Protocol for recombinant RBD-based SARS vaccines: protein preparation, animal vaccination and neutralization detection.
Identifieur interne : 002062 ( Main/Exploration ); précédent : 002061; suivant : 002063Protocol for recombinant RBD-based SARS vaccines: protein preparation, animal vaccination and neutralization detection.
Auteurs : Lanying Du [États-Unis] ; Xiujuan Zhang ; Jixiang Liu ; Shibo JiangSource :
- Journal of visualized experiments : JoVE [ 1940-087X ] ; 2011.
Descripteurs français
- KwdFr :
- Animaux, Anticorps neutralisants (biosynthèse), Anticorps neutralisants (immunologie), Femelle, Glycoprotéine de spicule des coronavirus, Glycoprotéines membranaires (génétique), Glycoprotéines membranaires (immunologie), Humains, Plasmides (génétique), Protéines de l'enveloppe virale (génétique), Protéines de l'enveloppe virale (immunologie), Souris, Souris de lignée BALB C, Structure tertiaire des protéines, Transfection, Vaccins antiviraux (administration et posologie), Vaccins antiviraux (génétique), Vaccins antiviraux (immunologie), Vaccins antiviraux (sang), Vaccins synthétiques (administration et posologie), Vaccins synthétiques (génétique), Vaccins synthétiques (immunologie), Vaccins synthétiques (sang), Virus du SRAS (génétique), Virus du SRAS (immunologie).
- MESH :
- administration et posologie : Vaccins antiviraux, Vaccins synthétiques.
- biosynthèse : Anticorps neutralisants.
- génétique : Glycoprotéines membranaires, Plasmides, Protéines de l'enveloppe virale, Vaccins antiviraux, Vaccins synthétiques, Virus du SRAS.
- immunologie : Anticorps neutralisants, Glycoprotéines membranaires, Protéines de l'enveloppe virale, Vaccins antiviraux, Vaccins synthétiques, Virus du SRAS.
- sang : Vaccins antiviraux, Vaccins synthétiques.
- Animaux, Femelle, Glycoprotéine de spicule des coronavirus, Humains, Souris, Souris de lignée BALB C, Structure tertiaire des protéines, Transfection.
English descriptors
- KwdEn :
- Animals, Antibodies, Neutralizing (biosynthesis), Antibodies, Neutralizing (immunology), Female, Humans, Membrane Glycoproteins (genetics), Membrane Glycoproteins (immunology), Mice, Mice, Inbred BALB C, Plasmids (genetics), Protein Structure, Tertiary, SARS Virus (genetics), SARS Virus (immunology), Spike Glycoprotein, Coronavirus, Transfection, Vaccines, Synthetic (administration & dosage), Vaccines, Synthetic (blood), Vaccines, Synthetic (genetics), Vaccines, Synthetic (immunology), Viral Envelope Proteins (genetics), Viral Envelope Proteins (immunology), Viral Vaccines (administration & dosage), Viral Vaccines (blood), Viral Vaccines (genetics), Viral Vaccines (immunology).
- MESH :
- chemical , administration & dosage : Vaccines, Synthetic, Viral Vaccines.
- chemical , biosynthesis : Antibodies, Neutralizing.
- chemical , blood : Vaccines, Synthetic, Viral Vaccines.
- chemical , genetics : Membrane Glycoproteins, Vaccines, Synthetic, Viral Envelope Proteins, Viral Vaccines.
- chemical , immunology : Antibodies, Neutralizing, Membrane Glycoproteins, Vaccines, Synthetic, Viral Envelope Proteins, Viral Vaccines.
- genetics : Plasmids, SARS Virus.
- immunology : SARS Virus.
- Animals, Female, Humans, Mice, Mice, Inbred BALB C, Protein Structure, Tertiary, Spike Glycoprotein, Coronavirus, Transfection.
