DK175114B1 - Pseudo-rabies virus protein - Google Patents

Abstract

A novel recombinant DNA molecule comprises a DNA sequence coding for a polypeptide displaying pseudorabies virus (PRV) glycoprotein gp50, gp63 or gI immunogenicity, the DNA sequence being operatively linked to an expression control sequence. PRV genomic DNA derived from PRV Rice strain was isolated from the cytoplasm of PRV-infected Vero cells (ATCC CCL 81). The genomic DNA was fragmented by sonication and then cloned into lambda gt11 to produce lambda/PRV recombinant (lambda PRV) DNA library. Antisera for screening the lambda PRV library were produced by inoculating mice with proteins isolated from cells infected with PRV (infected cell proteins or ICP's) that had been segregated according to size on SDS gels and then isolating the antibodies. The lambda PRV phages to be screened were plated on a lawn of E.coli Foreign DNA's inserted into a unique cloning site in the 3' end of the lacZ gene in the proper orientation and reading frame produce, on expression, polypeptides fused to beta-galactosidase.

Description

in DK 175114 B1

The present invention relates to a recombinant DNA molecule, a host cell transformed therewith, and a method for producing a polypeptide using the recombinant DNA molecule. The polypeptide is useful

V

5 for vaccinating animals against PRV, and the recombinant DNA molecule is suitable for expression of the PRV glycoproteins encoded therefrom.

Pseudorabies virus (PRV) is a disease that attacks many animal species worldwide. PRV infections 10 are referred to as alternating paralysis bulbaris infectiosa,

Aujeszky's disease and "food itch". Infections are known in 1 important domestic animals, such as pigs, cattle, dogs, cats, sheep, rats and mink. The host range is very wide and includes most mammals and, at least experimentally, 15 many bird species (for a detailed list of host animals, see DP Gustafson, "Pseudorabies", in Diseases of Swine, 5th ed., AD Leman et al. al., eds., (1981)). For most infected animals, diseases are fatal. However, adult pigs and possibly rats do not die from the disease and are therefore carriers.

Pig herds are particularly vulnerable to PRV. Although adult pigs rarely exhibit symptoms or die of the disease, piglets become acute; ill when infected and usually die within 24-48 hours, often without specific clinical signs (TC Jones and RD Hunt, Veterinary Pathology, 5th ed., Lea & Febiger (1983)).

PRV vaccines have been produced by various methods and vaccination in endemic areas of Europe has been practiced for more than 15 years. The losses have been reduced by the vaccination, but the vaccination has retained the virus in the environment. No vaccine has been produced that will prevent infection. Vaccinated animals exposed to virulent virus survive the infection and then secrete more virulent virus. Vaccinated animals 35 can therefore harbor a latent infection that can flare up again (see D.P. Gustafson, supra).

2 DK 175114 B1

Live attenuated and inactivated PRV vaccines are commercially available in the United States and have been approved by the USDA (See C.E. Aronson, ed., Veterinary Pharmaceuticals & Biologicals, (1983)).

5 PRV is a herpes virus. Herpes viruses are generally among the most complex of animal viruses. Their genomes encode at least 50 virus-specific proteins and contain over 150,000 nucleotides. Among the most immunologically reactive proteins of herpes viruses, the glycoproteins 10 are found, among others, in virion membranes and the membranes of infected cells. The literature on PRV glycoproteins refers to at least four viral glycoproteins (T. Ben - Porat and A.S. Kaplan, Virology £ 1, 265-73 (1970); A.S. Kaplan and T. Ben-Porat, Proc. Natl. Acad. Sci.

USA 66, 799-806 (1970)).

W., Wathen and L.K. Wathen, J. Virol. 51, 57-62 (1984) refer to a PRV containing a mutation in one viral glycoprotein (gp50) and a method of selecting the mutant using neutralizing monoclonal antibody directed against gp50. Wathen and Wathen also state that a monoclonal antibody directed against gp50 is a strong neutralizing factor for PRV, with or without the aid of complement, and that polyvalent immune serum is highly reactive to gp50, 25g therefore concludes that gp50 may be one of the important PRV immunogens. On the other hand, it has been reported that monoclonal antibodies that react with the coat glycoprotein with a molecular weight of 98,000 neutralize PRV infectivity, but that monoclonal antibodies directed against some of the other membrane glycoproteins have very little neutralizing effect. activity (H. Hampi et al., J. Virol. 52, 583-90 (1984); and T. Ben-Porat and A. S. Kaplan, "Molecular Biology of Pseudorabies Virus", in B. Roizman ed., The Herpesviruses, 35, pp. 105-73 (1984)).

3 DK 175114 B1 L.M.K. Wathen et al., Virus Research 4, 19-29 (1985) describe the preparation and characterization of monoclonal antibodies directed against PRV glycoproteins identified as gp50 and gp83 and their use for passive immunization of mice against PRV infection.

A. K. Robbins et al., "Localization of a Pseudorabies Virus Glycoprotein Gene Using an E. coli Expression Plasmid Library", in Herpesvirus, pp. 551-61 (1984) describes the construction of a library of 10 E. coli plasmids containing PRV DNA . They also describe the identification of a PRV gene encoding glycoproteins having a molecular weight of 74,000 and 92,000. They do not describe the glycoproteins prepared according to the present invention.

15 A.K. Robbins et al., EP patent application no.

85400704.4 (Publication No. 162,738) discloses the isolation, cloning and expression of PRV glycoproteins identified as gll and gill. They do not describe the PRV glyco proteins made in accordance with the present invention.

20 T.C. Mettenleiter et al., "Mapping of the Structural Gene of Pseudorabic Virus Glycoprotein A and Identification of Two Non-Glycosylated Precursor Polypeptides", J. Virol. 53, 52-57 (1985) describe mapping of the coding region of glycoprotein gA 25 (which they equate to gI) to the BamHI-7 fragment of PRV DNA. They also state that the BamHI-7 fragment encodes at least three other viral proteins with a molecular weight of 65,000, 60,000 and 40,000. They do not describe or imply the DNA sequence encoding the glycoproteins of the present invention, or the preparation of such polypeptides by recombined DNA methods.

