Molecular cloning, sequencing and expression in Escherichia coli of the capsid protein gene from rabbit haemorrhagic disease virus (Spanish isolate AST/89)

Abstract

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 flgalactosidase 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

Horizontal Transmissible Protection against Myxomatosis and Rabbit Hemorrhagic Disease by Using a Recombinant Myxoma Virus

“…A hyperimmune antiserum against RHDV recognized a protein with an apparent molecular mass of 60 kDa in 6918VP60-infected cell lysates, which corresponded to the monomeric form of the VP60 capsid protein, in agreement with published values (4,8,20,28,32,41,56). This polypeptide was not detected in lysates of cells infected by parental 6918 virus.…”

“…The resulting construct was BamHI/HindIII digested and subsequently ligated to a 1,731-bp BamHI/HindIII fragment from plasmid pT35 (8), resulting in plasmid pTKVP60. A 39-bp synthetic DNA fragment, coding for the DA3 tag, was generated by annealing oligonucleotides 5Ј-GATCTAGAAAATTATACAGAT GTGTTTGATGACACACAG-3Ј and 5Ј-GATCCTGTGTGTCATCAAACAC ATCTGTATAATTTTCTA-3Ј and then inserted into the unique BamHI site of pTKVP60.…”

“…In recent years, the RHDV capsid protein gene has been successfully expressed in several heterologous systems. To date, VP60 protein has been produced in Escherichia coli (8); Saccharomyces cerevisiae (9), recombinant virus-based systems such as baculovirus (28,32,41,56) and poxvirus (4,5,20), and plants (12). The recombinant VP60 obtained in all these systems has been shown to induce protection of rabbits against a lethal challenge with RHDV.…”

A single dose immunization with rabbit haemorrhagic disease virus major capsid protein produced in Saccharomyces cerevisiae induces protection.

“…To obviate the need to handle infectious material for vaccine preparation, several authors have expressed the capsid protein in different heterologous systems. To date, the RHDV VP60 has been produced in Escherichia coli and in Sf9 insect cells ; the recombinant protein obtained in either system is able to induce protection in rabbits against a lethal challenge with RHDV (Boga et al, 1994 ;Laurent et al, 1994 ;Marı! n et al, 1995 ;Sibilia et al, 1995).…”

“…In brief, an 884 bp fragment corresponding to the 5h region of the VP60-coding sequence, amplified by PCR (Boga et al, 1994), was digested with BglII and BstZI. The resulting 415 bp fragment was inserted into the cDNA clone pT35 (Boga et al, 1994), previously digested with BglII and BstZI. The resulting vector, pT35∆4955-5305, was digested with HindIII, blunt-ended by Klenow and ligated to BglII linkers.…”