Abstract
Based on their safety profile and ability to induce potent immune responses against infections, subunit vaccines have been used as candidates for a wide variety of pathogens. Since the mammalian cell system is capable of post-translational modification, thus forming properly folded and glycosylated proteins, recombinant proteins expressed in mammalian cells have shown the greatest potential to maintain high antigenicity and immunogenicity. Although no new cases of SARS have been reported since 2004, future outbreaks are a constant threat; therefore, the development of vaccines against SARS-CoV is a prudent preventive step and should be carried out. The RBD of SARS-CoV S protein plays important roles in receptor binding and induction of specific neutralizing antibodies against virus infection. Therefore, in this protocol, we describe novel methods for developing a RBD-based subunit vaccine against SARS. Briefly, the recombinant RBD protein (rRBD) was expressed in culture supernatant of mammalian 293T cells to obtain a correctly folded protein with proper conformation and high immunogenicity. The transfection of the recombinant plasmid encoding RBD to the cells was then performed using a calcium phosphate transfection method with some modifications. Compared with the lipid transfection method, this modified calcium phosphate transfection method is cheaper, easier to handle, and has the potential to reach high efficacy once a transfection complex with suitable size and shape is formed. Finally, a SARS pseudovirus neutralization assay was introduced in the protocol and used to detect the neutralizing activity of sera of mice vaccinated with rRBD protein. This assay is relatively safe, does not involve an infectious SARS-CoV, and can be performed without the requirement of a biosafety-3 laboratory. The protocol described here can also be used to design and study recombinant subunit vaccines against other viruses with class I fusion proteins, for example, HIV, respiratory syncytial virus (RSV), Ebola virus, influenza virus, as well as Nipah and Handra viruses. In addition, the methods for generating a pseudovirus and subsequently establishing a pseudovirus neutralization assay can be applied to all these viruses.
DOI: 10.3791/2444
PubMed: 21587153
Affiliations:
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Le document en format XML
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<term>Viral Vaccines (immunology)</term>
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<term>Femelle</term>
<term>Glycoprotéine de spicule des coronavirus</term>
<term>Glycoprotéines membranaires (génétique)</term>
<term>Glycoprotéines membranaires (immunologie)</term>
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<term>Plasmides (génétique)</term>
<term>Protéines de l'enveloppe virale (génétique)</term>
<term>Protéines de l'enveloppe virale (immunologie)</term>
<term>Souris</term>
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<front><div type="abstract" xml:lang="en">Based on their safety profile and ability to induce potent immune responses against infections, subunit vaccines have been used as candidates for a wide variety of pathogens. Since the mammalian cell system is capable of post-translational modification, thus forming properly folded and glycosylated proteins, recombinant proteins expressed in mammalian cells have shown the greatest potential to maintain high antigenicity and immunogenicity. Although no new cases of SARS have been reported since 2004, future outbreaks are a constant threat; therefore, the development of vaccines against SARS-CoV is a prudent preventive step and should be carried out. The RBD of SARS-CoV S protein plays important roles in receptor binding and induction of specific neutralizing antibodies against virus infection. Therefore, in this protocol, we describe novel methods for developing a RBD-based subunit vaccine against SARS. Briefly, the recombinant RBD protein (rRBD) was expressed in culture supernatant of mammalian 293T cells to obtain a correctly folded protein with proper conformation and high immunogenicity. The transfection of the recombinant plasmid encoding RBD to the cells was then performed using a calcium phosphate transfection method with some modifications. Compared with the lipid transfection method, this modified calcium phosphate transfection method is cheaper, easier to handle, and has the potential to reach high efficacy once a transfection complex with suitable size and shape is formed. Finally, a SARS pseudovirus neutralization assay was introduced in the protocol and used to detect the neutralizing activity of sera of mice vaccinated with rRBD protein. This assay is relatively safe, does not involve an infectious SARS-CoV, and can be performed without the requirement of a biosafety-3 laboratory. The protocol described here can also be used to design and study recombinant subunit vaccines against other viruses with class I fusion proteins, for example, HIV, respiratory syncytial virus (RSV), Ebola virus, influenza virus, as well as Nipah and Handra viruses. In addition, the methods for generating a pseudovirus and subsequently establishing a pseudovirus neutralization assay can be applied to all these viruses.</div>
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