B. Lomniczi et al., "Deletions in the Genomes of Pseudorabies Virus Vaccine Strains and the Existence of Four Isomers of the Genomes", J. Virol. 4, 970-79 5 (1984) discloses PRV vaccine strains which have omissions in the particular short sequence between 0.855 and 0.882 short units. This is near the gi gene. T. C. Metten-leiter et al., "Pseudorabic Virus Avirulent Strains Fail to Express a Major Glycoprotein", J. Virol. 56, 307-11 (1985), have demonstrated that three commercial PRV-5 vaccine strains lack glycoprotein gi. It has also recently been found that the Bartha vaccine strain contains an omission of most of the gp63 gene.

T.J. Rea et al., J. Virol. 5 ±, 21-29 (1985), describes mapping and sequencing of the gene for the PRV glycoprotein, which accumulates in the medium of infected cells (gX). Among the flanking sequences of the gX gene shown therein is a small portion of the gp50 sequence that specifically begins at base number 1682 of FIG. 6 therein. However, this sequence was not identified as the gp50 sequence. Furthermore, errors in the sequence published by Rea et al. The bases 1586 and 1603 must be omitted. The bases must be inserted between bases 1708 and 1709, bases 1737 and 1738, bases 1743 and 1744 and bases 1753 and 1754. The consequence of these errors 2o in the published partial sequence of gp50 is a reading frame offset. Translation of the open reading frame beginning at the AUG start site will give an incorrect | amino acid sequence for the gp50 glycoprotein.

European Patent Publication No. 133,200 discloses a diagnostic antigenic factor to be used with certain lectin-bound PRV glycoprotein subunit vaccines to differentiate between carriers of PRV and non carriers of PRV.

According to the present invention, there is provided a recombinant DNA molecule containing a DNA sequence encoding a polypeptide exhibiting pseudorabies virus (PRV) glycoprotein gp50 immunogenicity, wherein said DNA sequence is operably linked to an expression control sequence. wherein the DNA sequence encoding the polypeptide is selected from the sequence encoding gp50 and which is 35

'AIG CTG CTC GCA GCG CIA TTG GCG GCG CTG GTC GCC CGG ACG ACG CTC OGT GCG

GAC CTG GAC GCC GIG CCC GCG · CCG ACC TTC CCC CCG CCC GCG TAC CCG TAC ACC

GAG TCG TGG CAG CTG ACG CTG ACG ACG GTC CCC TCG CCC TTC CTC GGC CCC GCG

GAC CTC TAC CAC ACG CGC CCG CTG GAG GAC CCG TGC GCG GTG CTG GCG CTG ATC

, TCC GAC CCG CAG GTG GAC CGG CTG CTG AAC GAG GCG GTG GCC CAC CGG CGG CCC

ACG TAC CGC GCC CAC CTG GCC TGG TAC CGC ATC GCG GAC GGG TGC GCA CAC CTG

CTG TAC TTT ATC GAG TAC GCC GAC TGC GAC CCC AGG CAG GTC TTT GGG CGC TGC

CGG CGC CGC ACC ACG CCG ATG TGG TGG ACC CCG TCC GCG GAC TAC ATG TTC CCC

1Q ACG GAG GAC GAG CTG GGG CTG CTC ATG CTG GCC CCG GGG CGG TTC AAC GAG GGC

CAG TAC CGG CGC CTG GTG TCC CTC GAC GGC CTG AAC ATC CTC ACC GAC TTC ATG

CTG GCG CTC CCC GAG GGG CAA GAG TGC CCG TTC GCC CGC GTG GAC CAG CAC CGC

ACG TAC AAG TTC GGC GCG TGC TGG AGC GAC GAC AGC TTC AAG CGG GGC CTG GAC

CTG ATG CGA TTC CTG ACG CCG TTC TAC CAG CAG CCC CCG CAC CGC GAG GTG CTG

15 AAC TAC TGG TAC CGC AAG AAC GGC CGG ACG CTC CCG CGG GCC CAC GCC GCC GCC ·

ACG CCG TAC GCC ATC GAC CCC GCG CGG CCC TCG GCG GGC TCC CCG ACG CCC CGC

CCC CGG CCC CCG CCC CGG CCC CGG CCG AAG CCC GAG CCC GCC CCG GCG ACG CCC

'GCG CCC CCC GAC OGC CTG CCC GAG CCG GCG ACG CGG GAC CAC GCC GCC GGG GGC

CGC CCC ACG CCG CGA CCC CCG AGC CCC GAG ACG CCG CAC OGC CCC TTC GCC CCG

2 0 CCG GCC GTC GTG CCC AGC GGG TGG CCG CAG CCC GCG GAG CCG TTC CAG CCG CGG

ACC CCC GCC GCG CCG GGC GTC TCG CGC CAC CGC TCG GTG ATC CTC GGC ACG CGC

ACC GCG ATG GGC GCG CTC CTG CTG GGC GTG TGC GTC TAC ATC TIC TTC CGC CTG

AGG GGG GCG AAG GGG TAT CGC CTC CTG GGC GCT CCC GCG GAC GCC GAC GAG CIA

AAA GCG CAG CCC GGT CCG TAG, 25 and fragments thereof encoding polypeptides exhibiting PRV antigenicity.

In addition, according to the present invention, there is provided a host cell transformed with a recombinant DNA molecule of the invention.

The present invention further provides a method for producing a polypeptide exhibiting PRV-gp50 antigenicity, comprising: (a) producing a recombinant DNA molecule, wherein the molecule contains a DNA sequence encoding a polypeptide exhibiting PRV-gp50 antigenicity, wherein said DNA sequence is operably linked to an expression control sequence, (b) transforming an appropriate host cell with said recombinant DNA molecule, (c) culturing said said host cell, and 5 (d) collecting the polypeptide, wherein the DNA sequence is selected from the gp50 sequence which is

