzyme digestion on |
| + | VP1-LTB |
| + | and VP1, which create sticky ends at both ends of these DNA fragments.</p> |
| + | <p>We will then carry out enzyme digestion on the plasmid pGEX-6P-1 to cut off the DNA fragment. Then, we |
| + | will |
| + | ligate link VP1 and VP1-LTB fragments respectively with the plasmid pGEX-6P-1. Finally, we will get two |
| + | plasmids: pGEX-6P-1-VP1 and pGEX-6P-1-VP1-LTB.</p> |
| + | <p>Next, we will transform these two plasmids into chemically competent cells, E. coli DH5α, and allow these |
| + | bacteria to grow and replicate. After this step, a massive number of plasmids pGEX-6P-1-VP1 and |
| + | pGEX-6P-1-VP1-LTB could be extracted from the bacteria. A small amount of this sample will be sent to |
| + | genetic sequencing agencies to test if the transformation of the plasmid has been successful. The rest |
| + | of |
| + | the sample will be processed in another transformation process. pGEX-6P-1-VP1 and pGEX-6P-1-VP1-LTB will |
| + | be |
| + | transformed into two other types of bacteria: E. coli BL21 and L. casei ATCC 334. Then, we will carry |
| + | out |
| + | IPTG induction for E. coli BL21 and L. casei ATCC 334. After this, we will manipulate SDS-PAGE, |
| + | Coomassie |
| + | brilliant blue staining, and Western blot to detect the expression of VP1 and VP1-LTB proteins.</p> |
| + | <p>All the above processes are shown in figures 2 and 3.</p> |
| + | </section> |
| + | <div class="img-container"> |
| + | <img src="https://static.igem.org/mediawiki/2021/7/72/T--Shanghai_Metropolis--img_desc_3.jpg" alt="" style="width: 80%"> |
| + | <span>Figure-2 General concept of the experiments (1)</span> |
| + | </div> |
| + | <div class="img-container"> |
| + | <img src="https://static.igem.org/mediawiki/2021/f/fe/T--Shanghai_Metropolis--img_desc_4.jpg" alt="" style="width: 80%"> |
| + | <span>Figure-3 General concept of the experiments (2)</span> |
| + | </div> |
| + | </section> |
| | | |
− | <h2 class="title2 blue">Design</h2>
| |
| <section> | | <section> |
− | <p>First of all, we will manipulate PCR on two types of plasmids: pUC57-VP1 and pUC57-LTB. After each PCR | + | <h1 class="title">III.Expected Result</h1> |
− | process, we will manipulate electrophoresis on the samples that we have got, in order to examine if our
| + | <ol> |
− | PCR
| + | <li> |
− | is successful and to extract the DNA fragments we want. Therefore, from the first step, we will obtain
| + | <p>The plasmid pGEX-6P-1-VP1 and pGEX-6P-1-VP1-LTB are successfully constructed, which are identified by enzyme double digestion and enzyme single digestion.</p> |
− | DNA
| + | </li> |
− | fragments VP1, VP1-Lkr, and LTB. With the linker, we are able to manipulate overlap extension PCR (OE
| + | <li> |
− | PCR)
| + | <p>VP1 protein products with biological functions could be obtained from E. coli BL21 and L. casei ATCC 334. SDS-PAGE, Coomassie brilliant blue staining, and Western blot analysis show that the protein has good antigenicity.</p> |
− | to connect VP1-Lkr and LTB together to form VP1-LTB. After this, we will conduct enzyme digestion on
| + | </li> |
− | VP1-LTB
| + | <li> |
− | and VP1, which create sticky ends at both ends of these DNA fragments.</p>
| + | <p>We hope that our project could pave the way for a much safer, more convenient and efficient oral vaccine for children to defend against HFMD.</p> |
− | <p>We will then carry out enzyme digestion on the plasmid pGEX-6P-1 to cut off the DNA fragment. Then, we | + | </li> |
− | will | + | </ol> |
− | ligate link VP1 and VP1-LTB fragments respectively with the plasmid pGEX-6P-1. Finally, we will get two
| + | |
− | plasmids: pGEX-6P-1-VP1 and pGEX-6P-1-VP1-LTB.</p>
| + | |
− | <p>Next, we will transform these two plasmids into chemically competent cells, E. coli DH5α, and allow these
