RESUMO
To seek a new delivery system of vaccine for infectious bronchitis virus (IBV), transgenic potato expressing full-length spike (S) protein of IBV was produced and its immunogenicity in chickens was investigated. One to three copies of S gene of IBV were randomly and stably inserted into potato (Solanum tuberrosum cv. Dongnong 303) genome by Agrobacterium tumefaciens-mediated transformation. Transcription and translation of S gene for IBV were confirmed by Northern blot and Western blot analyses in transgenic plantlets. Chickens immunized orally and intramuscularly with transgenic potato tubers expressing S protein generated the detectable levels of serum neutralizing antibodies and were protected against the challenge with the virulent IBV. In vitro secretion of interleukin 2 and T lymphocyte proliferation of spleen cells from the immunized chickens varied with the dose and the manner of vaccination with S protein derived from transgenic plants. The results indicated that S protein expressed in transgenic plants might be a new source for the production of Coronaviridae IBV vaccine.
Assuntos
Vírus da Bronquite Infecciosa/genética , Vírus da Bronquite Infecciosa/metabolismo , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Solanum tuberosum/genética , Solanum tuberosum/metabolismo , Proteínas do Envelope Viral/biossíntese , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Animais , Galinhas , Clonagem Molecular , Glicoproteínas de Membrana/química , Plantas Geneticamente Modificadas/metabolismo , Engenharia de Proteínas/métodos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Glicoproteína da Espícula de Coronavírus , Proteínas do Envelope Viral/químicaRESUMO
To check the feasibility of expression of the immunogenic gene of avian coronavirus infectious bronchitis virus (IBV) in plants, the transformation of S1 gene of IBV into potato and the immunogenicity of its expression product was studied. The S1 gene of IBV-ZJ971 strain was inserted into plasmid pBI121 under the control of 35 S promoter. Agrobacterium fumefaciens EHA105 with the recombinant vector pBI121 was obtained by tri-parental mating method. So, an efficient potato transformation system mediated by Agrobacterium fumefaciens was established. The rates of calli and shoots differentiation were 100%, and more than 95% respectively, for transgenic potato with S1 gene of IBV. PCR and Southern blot analyses showed that IBV S1 gene was integrated into genomic DNA of the potato plant and most transgenic plants had two copies of S1 gene of IBV. In our experiments, 47 transgenic plantlets have been obtained. Northern blot and ELISA analyses indicated that most transgenic plants could normally transcribe and translate S1 gene of IBV, though the levels of transcription and translation were different in various transgenic plants. Immunity assay with BALB/C mice showed that expression products of transgenic potato with S1 gene of IBV were immunogenic, and ELISA antibody titer reached 1:20 to 1:40 and 1:80 to 1:160 with doses of 0.5 g and 1 g, respectively. Virus neutralization (VN) antibodies were detected by tracheal organ cultures, and the results showed that VN titers reached respectively 1:160 to 1:320 and 1:320 to 1:2048 with doses of 0.5 g and 1 g.
Assuntos
Vírus da Bronquite Infecciosa/genética , Solanum tuberosum/genética , Animais , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Northern Blotting , Southern Blotting , Técnicas de Cultura , DNA de Plantas/genética , Expressão Gênica , Vírus da Bronquite Infecciosa/imunologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Testes de Neutralização , Plantas Geneticamente Modificadas , RNA de Plantas/genética , Glicoproteína da Espícula de Coronavírus , Transformação Genética , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologiaRESUMO
The expression of infectious bronchitis virus (IBV) S1 glycoprotein in potatoes and its immunogenicity in mice and chickens were investigated. Potato plants were genetically transformed with a cDNA construct encoding the IBV S1 glycoprotein with the Agrobacterium system. Genomic DNA and mRNA analyses of the transformed plantlets confirmed the integration of the foreign cDNA into the potato genome, as well as its transcription. Mice and chickens vaccinated with the expressed IBV S1 glycoprotein produced antibodies that neutralized IBV infectivity. After three immunizations, vaccinated chickens were completely protected from virulent IBV infection. These results demonstrate that transgenic potatoes expressing IBV S1 glycoprotein can be used as a source of recombinant antigen for vaccine production.