10 ATG CTG CTC GCA GCG CTA TTC GCC GCC CTC CTC GCC CGC ACG ACG CTC GCT GCC

GAC GTG GAC GCC GTG CCC GCG CCG ACC TTC CCC CCG CCC GCG XAC CCG TAC ACC

GAG TCG TGC CAG CTG ACG CTG ACG ACG GTC CCC TCG CCC TTC GTC GGC CCC GCG

GAC GTC TAC CAC ACG CGC CCG CTG GAG GAC CCG TGC GCG GTG GTG GCG CTG AIC

TCC GAC CCG CAG GTG GAC CGG CTG CTG MC GAG GCG GTG GCC CAC CGC CCG CCC

15 ACG TAC CGC GCC CAC GTG GCC TCG TAC CGC AIC GCG GAC GGG TCC GCA CAC CTG

CTG TAC TIT AIC GAG TAC GCC GAC TGC GAC CCC ACG CAG GTC ITT GGG CGC TGC

CGG CGC CGC ACC ACG CCG ATG TGG TCG ACC CCG TCC GCG GAC TAC ATG TTC CCC

ACG GAG GAC GAG CTG GGG CTG CTC ATG GTG GCC CCG GGG CGG TTC AAC GAG GGC

CAG TAC CGG CGC CTG GTG TCC CTC GAC GGC CTG AAC AIC CTC ACC GAC TTC ATG

20 GTG CCG GTC CCC GAG GGG CAA GAG TGC CCG TTC GCC CGC GTG GAC CAG CAC CGC

ACG TAC AAG TTC GGC GCG TGC TGG AGC GAC GAC ACC TTC AAC CGG GGC GTG GAC

GTG ATG OGA TTC CTG ACG CCG TTC TAC CAG CAG CCC CCG CAC CGG GAG GTG GTG

AAC TAC TGG TAC CGC AAG AAC GGC CGG ACG CTC CCC CGG GCC CAC GCC GCC GCC

ACG CCG TAC GCC AIC GAC CCC GCG CGG CCC TCG GCG GGC TCG CCG AGG CCC CGG

25 CCC CGG CCC CGG CCC CGG CCC OGG CCG AAG CCC GAG CCC GCC CCG GCG ACG CCC

GCG CCC CCC GAC CGC CTG CCC GAG CCG GCG ACC CGG GAC CAC GCC GCC GGG GGC

CGC CCC ACG CCG CGA CCC CCG AGG CCC GAG ACG CCG CAC CGC CCC TTC GCC CCG

CCG GCC GTC GTG CCC AGC GGG TGG CCG CAG CCC GCG GAG CCG TTC CAG CCG CGG

30 ACC CCC GCC GCG CCG GGC GTC TCG CGC CAC CGC TCG GTG ATC GTC GGC ACG GGC

ACC GCG ATG CGC GCG CTC CTG CTG GGC GIG TCC GTC TAC ATC TTC TTC CGC CTG

AGG GGG GCG AAG GGG TAT CGC CTC CTG GGC GGT CCC GCG GAC GCC GAC GAG CTA

AAA GCG CAG CCC GGT CCG TAG, and fragments thereof encoding polypeptides exhibiting 35 pseudorabies virus antigenicity.

More specifically, according to the present invention, polypeptides of the formulas listed on leaves A, B and C, immunogenic fragments thereof and immunologically functional equivalents thereof, are provided.

The polypeptides prepared according to the present invention encode the pseudorabies virus glycoprotein gp50 or immunogenic fragments and are useful for protecting animals against PRV infection by vaccinating them with these polypeptides.

The present recombinant DNA molecule, host cell and method disclose, for the first time, production by recombinant DNA technique of gp50 polypeptide and fragments thereof useful for the preparation of vaccines, and the possibilities and advantages are obtained. , which is discussed later in the description (page 9).

The existence and location of the gene encoding the glycoprotein gp50 of PRV has been demonstrated by M.W. Wathen and L.M. Wathen, see above.

The glycoprotein encoded by the gene is defined as a glycoprotein that reacts with a particular monoclonal antibody. This glycoprotein does not match any of the known PRV glycoproteins. Wathen and Wa-then <have mapped a mutation that is resistant to the monoclonal antibody, which, on the basis of precedents, in the herpes simplex virus (e.g., TC Holland et al., J. Virol 52, 566- 74 (1984)), map the location of the gp50 structural gene. Wathen and Wathen have mapped the gp50 gene to the smaller SalI / BamHI fragment within the BamHI-7 fragment of PRV. Rea et al., Supra, have mapped the PRV glycoprotein gX gene to the same region.

As mentioned above, the gene encoding gp50 is placed in the BamHI-7 fragment of PRV DNA. The Bam HI-7 fragment of PRV can be derived from the plasmid pPRXhl (also known as pUC1129), and fragments convenient for DNA sequence analysis can be obtained by standard subcloning procedures. Plasmid pUC1129 is available from E. coli HB101, NRRL B-15772. This culture is available from the permanent collection at the Nothern Regional Research Center Fermentation Laboratory (NRRL), U.S. Department of Agriculture, Peoria, Illinois, U.S.A.

E. coli HB101 containing pUC1129 can be grown in 5 L nutrient medium by well known procedures. Typically, cultures are grown to an optical density of 0.6, then chloramphenicol is added and the culture is left to shake overnight. The culture is then lysed e.g. using high salt concentration SDS, and the above liquid is subjected to a casium chloride / ethidium bromide equilibrium density gradient centrifugation to obtain the plasmids.

The availability of this gene sequence allows for direct manipulation of the gene and gene sequence, allowing 15 modifications to the regulation of the expression and / or structure of the protein encoded by the gene or a fragment thereof. Knowledge of this gene sequence also allows cloning of the corresponding gene or fragment thereof from any strain of PRV using the known sequence as a hybridization probe and to express the entire protein or fragment thereof by recombinant methods known in the art.

Knowledge of this gene sequence has allowed the amino acid sequence of the corresponding polypeptide 25 (map A) to be deduced. As a result, fragments of these polypeptides having PRV immunogenicity can be prepared by standard methods for protein synthesis or recombinant DNA methods. As used in the present specification, the terms immunogenicity and antigenicity are used interchangeably for the ability to stimulate any type of adaptive immune response, ie. the terms antigen and antigenicity are not limited to substances that stimulate the production of antibodies.

The primary structure (sequence) of the gene encoding gp50 is also listed in map A.

The gene or fragments thereof can be recovered from pUC1129 by digestion of the plasmid DNA from a culture of appropriate endonuclease restriction enzymes.

Eg. For example, the BamHI-7 fragment can be isolated by digestion of a preparation of pUC1129 with BamHI and isolated by gel electrophoresis.