| + | |
− | bacteria to grow and replicate. After this step, a massive number of plasmids pGEX-6P-1-VP1 and | + | |
− | pGEX-6P-1-VP1-LTB could be extracted from the bacteria. A small amount of this sample will be sent to
| + | |
− | genetic sequencing agencies to test if the transformation of the plasmid has been successful. The rest
| + | |
− | of
| + | |
− | the sample will be processed in another transformation process. pGEX-6P-1-VP1 and pGEX-6P-1-VP1-LTB will
| + | |
− | be
| + | |
− | transformed into two other types of bacteria: E. coli BL21 and L. casei ATCC 334. Then, we will carry
| + | |
− | out
| + | |
− | IPTG induction for E. coli BL21 and L. casei ATCC 334. After this, we will manipulate SDS-PAGE,
| + | |
− | Coomassie
| + | |
− | brilliant blue staining, and Western blot to detect the expression of VP1 and VP1-LTB proteins.</p>
| + | |
− | <p>All the above processes are shown in figures 2 and 3.</p>
| + | |
| </section> | | </section> |
− | <div class="img-container">
| |
− | <img src="https://static.igem.org/mediawiki/2021/7/72/T--Shanghai_Metropolis--img_desc_3.jpg" alt="" style="width: 80%">
| |
− | <span>Figure-2 General concept of the experiments (1)</span>
| |
− | </div>
| |
− | <div class="img-container">
| |
− | <img src="https://static.igem.org/mediawiki/2021/f/fe/T--Shanghai_Metropolis--img_desc_4.jpg" alt="" style="width: 80%">
| |
− | <span>Figure-3 General concept of the experiments (2)</span>
| |
− | </div>
| |
− | </section>
| |
| | | |
− | <section>
| + | <h1 class="title">IV.Reference</h1> |
− | <h1 class="title">III.Expected Result</h1> | + | <section class="align-normal-all"> |
− | <ol>
| + | <ol> |
− | <li>
| + | <li> |
− | <p>The plasmid pGEX-6P-1-VP1 and pGEX-6P-1-VP1-LTB are successfully constructed, which are identified by enzyme double digestion and enzyme single digestion.</p>
| + | <p>Buch MH, Liaci AM, O'Hara SD, Garcea RL, Neu U, Stehle T (October 2015). "Structural and |
− | </li>
| + | Functional Analysis of Murine Polyomavirus Capsid Proteins Establish the Determinants of Ligand |
− | <li>
| + | Recognition and Pathogenicity". PLoS Pathogens. 11 (10): e1005104. |
− | <p>VP1 protein products with biological functions could be obtained from E. coli BL21 and L. casei ATCC 334. SDS-PAGE, Coomassie brilliant blue staining, and Western blot analysis show that the protein has good antigenicity.</p>
| + | doi:10.1371/journal.ppat.1005104</p> |
− | </li>
| + | </li> |
− | <li>
| + | <li> |
− | <p>We hope that our project could pave the way for a much safer, more convenient and efficient oral vaccine for children to defend against HFMD.</p>
| + | <p>Ramqvist T, Dalianis T (August 2009). "Murine polyomavirus tumour specific transplantation |
− | </li>
| + | antigens and viral persistence in relation to the immune response, and tumour development". |
− | </ol>
| + | Seminars in Cancer Biology. 19 (4): 236–43. doi:10.1016/j.semcancer.2009.02.001</p> |
− | </section>
| + | </li> |
− | | + | <li> |
− | <h1 class="title">IV.Reference</h1>
| + | <p>Nassef, C., Ziemer, C., & Morrell, D. S. (2015). Hand-foot-and-mouth disease: a new look at a |
− | <section class="align-normal-all">
| + | classic viral rash. Current opinion in pediatrics, 27(4), 486–491. |
− | <ol>
| + | https://doi.org/10.1097/MOP.0000000000000246</p> |
− | <li>
| + | </li> |
− | <p>Buch MH, Liaci AM, O'Hara SD, Garcea RL, Neu U, Stehle T (October 2015). "Structural and
| + | <li> |
− | Functional Analysis of Murine Polyomavirus Capsid Proteins Establish the Determinants of Ligand
| + | <p>Who.int. 2021. How do vaccines work?. [online] Available at: <https://www.who.int/news-room/feature-stories/detail/how-do-vaccines-work?gclid=EAIaIQobChMIn4OC7YOh8gIVsG1vBB0wYgcmEAAYAyAAEgIBFvD_BwE> |
− | Recognition and Pathogenicity". PLoS Pathogens. 11 (10): e1005104.
