High-level expression and immunogenic properties of the recombinant rabbit hemorrhagic disease virus VP60 capsid protein obtained in Pichia pastoris.
The VP60 capsid protein from rabbit hemorrhagic disease virus (RHDV) (Spanish isolate AST/89) was cloned and expressed in Pichia pastoris. The transformed yeast was grown at high cell density and an expression level of about 1.5 g VP60L(-1) culture was obtained. The protein was detected associated with the cell debris fraction of the recombinant yeast after mechanical disruption. It was purified by a simple method and was obtained N-glycosylated with purity of approximately 70% as deduced from densitometry scan analysis. The recombinant product was antigenically similar to the native capsid protein as determined with polyclonal antibodies obtained from rabbits vaccinated with VP60 protein purified from native virus. The immunogenicity of VP60 protein purified from P. pastoris was demonstrated by ELISA in a vaccination experiment conducted with two groups of rabbits subcutaneously immunized. Animals vaccinated with VP60 in Freund's incomplete adjuvant developed a significant (p<0.01) virus-specific antibody response while the group injected with placebo remained seronegative. Preliminary results showed that the antigen administered within the cell debris fraction of the transformed yeast protected rabbits immunized by the oral route against an intramuscular challenge with 100 LD50 (16,000 hemagglutination units) of homologous virus.
Farnós
Omar
O
Animal Biotechnology Division, Center for Genetic Engineering and Biotechnology, P.O. Box 6162, CP 10600, Havana, Cuba. omar.farnos@cigb.edu.cu
Boué
Oscar
O
Parra
Francisco
F
Martín-Alonso
José Manuel
JM
Valdés
Odaysa
O
Joglar
Marisdania
M
Navea
Leonor
L
Naranjo
Paula
P
Lleonart
Ricardo
R
D000818:Animals; D000956:Antigens, Viral / Q000235:genetics / Q000276:immunology; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D005620:Freund's Adjuvant / Q000276:immunology; D015967:Gene Expression Regulation, Viral; D017929:Hemorrhagic Disease Virus, Rabbit; D007279:Injections, Subcutaneous; D008297:Male; D010843:Pichia / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D011817:Rabbits; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D015678:Viral Structural Proteins / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism*; D014765:Viral Vaccines / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology*
D000956:Antigens, Viral; D011994:Recombinant Proteins; D015678:Viral Structural Proteins; D014765:Viral Vaccines; C081149:viral protein 60, rabbit hemorrhagic disease virus; D005620:Freund's Adjuvant