5. All of the restriction endonucleases disclosed in the present specification are commercially available and their use is well known. Instructions for use are generally provided by the commercial suppliers of the restriction enzymes.

The cut gene or fragments thereof can be read into various cloning carriers or vectors for use in transforming a host cell. The vectors preferably contain DNA sequences to initiate, control, and complete transcription and translation (which together constitute expression) of the PRV glycoprotein genes and are therefore operably linked therewith. These "expression control sequences" are preferably compatible with the host cell to be transformed. When the host cell is a cell of a higher animal, e.g. a mammalian cell, the naturally occurring expression control sequences of the glycoprotein genes can be used alone or together with heterologous expression control sequences. Heterologous sequences can also be used alone. The vectors further preferably contain a marker gene (e.g., antibiotic resistance) to provide a phenotypic trait for selection of transformed host cells. In addition, a replicating vector will contain a replicon.

Typical vectors are plasmids, phages and viruses that infect animal cells. Essentially, any DNA sequence capable of transforming a host cell can be used.

By the term "host cell" as used herein is meant a cell capable of being transformed with the DNA sequence encoding a polypeptide exhibiting PRV glycoprotein antigenicity.

Preferably, the host cell is capable of expressing the PRV polypeptide or fragments thereof. The host cell may be prokaryotic or eukaryotic. Illustrative pro-

DK 175114 B1 I

io I

karyotic cells are bacteria such as E. coli, B. sub-I

tilis, pseudomonas and B. stearothermophilus. Illustrations- I

eukaryotic cells are yeast cells or cells of I

higher animals, such as cells from insects, plants or pathogens

5 ted animals. Mammalian cell systems will often be in Form I

of monolayers of cells, although mammalian cell suspensions I

can also be used. Mammalian cell lines include e.g. IN

In Vero and HeLa cells, Chinese hamster ovary cell lines I

I (ChO cell lines), WI38, BHK, COS-7 or MDCK-I

In 10 cell lines. Insect cell lines include the Sf9 line of I

In Spodoptera frugiperda (ATCC CRL1711). A summary I

I of some available eukaryotic plasmids, host I

In cells and methods for using them for cloning I

I and expression of PRV glycoproteins can be found in I

In 15 K. Esser et al., Plasmids of Eukaryotes (Fundamentals I

I and Applications), Springer-Verlag (1986). IN

As noted above, the vector, e.g. IN

In a plasmid used to transform the host cell, I

Preferably, expression control sequences for I

I 20 I

In expression of the PRV glycoprotein gene or fragments thereof- I

I af. Therefore, the expression control sequences are operative I

In association with the gene or fragment. When the host cells I

You are bacteria, including illustratively useful excipients

In the pressure control sequences, the trp promoter and operator I

I (Goeddel et al., Nucl. Acids Res. 8, 4057 (1980)); lac- I

I promoter and operator (Chang et al., Nature 275, 615 I)

I (1978)); outer membrane protein promoter (EMBO J. 1, I

I 771-775 (1982)); bacteriophage / 1 promoters and op I

In rators (Nucl. Acids Res. 11, 4677-4688 (1983)); α-amy- I

I 2 0 I

In lase (B. subtilis) promoter and operator. Termination I

In sequences and other expression promotion and control I

In running sequences that are compatible with the selected host I

In cell. When the host cell is yeast, illustrative I

In useful expression control sequences, e.g. α-feed I

I 35 I

11 DK 175114 B1 factor. For insect cells, the polyhedrin promoter of baculoviruses can be used (Mol. Cell. Biol. 3, pp. 2156-65 (1983)). When the host cell originates from insects or mammals, illustratively useful expression control sequences include e.g. SV-40 promoter (Science 222, 524-527 (1983)) or e.g. metallothionein promoter (Nature 296, 39-42 (1982)) or a heat shock promoter (Voellmy et al., Proc. Natl. Acad. Sci.

United States 8_2, pp. 4949-53 (1985)). As noted above, when the host cell is a mammalian cell, the expression control sequences of the PRV glycoprotein gene may be used, but preferably in combination with heterologous expression control sequences.

The plasmid or replicating or integrating DNA material containing the expression control sequences is cleaved using restriction enzymes, size-adjusted as necessary or desirable, and ligated to the PRV glycoprotein gene or fragments thereof by well known methods. When yeast host cells 20 or higher animal host cells are used, polyadenylation or terminator sequences from known yeast or mammalian genes can be incorporated into the vector. For example, For example, the bovine growth hormone polyadenylation sequence is used as set forth in European Patent Publication No. 93,619. In addition, gene sequences for controlling the replication of the host cell can be incorporated into the vector.

The host cells are competent or made competent for transformation in various ways. When bacterial cells are host cells, they can be made competent by treatment with salts, typically a calcium salt, as generally described by Cohen, PNAS, 69, 2110 (1972). A yeast host cell is generally rendered competent by removal of its cell wall or otherwise, e.g. by ionic treatment (J. Bacteriol. 153, 163-168 (1983)).

35 12 DK 175114 B1

There are several well-known methods for introducing DNA into animal cells, including e.g. calcium phosphate precipitation, fusion of the receiving cells with bacteria - protoplasts containing the DNA, treatment of the 5 receiving cells with liposomes containing the DNA, and microinjection of the DNA directly into the cells.

The transformed cells are propagated by well known methods (Molecular Cloning, T. Maniatis et al.,

Cold Spring Harbor Laboratory, (1982); Biochemical 10 Methods in Cell Culture And Virology, R.J. Kuchler,

Dowden, Hutchinson, and Ross, Inc., (1977); Methods In Yeast Genetics, F. Sherman et al., Cold Spring Harbor Laboratory, (1982)) and the expressing PRV glyco protein or fragment thereof are harvested from the cell medium 15 in the systems where the protein is secreted from the host cells or from the cell suspension after breaking the host cell system by e.g. well-known mechanical or enzymatic methods.

As noted above, the amino acid sequence of the PRV glyco protein derived from the gene structure is listed in map A. Poly peptides exhibiting PRV glycoprotein antigen include the sequence listed in map A and any portion of the polypeptide sequence contained in capable of eliciting an immune response in an animal, e.g. a mammal that has received an injection of the polypeptide sequence.

