Molecular cloning, sequencing and expression in Escherichia coli of the capsid protein gene from rabbit haemorrhagic disease virus (Spanish isolate AST/89).
We describe the cloning, nucleotide sequencing and expression in Escherichia coli of the major capsid component (VP60) from the Spanish field isolate AST/89 of rabbit haemorrhagic disease virus (RHDV). The sequence of the 3'-terminal 2483 nucleotides of the genome was found to be 95.4% identical to the German RHDV strain, showing ten changes in the deduced VP60 amino acid sequence. The gene coding for this structural polypeptide has been expressed in bacteria as a beta-galactosidase fusion protein or using a T7 RNA polymerase-based system. The VP60 fusion protein showed only partial antigenic similarity with native VP60 and did not confer protective immunity. The recombinant VP60 produced in the T7 RNA polymerase-based system was antigenically similar to the viral polypeptide as determined using polyclonal and monoclonal antibodies. When used to immunize rabbits the recombinant VP60 was able to protect the animals against a lethal challenge using purified RHDV.
Boga
J A
JA
Departamento de Biología Funcional, Facultad de Medicina, Universidad de Oviedo, Spain.
Casais
R
R
Marin
M S
MS
Martin-Alonso
J M
JM
Carmenes
R S
RS
Prieto
M
M
Parra
F
F
D000818:Animals; D001483:Base Sequence; D002213:Capsid / Q000096:biosynthesis* / Q000302:isolation & purification; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D015870:Gene Expression; D005814:Genes, Viral; D005822:Genetic Vectors; D005858:Germany; D017929:Hemorrhagic Disease Virus, Rabbit / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D015183:Restriction Mapping; D013030:Spain; D015678:Viral Structural Proteins / Q000096:biosynthesis* / Q000302:isolation & purification
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D015678:Viral Structural Proteins; C081149:viral protein 60, rabbit hemorrhagic disease virus