RESUMO
The newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is causing a spread of coronavirus disease 2019 (COVID-19) globally. In order to end the COVID-19 pandemic, an effective vaccine against SARS-CoV-2 must be produced at low cost and disseminated worldwide. The spike (S) protein of coronaviruses plays a pivotal role in the infection to host cells. Therefore, targeting the S protein is one of the most rational approaches in developing vaccines and therapeutic agents. In this study, we optimized the expression of secreted trimerized S protein of SARS-CoV-2 using a silkworm-baculovirus expression vector system and evaluated its immunogenicity in mice. The results showed that the S protein forming the trimeric structure was the most stable when the chicken cartilage matrix protein was used as the trimeric motif and could be purified in large amounts from the serum of silkworm larvae. The purified S protein efficiently induced antigen-specific antibodies in mouse serum without adjuvant, but its ability to induce neutralizing antibodies was low. After examining several adjuvants, the use of Alum adjuvant was the most effective in inducing strong neutralizing antibody induction. We also examined the adjuvant effect of paramylon from Euglena gracilis when administered with the S protein. Our results highlight the effectiveness and suitable construct design of the S protein produced in silkworms for the subunit vaccine development against SARS-CoV-2.
Assuntos
Compostos de Alúmen/farmacologia , Hidróxido de Alumínio/farmacologia , Bombyx/genética , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Adjuvantes Imunológicos/farmacologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/imunologia , Vacinas contra COVID-19/imunologia , Linhagem Celular , Galinhas/genética , Galinhas/imunologia , Chlorocebus aethiops , Euglena gracilis/imunologia , Infecções por Euglenozoa/imunologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Pandemias/prevenção & controle , SARS-CoV-2/imunologia , Vacinação/métodos , Células VeroRESUMO
Human α1-antitrypsin (AAT) is the most abundant serine proteinase inhibitor (serpin) in the human plasma. Commercially available AAT for the medications of deficiency of α1-antitrypsin is mainly purified from human plasma. There is a high demand for a stable and low-cost supply of recombinant AAT (rAAT). In this study, the baculovirus expression vector system using silkworm larvae as host was employed and a large amount of highly active AAT was recovered from the silkworm serum (~ 15 mg/10 ml) with high purity. Both the enzymatic activity and stability of purified rAAT were comparable with those of commercial product. Our results provide an alternative method for mass production of the active rAAT in pharmaceutical use.
Assuntos
Baculoviridae/genética , Bombyx/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , alfa 1-Antitripsina/química , alfa 1-Antitripsina/genética , Animais , Bombyx/metabolismo , Linhagem Celular , Clonagem Molecular , Humanos , Larva/genética , Larva/metabolismo , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , alfa 1-Antitripsina/isolamento & purificação , alfa 1-Antitripsina/metabolismoRESUMO
Porcine circovirus type 2 (PCV2) is a primary causative agent of postweaningmultisystemic wasting syndrome (PMWS), which has a significant economic impact on the swine industry. The capsid protein (Cap) encoded by ORF2 of the viral genome has been used effectively as a vaccine against PCV2 infection. The Cap protein can spontaneously assemble into virus-like particles (VLPs) that are safe and highly immunogenic for vaccine applications. Several expression systems, including bacteria, yeast and insect cells, have been utilized to produce PCV2 VLPs. However, in some cases, the recombinant Cap (rCap) proteins produced in bacteria and yeast do not assemble spontaneously. In this study, we expressed rCap protein using a silkworm-baculovirus expression vector system (silkworm-BEVS) for mass production of PCV2 VLPs and established a simple three-step protocol for its purification from pupae: extraction by detergent, ammonium sulfate precipitation and anion exchange column chromatography. Size-exclusion chromatography (SEC) analysis and transmission electron microscope (TEM) observation showed that purified rCap proteins formed VLPs with a similar morphology to that of the original virus. Furthermore, the VLPs produced in silkworms were capable of inducing neutralizing antibodies against PCV2 in mice. Our results demonstrated that the silkworm system is a powerful tool for the production of PCV2 VLPs and will be useful for the development of a reliable and cost-effective PCV2 vaccine.
Assuntos
Bombyx/virologia , Proteínas do Capsídeo/isolamento & purificação , Circovirus/efeitos dos fármacos , Síndrome Definhante Multissistêmico de Suínos Desmamados/prevenção & controle , Vacinas de Partículas Semelhantes a Vírus/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes/biossíntese , Anticorpos Antivirais/biossíntese , Baculoviridae/genética , Baculoviridae/metabolismo , Bombyx/genética , Bombyx/crescimento & desenvolvimento , Proteínas do Capsídeo/administração & dosagem , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Circovirus/genética , Circovirus/imunologia , Clonagem Molecular , Feminino , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Imunogenicidade da Vacina , Camundongos , Camundongos Endogâmicos BALB C , Síndrome Definhante Multissistêmico de Suínos Desmamados/imunologia , Síndrome Definhante Multissistêmico de Suínos Desmamados/virologia , Pupa/genética , Pupa/metabolismo , Pupa/virologia , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Suínos , Vacinas de Partículas Semelhantes a Vírus/administração & dosagem , Vacinas de Partículas Semelhantes a Vírus/biossíntese , Vacinas de Partículas Semelhantes a Vírus/genética , Vacinas Virais/administração & dosagem , Vacinas Virais/biossíntese , Vacinas Virais/genéticaRESUMO
Tetracycline-inducible gene expression (Tet-on) system has become one of the first choices for the control of transgenes expression in mammal and drosophila. However, the Tet-on systems that have been established in mammalian system or tuned into drosophila do not function in the silkworm, Bombyx mori. To construct a functional Tet-on system in B. mori, we modified rtTA by introducing a transcription activation domain of immediate-early gene 1 of Autographa californica nuclear polyhedrosis virus and nuclear localization signal of SV40 large T-antigen. The modified rtTA can activate the transcription from 9 x tetO promoter in the silkworm cells up to 250-fold in the presence of doxycycline.
