Baculovirus expression vector
The invention concerns a baculovirus expression vector for recombinantly expressing a Foot-and-mouth disease virus (FMDV) capsid precursor protein under control of a promoter, the expression vector comprising a nucleic acid sequence encoding the FMDV capsid precursor protein, wherein the ATG start codon of an open reading frame encoding the FMDV capsid precursor protein is preceded at position −4 to −1 by the nucleic acid sequence 5′-AAAT-3′. The invention further relates to a host cell comprising the baculovirus expression vector, a method of producing FMDV virus-like particles (VLPs), and a method of producing a vaccine.
1 . A baculovirus expression vector for recombinantly expressing a Foot and mouth disease virus (FMDV) capsid precursor protein, the expression vector comprising a nucleic acid sequence encoding the FMDV capsid precursor protein under the control of a promoter, wherein the ATG start codon of an open reading frame encoding the FMDV capsid precursor protein is preceded at position −4 to −1 by the nucleic acid sequence 5′-AAAT-3′ and wherein the FMDV is of the Asia1 or SAT2 serotype.
2 . The baculovirus expression vector according to claim 1 , the vector further comprising one or more restriction sites for cleavage by one or more restriction enzymes between the nucleic acid sequence at position −4 to −1 of 5′-AAAT-3′ and the promoter.
3 . The baculovirus expression vector according to claim 1 , having the nucleic acid sequence 5′-GGTAACCAAAT-3′ (SEQ ID NO. 1) at position −11 to −1.
4 . The baculovirus expression vector according to claim 1 , wherein expression of the FMDV capsid precursor protein is achieved under the control of the baculovirus polyhedrin promoter (polh) or p10 promoter.
5 . The baculovirus expression vector according to claim 1 , wherein the capsid precursor protein comprises the capsid precursor P1.
6 . The baculovirus expression vector according to claim 1 , the vector further comprising a nucleic acid sequence encoding a protease capable of cleaving the capsid precursor protein into one or more capsid proteins.
7 . The baculovirus expression vector according to claim 6 , wherein the capsid precursor protein comprises the capsid precursor P1 and the peptide 2A and the protease is 3C.
8 . A host cell comprising the baculovirus expression vector according to claim 1 .
9 . The host cell according to claim 8 , which is an insect cell.
10 . A method of producing FMDV capsid precursor protein, the method comprising the steps of:
(i) infecting a host cell with the baculovirus expression vector according to claim 1 , and
(ii) harvesting FMDV capsid precursor protein produced by the host cell.
11 . A method of producing FMDV virus-like particles (VLPs), the method comprising the steps of:
(i) infecting a host cell with the baculovirus expression vector according to claim 6 , and
(ii) harvesting FMDV VLPs produced by the host cell.
12 . A method of producing a vaccine, which comprises the steps of:
(i) producing FMDV virus-like particles (VLPs) by the method according to claim 11 and
(ii) incorporating the FMDV VLPs into a vaccine by addition of a pharmaceutically acceptable carrier.
13 . A method of protecting a subject against an infection with FMDV, which comprises the step of expressing an FMDV capsid precursor protein from the baculovirus expression vector according to claim 1 in a host cell to produce a VLPs, incorporating the VLPs into a vaccine by addition of a pharmaceutically acceptable carrier and administering the vaccine to the subject.