As noted above, the entire gene encoding the PRV glycoprotein can be used to construct the vectors and transform the host cells to express the PRV glycoprotein, or fragments of the gene encoding the PRV glycoprotein can be used, whereby the resulting host cell will expressing polypeptides exhibiting PRV antigenicity. Any fragment of the PRV glycoprotein gene can be used, resulting in expression of a polypeptide which is an immunogenic fragment of the PRV glycoprotein or an analog thereof. As is well known in the art, the degeneration of the genetic code readily allows substitution of base pairs to form functionally equivalent genes or fragments thereof encoding polypeptides exhibiting PRV glycoprotein antigenicity. These functional equivalents are also encompassed by the invention.

Maps D-L are listed to illustrate the structures of the Examples. Certain conventions are used to illustrate plasmids and DNA fragments as follows: (1) The single-line figures show both circular and linear double-stranded DNA.

15 (2) Stars show that the molecule shown is circular.

If no star is listed, the molecule is linear.

(3) Endonuclease restriction sites of interest are listed above the line.

(4) Genes are listed below the line.

(5) The distances between genes and restriction sites are not properly scaled. The figures show only the relative positions, unless otherwise stated.

Most of the recombinant DNA methods used in the practice of the present invention are standard procedures well known to those skilled in the art and e.g. described in detail in Molecular Cloning, T.

Maniatis et al., Cold Spring Harbor Laboratory, (1982) and B. Perbal, A Practical Guide to Molecular Cloning,

John Wiley & Sons (1984).

30 35 DK 175114 B1

Example 1

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14 In this example, the sequencing, cloning and expression of the PRV glycoprotein gp50 is described.

5 1. Sequencing of the gp50 gene.

The BamHI-7 fragment of PRV-Rice strain DNA (map D) encoding the gp50 gene is isolated from pPRXhl (NRRL B-15772), above, and subcloned into the BamHI site of plasmid pBR322 (Maniatis et al. , above).

Referring to Map E, the subclones are further fragmented using standard procedures for degrading BamHI-7 with PvuII, isolating the two BamHI / PvuII fragments (1.5 and 4.9 kb), and subcloning them between BamHl and the PvuII sites of pBR322 to form plasmids pPR28-4 and pPR28-1, containing the 1/5 kb and 4.9 kb fragments, respectively (see also Rea et al., supra). These subclones are used as sources of DNA for DNA sequencing experiments.

Map F shows various restriction enzyme cleavage sites located in the gp50 gene and the flanking regions. The above subcloned fragments of 1.5 and 4.9 kb are broken down by these restriction enzymes. Each of the ends formed by the restriction enzymes is labeled with 32 J · P-ATP using polynucleotide kinase 25 and subjected to sequencing by the method of

Maxam and Gilbert, Methods Enzymol. 65, 499-560 (1980).

The whole gene is sequenced at least twice on both strands. The DNA sequence of gp50 is listed in Map A. This DNA can be used to detect animals actively infected with PRV. One can, for example. take a nose or throat swab and then perform DNA / DNA hybridization by standard methods for detecting the presence of PRV.

35

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15 DK 175114 B1 2. Expression of gp50.

Referring to map G, a Narl cleavage site is located 35 base pairs above the gp50 gene start cocoon.

The first step of the expression is the insertion of the target BamHI cleavage site at the site of the NarI cleavage site. The above plasmid pPR28-4 is digested with the restriction endonuclease Narl to form DNA fragment 3 containing the N-terminus coding end of the gp50 gene and a portion of the gX gene. BamHI linkers are added to fragment 3 and the fragment is degraded with BamHI

to remove the gX sequence to give fragment 4. The BamHI ends are then ligated to generate the plasmid pPR28-4 Nar2.

Map H shows the assembly of the complete 15 gp50 gene. pPR28-4 Nar2 is digested with BamHI and PvuII to form fragment 5 (160 bp) containing the N-terminal coding portion of the gp50 gene. Plasmid pPR28-1 above is also digested with PvuII and BamHI to form a 4.9 kb fragment containing the C-terminal coding portion 20 of the gp50 gene (fragment 6). Plasmid pPGX1 (constructed as disclosed in U.S. Patent Application No. 760,130) or, alternatively, plasmid pBR322 is digested with BamHI, treated with bacterial basic phosphatase (BAP) and then ligated with fragments 5 and 6 to form plasmid pBGP50-23, which contains the complete gp50 gene.

In Map I, the preparation of the plasmid pD50 is shown. The plasmid pBG50-23 is digested with the restriction enzyme Maelll (K. Schmid et al., Nucl. Acids Res., 12, 26,1919 (1984)) to form a mixture of fragments.

The MaellI ends are blunted with T4 DNA polymerase and EcoRI linkers are added to the blunt ends and then an EcoRI degradation is performed. The resulting fragments are cleaved with BamHI and a 1.3 kb BamHI / EcoRI fragment

ISLAND

16 DK 175114 B1 containing the gp50 gene (fragment 7) is isolated. The plasmid pSV2dhfr (from the American Type Culture Collection,

Bethesda Research Laboratories, or synthesized by the method of S, Subramani et al., Mol. Cell. Biol.

5 2, pp. 854-64 (1981)) is digested with BamHI and EcoRI, and the large fragment (5.0 kb) is isolated to form fragment 8 containing the dihydrofolate reductase marker (dhfr). Fragments 7 and 8 are then ligated to generate the plasmid pD50 containing the gp50 gene and the dhfr-10 marker.

Referring to map J, the immediate early promoter of human cytomegalovirus, Towne strain, is added above the gp50 gene. pD50 is digested with BamHI and treated with bacterial basic phosphatase 15 to form fragment 9. A 760 bp Sau3A fragment containing the immediate early promoter of human cytomegalovirus (Towne) is isolated by the procedure disclosed in U.S. Patent Application No. 758,517 to generate the fragment 10 (see also DR Thomsen et al., Proc. Natl.

20 Acad. Sci. USA 83, pp. 659-63 (1984)). These fragments are then ligated by a BamHI / Sau3A fusion to generate the plasmid pDIE50. To confirm that the promoter is in the correct orientation for transcription of the gp50 gene, the plasmid is digested with SacI and PvuII and a 185 bp fragment is generated.