| + | [Accessed 8 August 2021]. |
− | doi:10.1371/journal.ppat.1005104</p>
| + | </p> |
− | </li>
| + | </li> |
− | <li>
| + | <li> |
− | <p>Ramqvist T, Dalianis T (August 2009). "Murine polyomavirus tumour specific transplantation
| + | <p>Yee, Pinn & Poh, Chit. (2015). Development of Novel Vaccines against Enterovirus-71. Viruses. 8. |
− | antigens and viral persistence in relation to the immune response, and tumour development".
| + | 1. 10.3390/v8010001.</p> |
− | Seminars in Cancer Biology. 19 (4): 236–43. doi:10.1016/j.semcancer.2009.02.001</p>
| + | </li> |
− | </li>
| + | <li> |
− | <li>
| + | <p>Orlando, A.; Refolo, M. G.; Messa, C.; Amati, L.; Lavermicocca, P.; Guerra, V.; Russo, F. |
− | <p>Nassef, C., Ziemer, C., & Morrell, D. S. (2015). Hand-foot-and-mouth disease: a new look at a
| + | (October 2012). "Antiproliferative and Proapoptotic Effects of Viable or Heat-Killed IMPC2.1 and |
− | classic viral rash. Current opinion in pediatrics, 27(4), 486–491.
| + | GG in HGC-27 Gastric and DLD-1 Colon Cell Lines". Nutrition and Cancer. 64 (7): 1103–1111. |
− | https://doi.org/10.1097/MOP.0000000000000246</p>
| + | doi:10.1080/01635581.2012.717676</p> |
− | </li>
| + | </li> |
− | <li>
| + | </ol> |
− | <p>Who.int. 2021. How do vaccines work?. [online] Available at: <https://www.who.int/news-room/feature-stories/detail/how-do-vaccines-work?gclid=EAIaIQobChMIn4OC7YOh8gIVsG1vBB0wYgcmEAAYAyAAEgIBFvD_BwE>
| + | |
− | [Accessed 8 August 2021].
| + | |
− | </p>
| + | |
− | </li>
| + | |
− | <li>
| + | |
− | <p>Yee, Pinn & Poh, Chit. (2015). Development of Novel Vaccines against Enterovirus-71. Viruses. 8.
| + | |
− | 1. 10.3390/v8010001.</p>
| + | |
− | </li>
| + | |
− | <li>
| + | |
− | <p>Orlando, A.; Refolo, M. G.; Messa, C.; Amati, L.; Lavermicocca, P.; Guerra, V.; Russo, F.
| + | |
− | (October 2012). "Antiproliferative and Proapoptotic Effects of Viable or Heat-Killed IMPC2.1 and
| + | |
− | GG in HGC-27 Gastric and DLD-1 Colon Cell Lines". Nutrition and Cancer. 64 (7): 1103–1111.
| + | |
− | doi:10.1080/01635581.2012.717676</p>
| + | |
− | </li>
| + | |
− | </ol>
| + | |
| | | |
| | | |
| + | </section> |
| </section> | | </section> |
− | </section> | + | </div> |
− | </div> | + | |
| | | |
− | <!--Footer--> | + | <!--Footer--> |
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− | <div class="content">
| + | <div class="content"> |
− | <hr>
| + | <hr> |
− | <span>Email us at: zhixuansong1022@qq.com</span>
| + | <span>Email us at: zhixuansong1022@qq.com</span> |
− | <span>Search‘EV71terminator’ in Wechat to find more about us</span>
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