Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Amino acid sequence disclosed in whole or in part; or...
Reexamination Certificate
2002-05-17
2003-07-15
Salimi, Ali R. (Department: 1648)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Amino acid sequence disclosed in whole or in part; or...
C424S201100, C435S006120, C435S069100, C435S975000
Reexamination Certificate
active
06592870
ABSTRACT:
FIELD OF THE INVENTION
This invention, in general, refers to recombinant proteins suitable for the diagnosis of a porcine pathogen, and in particular, to recombinant fusion proteins which comprise the protein of the nucleocapsid of the porcine reproductive and respiratory syndrome virus (PRRSV), obtained both from European isolates and from American isolates, and its use in the diagnosis of PRRSV.
BACKGROUND OF THE INVENTION
The causative agent of the porcine reproductive and respiratory syndrome (PRRS) is a new arterivirus (PRRSV) isolated for the first time in Europe [Wensvoort et al., Vet. Quarterly, 13, (1991), 121-130] and later in the United States [Collins et al., J. Vet. Diagn. Invest., 4, (1992), 117-126].
PRRSV causes an important pathology in the porcine livestock, characterised by reproductive disorders in the female pigs (miscarriages, piglets born dead or weak), and an increase of the perinatal mortality as well as dyspnea and piglet pneumonia [Terpstra et al., Vet. Quarterly, 13, (1991), 131-136).
PRRSV is an enveloped RNA virus, of approximately 62 nm of diameter, the viral genome of which comprises a single-stranded RNA of positive polarity of approximately 15 kilobases (kb) in length. The genome contains eight overlapping open reading frames (ORF) (Meulenberg et al., Virology, 192, (1993), 62-72). The virion contains 6 structural proteins encoded by ORFs 2 to 7 (Meulenberg et al., Virology, 206, (1995), 155-163; van Nieuwstadt et al., J. Virol., 70, (1996), 4767-4772]. The ORF7 encodes the nucleocapsid protein (protein N), which is the most immunogenic protein of PRRSV [Sanz et al., Second Symposium on Aujeszky and PRRS viruses, Copenhagen, Denmark (1995)], making it a good candidate for the detection of specific antibodies to the virus, and therefore, for the diagnosis of the disease.
Two different antigenic groups of PRRSV have been described, which correspond to the American and European isolates. The Japanese PRRSV isolates, as well as other Asiatic isolates are antigenicaly related with the American isolate. A characteristic example of a European PRRSV isolate is that deposited at the ECACC under the deposit number V93070108 [Spanish Patent ES 2.074.950], whereas a representative example of an American isolate of PRRSV is that identified as VR-2332 [European Patent EP 0 529 584]. The European and American isolates of PRRSV exhibit a different genotype [Meng et al., Archives of Virology, 140(1995), 745-755] with important antigenic differences [Wensvoort et al., J. Vet., Diagn. Inves., 4, (1992), 134138].
The serological differences between the European and American isolates complicate the diagnosis of the disease, as in many cases there is no cross-reactivity between the respective porcine sera (perhaps due to the absence of linear immunodominant epitopes). Additionally, the recent introduction in Europe of vaccines based on the American isolate represents an additional complication in the serological analysis of infected and/or vaccinated animals. Therefore, it is necessary to develop a diagnostic method which may allow to discriminate between both isolates, both in individual and in mixed populations, with the object of performing an accurate distinction with regard to the origin of the virus.
There are kits for the detection of the presence of PRRSV or of antibodies which recognise PRRSV, specific for the European and American isolates.
The usual methods for the diagnosis of PRRSV comprise carrying out an assay based on immunoperoxidase (IPMA) or the performance of an ELISA type assay based on pig's lung alveolar macrophages or on antigenic recombinant proteins.
The techniques based on IPMA are expensive and bloody techniques, which require the use of pig's lung alveolar ffacrophages coming from the sacrifice of animals, being it possible that said macrophages could be contaminated or infected with other pathogens [unless gnotobiotic or specific pathogen free (SPF) animals are used], and are not susceptible to automation as they require visual inspection under the microscope.
ELISA techniques based on macrophages require the use of macrophage extracts and therefore, present the same problems, in that sense, as the techniques based on IPMA. Additionally, the use of antigen in the form of a cell extract requires an internal reference with the same cell extracts, but without infection, in case there was any serum from an animal from the field which had natural reactivity, i.e. which had natural antibodies, which could mask the result.
The ELISA techniques based upon PRRSV recombinant proteins require the production of viral antigen in appropriate amounts and in an active form. The production of PRRSV antigens, for example the nucleocapsid protein, in tissue culture, is troublesome and expensive. On the other hand, the production of PRRSV recombinant proteins using recombinant baculovirus in permissive cells poses many problems, as the expression of said proteins in that system is costly and not very efficient, the proteins expressed are insoluble and only a small soluble fraction is recovered, which is the one used in the ELISA. Likewise, the products of ORF 3, 5 and 7 of PRRSV expressed by recombinant baculovirus in insect cells are difficult to purify. Additionally, reference uninfected insect cells must be included to rule out natural reactivity.
In general, diagnostic methods must be reliable, reproducible, sensitive, simple, cost-effective, of a wide spectrum and, advantageously, shall use active antigens the production of which, in unlimited amounts, is simple and cost-effective. Additionally, in the case of pathogens which have isolates with genetic, antigenic and pathogenic differences, it is convenient that they discriminate between the different isolates.
The currently available methods used for the diagnosis of PRRSV do not satisfactorily fulfill all the characteristics which have to be demanded from a diagnostic method and, therefore, there is still a need for other methods for the diagnosis of PRRSV which solve all or some of the problems mentioned above.
BRIEF SUMMARY OF THE INVENTION
The invention provides recombinant fusion proteins, which comprise the nucleocapsid protein of PRRSV obtained both from European and American isolates, of use for the diagnosis of PRRSV. The invention also provides methods and kits for the diagnosis of PRRSV which comprise the use of said recombinant fusion proteins. The nucleic acid sequences which essentially encode said recombinant fusion proteins constitute an additional object of the present invention. The invention presented here proposes a new process for constructing the thermocouple.
DETAILED DESCRIPTION OF THE INVENTION
The recombinant fusion proteins object of this invention comprise an amino acid sequence selected among the sequences identified (see the section regarding the LIST OF SEQUENCES) as SEQ. ID. No.: 1 and SEQ. ID. No.:2, or an active fragment of the same. The term “active fragment”, in the sense used in this description, refers to a protein fragment that is suitably recognised by positive sera against PRRSV, i.e. a fragment of ORF7 of PRRSV recognised in such a way that it allows the discrimination between PRRSV positive and negative sera.
SEQ. ID. No.: 1 shows the amino acid sequence corresponding to the nucleocapsid protein of a European isolate of PRRSV, specifically the one identified as Toledo 4/96 [Example 1], generally known as “European isolate” in this description, while SEQ. ID. No.: 2 shows the amino acid sequence corresponding to the nucleocapsid protein of an American isolate, specifically the one identified as Canada 14/96 (Example 2), generally known as “american isolate” in this description.
The recombinant fusion proteins also comprise an amino acid sequence, of variable length and composition, derived from the system chosen for the expression of the recombinant fusion protein, to which SEQ. ID. No.: 1, or SEQ. ID. No.: 2, or an active fragment of the same is fu
Alvarez Jose Ignacio Casal
Fernandez Antonio Sanz
Garcia Ma Jose Rodriguez
Harrison & Egbert
Inmunologia Y Genetica Aplicada, S. A.
Salimi Ali R.
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