Referring to map K, 0.6 kb of PvuII / EcoRI fragments containing bovine wash's thormone polyadenylation signal from the plasmid pGH2R2 (R.P.) are isolated.

Woychik et al., Nucl. Acids Res. .10, pp. 7197-7210 30 (1982) by digestion with PvuII and EcoRI or from pSVCOW7 (see above) to form fragment 11.

Fragment 11 is donated between the Eco RI and SmaI cleavage sites of pUC9 (from Pharmacia / PL or ATCC) to form pCOWT1. pCOWT1 is cleaved with SalI, the ends are blunted with T4 DNA polymerase added to EcoRI linkers, the DNA is cleaved with EcoRI and the 0.6 kb fragment (fragment 12) is isolated. This is the same as fragment 11 except that it has two Eco RI ends and a 5 polylinker sequence at one end.

Plasmid pDIE50 is digested with Eco RI and fragment 12 is cloned therein to form plasmid pD1B50PA. Degradation with BamHI and PvuII yields a 1.1 kb fragment when the polyadenylation signal has the correct orientation.

The plasmid can also be constructed by cloning into the polyadenylation sequence before the promoter.

The plasmid pDIE50PA is used for transfection of ChO dhfr cells (DXB-11, G. Urlaub and L.A. Chasin,

Proc. Natl. Acad. Sci. United States TJ_, pp. 4216-20 (1980)) by calcium phosphate co-precipitation with salmon milk carrier DNA (F.L. Graham and A.J. Van Der Eb, Virol. 52, pp. 456-67 (1973)). The dihydrofolate reductase-positive (dhfr +) transfected cells are selected in Dulbecco's modified Eagle's medium plus Eagle's non-essential amino acids plus 10% fetal calf serum. Selected dhfr + CHO cells generate gp50 as detected by immunofluorescence with anti-gp50 monoclonal antibody 14 3A-4 or by labeling with C-glucosamine and immunoprecipitation with 3A-4. The monoclonal antibody 3A-4 is prepared as described in U.S. Patent Application no.

817,429 (filed January 9, 1985). Immunoprecipitation reactions are performed as previously described (T.J. Rea et al., Supra), except for the following:

The extracts are first incubated with normal mouse serum, followed by washed staphylococcus aureiis cells, and centrifuged for 30 minutes in a Beckman SW50.1 rotor at 40,000 rpm after incubating the extracts with monoclonal or polyclonal antiserum plus S. aureus. cells, the cells are washed three times in 10 mM DK 175114 B1

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Tris-HCl, pH 7.0, 1 mM EDTA, 0.1 M NaCl, 1% NP40 and 0.5% deoxycholate. The analysis of the proteins is carried out on 11% SDS-polyacrylamide gels (L. Morse et al., J. ViroL 26, pp. 389-410 (1984)). Initial immunofluorescence assays, it is found that 3A-4 reacts with the pDIE50PA-transfected CHO cells but not with non-transfected CHO cells. When the transfected CHO-14 cells are labeled with C-glucosamine, 3A-4 immunoprecipitates a labeled protein from cells containing pDIE50PA, 10 but not from human renin-producing control cells.

The precipitated protein migrates on SDS-polyacrylamide gels together with the protein precipitated by 3A-4 from PRV-infected cells.

A clone of these transfected CHO cells producing gp50 can be grown in roller flasks, harvested in phosphate buffered saline plus 1 mM EDTA and mixed with complete Freund's adjuvant for use as a vaccine.

The gp5O gene can also be expressed in a Vaccinia vector. In this embodiment, after pBG50-23 20 is digested with Maelll and the ends blunted with T4 DNA polymerase, the DNA is digested with BamHI.

The 1.3 kp blunt-ended BamHI fragment containing the gp50 gene is isolated. The plasmid pGS20 (Mackett et al., J. Virol 9, pp. 857-64 (1984)) is digested with BamHI and 25 SmaI, and the larger 6.5 kb fragment is isolated by gel electrophoresis. These two fragments are ligated to form pW50. Plasmid pW50 is transfected into CV-1 cells (ATCC CCL 70) infected with the WR strain of Vaccinia virus (ATCC VR-119) and selected for thymidine kinase-30 negative recombinants by plating on 143 cells (ATCC CRL 8303). 5-Bromodeoxyuridine (BUdR) by the methods described by Mackett et al. in DNA Cloning, Volume II: A Practical Approach, D.M. Glover, ed., IRL Press,

Oxford (1985). The resulting virus, vaccinia-gp50, B1

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expresses gp50 in infected cells as determined by labeling the proteins of the infected cell with 14 C-glucosamine and immunoprecipitation with monoclonal antibody 3A-4.

5

Example 2 This example describes the protection of mice and pigs from exposure to PRV using gp50 of Example 10 as an immunogenic agent.

In Tables I-III below, the microneutralization assay is carried out as follows: Serial two-fold dilutions of serum samples are made in microtiter plates (Costar) using basal Eagle medium (BME) supplemented with 3% fetal calf serum and antibiotics. Ca.

1000 pfu (50 µl) of PRV is added to 50 µl of each dilution. Rabbit complement is included in the viral portion at a 1: 5 dilution for mouse serum assays, but not in pig serum assays. The samples are incubated for either 20 hours (pig serum) or 3 hours (mouse serum) at 37 ° C.

After the incubation period, a portion (50 µl) of pig kidney-15 cells (PK-15) (300,000 cells / ml) in Eagle's minimal essential medium is added to each serum per ml. PRV - try. The samples are then incubated at 37 ° C for 2 days.

The neutralizing titers represent the reciprocal values of the highest dilutions that protect 50% of the cells from cytopathic effects.

Table I shows the protection of mice from exposure to virulent PRV by immunization with gp50 produced in Vaccinia virus. The mice are immunized by tail scratching with 25 µl or by administration via the 50 µl step pads. The mice are immunized 28 days prior to virus exposure (mice that have received PR-Vac are immunized 14 days prior to virus exposure).

35

Table I

20 DK 175114 B1

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Immunization-Neutralizing Dose Vessel Wetting% Agent (PFU) _ titera) Survival0 5 gp50 3fOxlO7 tail 1024 93 gp50 6, OxlO7 treadmill 1024 100 gp50 7.5xl06 tail 512 93 vacci- 7.5xl06 tail <8 27 nia 10 BMEd) - tail <8 20

Pr-Vace ^ - step cushion 512 90

Neutralizing titer against PRV on day of exposure (+ complement).

B) Exposed to 10 x LD 2 q of PRV-Rice strain administered intraperitoneally.

c) Control virus.

d) Basal Eagle medium, negative control.

e) Norden Laboratories, Lincoln, NE, vaccine of inactivated PRV, positive control.

Table II shows the protection of mice from exposure to virulent PRV by immunization with gp50 produced in CHO cells. The mice are immunized 28 days, 18 days and 7 days 25 before virus exposure. The mice receive preparations with adjuvants subcutaneously at the first dose and saline intraperitoneally at the second and third doses. Each mouse receives 10 6 broken cells / dose.

30 35 21 DK 175114 B1

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Table II

Immunizing Neutralizing%. .

agent / adjuvant titera) _ survivalPJ

gp50 / CFAc) 512 100 (10/10) 5 gp50 / CFA (2 doses) not determined 80 (4/5) gp50 / IFAd) 1024 90 (9/10) gp50 / saline 256 100 (3/3) CHO- renin) / CFA <8 10 (1/10)

Not treated <8 0 (0/10) PR-Vacf) 4096 90 (9/10) a) Neutralizing titer against PRV on day of exposure to virus (+ complement).

b) Exposed to 30 times LDCn of PRV-Rice strain by administration via the cushion.

c) Complete Freund's adjuvant.

d) Incomplete Freund's adjuvant.

e) Control cells expressing renin.

f) Norden Laboratories, Lincoln, NE, vaccine of inactivated PRV, positive control.

Table III shows the protection of pigs from exposure to virulent PRV by immunization with gp50 produced in CHO cells. Pigs are immunized 21 and 7 days prior to exposure of 25 η to virus. The pigs receive 2 x 10 broken cells per dosage.

The first dose is mixed with complete Freund's adjuvant, while the second dose is suspended in saline. Both doses are administered intramuscularly.

30 35

Table III

22 DK 175114 B1

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Immunizing Geometric Mean%. x mean / adjuvant titer survival survival 5 gp50 / CFA 25 100 CHO renin / CFA <8 0 a) Neutralizing titer against PRV on day of virus exposure.

B) Exposure to PRV-Rice strain, 1 x 10 5 pfu / pig by intranasal administration.

These three tables show that gp50 can induce neutralizing antibodies and protect mice and pigs from exposure to a lethal dose of PRV.

In another aspect of the present invention, a fragment of glycoprotein gp50 is prepared by removing DNA encoding the C-terminal end of gp50.

The resulting polypeptide has a deletion for the amino acid sequence necessary to anchor gp50 in the cell membrane. When expressed in mammalian cells, this gp50 derivative is excreted in the medium. The purification of this gp50 derivative from the medium for use as a subunit vaccine is much simpler than whole cell fractionation. The removal of the anchor sequence for conversion of a membrane protein to a secreted protein was first demonstrated for the influenza rooster agglutinin gene (M.J.

Gething and J. Sambrook, Nature 300. pp. 598-603 (1982)).

Referring to map L, the plasmid pDIE50 above is digested with SalI and EcoRI. The 5.0 30 and 0.7 kb fragments are isolated. The 0.7 kb fragment encoding a portion of gp50 is digested with Sau3A and a 0.5 kb SalI / Sau3A fragment is isolated. For introduction of a stoocodon after the blunted gp50 gene, the following oligonucleotides are synthesized: the fragment and the condensed oligonucleotides are ligated to form the plasmid pDIE50T. Degradation with EcoRI and SalI gives a 580 bp fragment. pDIE50T is cleaved with EcoRI and the 0.6 kb EcoRI fragment containing the bGH-polyA site (from step 12) is cloned to form the plasmid pDIE50TPA. Degradation of pDIE50TPA with BamHI and PvuII yields a 970 bp fragment with the proper orientation polyadenylation signal.

pDIE50TPA is used for transfection of CHO- dhfr cells. Selected dhfr + -CHO cells produce a blunted form of gp50 which is secreted into the medium as detected by S-methionine labeling and immunoprecipitation.

The polypeptides of the present invention can also be expressed in insect cells as follows: By placing a BamHI linker on the EcoRI site of pD50 and degrading with BamHI, BaHI fragments containing gp50 genes are obtained. These BamHI fragments can be donated at a BamHI site below a polyhedrin promoter in pAC373 (Mol. Cell. Biol.

5, pp. 2860-65 (1985)). The plasmids 25 thus produced can be co-transfected with DNA from the baculovirus Autographa californica into Sf9 cells, and recombinant viruses can be isolated by the methods set forth in said article. These recombinant viruses produce gp50 after infection of Sf9 cells.

A vaccine prepared using a glycoprotein of the present invention or an immunogenic fragment thereof may consist of fixed host cells, a host cell extract, or one partially or fully purified PRV glycoprotein preparation from the host cells or produced by chemical synthesis. The PRV glycoprotein immunogen prepared according to the present invention is preferably free of PRV virus. Thus, the vaccine immunogen of the invention is composed essentially solely of the desired immunogenic PRV polypeptide and / or other PRV polypeptides exhibiting PRV antigenicity.

The immunogen can be prepared in vaccine dosage form by well known procedures. The vaccine may be administered intramuscularly, subcutaneously or intranasally. For parenteral administration, such as intramuscular injection, the immunogen can be combined with a suitable carrier. It can for example. administered in water, saline or buffered carriers with or without various excipients or immunomodulatory agents including aluminum hydroxide, aluminum phosphate, aluminum potassium sulphate (alum), beryllium sulphate, silica, kaolin, carbon, water-in-oil emulsions, oil-in-water emulsions, muramyl dipeptide, bacterial endotoxin, lipid X, Corynebacterium parvum (Propionobacterium aenes), Bordetella pertussis, polyribonucleotides, sodium alginate, lanolin, lysolecidine, vitamin A, saponin, liposomes, levanisol, levanisol, DEAE other synthetic auxiliaries. Such adjuvants are available commercially from various sources, e.g.

"Merck Adjuvant 65" (Merck and Company, Inc., Rahway, N.J.).

Another suitable adjuvant is Freund's incomplete adjuvant (Difco Laboratories, Detroit, Michigan).

The ratio of immunogen to adjuvant can be varied within a wide range, as long as both are present in effective amounts. Eg. For example, aluminum hydroxide may be present in an amount of approx. 0.5% of the vaccine mixture (based on alumina). On a single-dose basis, the concentration of the immunogen may be from ca. 1.0 Mg to approx. 100 mg per pig. A preferred range 35 is from ca. 100 mg to approx. 3.0 mg per pig. A suitable dose size is approx. 1-10 ml, preferably approx.

1.0 ml. Accordingly, a dose for intra-muscular injection, e.g. comprise 1 ml containing 1.0 mg of immunogen in admixture with 0.5% alumina.

Comparable dosage forms can also be prepared for parenteral administration to piglets, but the amount of immunogen per the dose will be smaller, e.g. ca. 0.25 to approx. 1.0 mg per dosage.

For vaccination of sows a 10 dose system can be used. The first dose can be given from several months to approx. 5-7 weeks before the pigs get pigs.

The second dose of the vaccine should then be administered a few weeks after the first dose, e.g. ca.

2 to 4 weeks later and the vaccine can then be given up to 15, but before the sows get pigs. Alternatively, the vaccine may be administered e.g. a single dose of 2 ml approx. 5 to 7 weeks before the pigs get pigs. However, a two-dose system is considered to be preferred for the most effective immunization of the piglets. Semi-annual revaccination is recommended for breeding animals. Orns can be revaccinated at any time. In addition, sows can be revaccinated before breeding. Pigs born of non-vaccinated sows can be vaccinated when they are approx. 3-10 days old, again when 4-6 months old and then annually or preferably 25 semi-annually.

The vaccine can also be combined with other vaccines against other diseases, so that combination vaccines are available. It can also be combined with other medicines, e.g. antibiotics. A pharmaceutically effective amount of the vaccine may be used with a pharmaceutically acceptable carrier or diluent for vaccinating animals such as pigs, cattle, sheep, goats and other mammals.

Other vaccines may be prepared according to methods well known to those skilled in the art and e.g. is described in 1st Tizard, An Introduction to Veterinary Immunology, 2nd ed. (1982).

26 DK 175114 B1

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SCHEME A

27 54 ATG CTG CTC GCA GCG CIA TTG GCG GCG CTG GTC GCC OGG ACG ACG CTC GGT GCG 5 Me C Leu Leu Ala Ala Leu Leu Ala Ala Leu Val Ala Arg Thr Thr Leu Gly Ala 81 108 GAC GTG GAC GCC CTG GCC GGG GCG ACC TTC GCC GCG CCC GCG TAC GOG IAC ACC Asp Val Asp Ala Val Pro Ala Pro Thr Rie Pro Pro Pro Ala Tyr Pro Tyr Thr 135 162 10 GAG TCG TGG CAG CTG ACG CTG ACG AGG GTC CCC TCG CCC TTC GTC GGC CCC GCG Glu Ser Trp Gin Leu Thr Leu Thr Thr Val Pro Ser Pro Rie Val Gly Pro Ala 189 216 GAC GTC TAC CAC ACG CGC CCG CTG GAG GAC CCG TGC GCG GTG GTG GCG CTG AIC Asp Val Tyr His Thr Arg Pro Leu Glu Asp Pro Cys Gly Val Val Ala Leu Ile 15 243 270 TCC GAC CCG CAG GTG GAC OGG CTG CTG AAC GAG GCG GTG GCC CAC CGG CGG CCC Ser Asp Pro Gin Val Asp Arg Leu Leu Asn Glu Ala Val Ala His Arg Arg Pro 297 324

ACG TAC OGC GCC CAC GTG GCC TGG TAC OGC ATC GCG GAC GGG TGC GCA CAC CTG

20

Thr Tyr Arg Ala His Val Ala Trp Tyr Arg Ile Ala Asp Gly Cys Ala His Leu 351 378 CTG TAC ITT ATC GAG TAC GCC GAC TGC GAC CCC AGG CAG GTC ΤΓΤ GGG CGC TGC Leu Tyr Rie Ile Glu Tyr Ala Asp Cys Asp Pro Arg Gin Val Rie Gly Arg Cys 405 432 25 CGG CGC CGC ACC ACG CCG ATG TGG TGG ACC CCG TCC GCG GAC TAC ATG TTC CCC Arg Arg Arg Thr Thr Pro With Trp Trp Thr Pro Ser Ala Asp Tyr With Rie Pro 459 486

ACG GAG GAC GAG CTG GGG CFG CTC ATG GTG GCC CCG GGG OGG TTC AAC GAG GGC

Thr Glu Asp Glu Leu Gly Leu Leu With Val Ala Pro Gly Arg Rie Asn Glu Gly 30 513 540 CAG TAC OGG CGC CTG GTG TCC GTC GAC GGC GTG AAC ATC CTC ACC GAC TTC ATG Gin Tyr Arg Arg Leu Val Ser Val Gly Val Asn Ile Leu Thr Asp Rie Met 567 594

35 GTG GCG CTC 0CC GAG GGG GAA GAG TCC CGG TTC GCC OGC GTG GAC CAG CAC CGC

Val Ala Leu Pro Glu Gly Gin Glu Cys Pro Rie Ala Arg Val Asp Gin His Arg 621 648 ACG TAC AAG TTC GGC GCG TGC TGG AGC GAC GAC AGC TTC AAG CGG GGC GIG GAC Thr Tyr List Rie Gly Ala Cys Trp Ser Asp Asp Ser Rie List Arg Gly Val Asp

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27 DK 175114 B1 SCHEDULE A (continued) 675 702 GTG ATG OGA TIC CTG ACG CCG TIC TAC CAG CAG CCC CCG CAC OGG GAG GTG GTG 5 Val With Arg Rie Leu Thr Pro Rie Tyr Gin Gin Pro His Arg Glu Val Val 729 756

AAC TAC TGG TAC OGC AAG AAC GGC OGG ACG CTC CCG CGG GCC CAC GCC GCC GCC

Asn Tyr Trp Tyr Arg List Asn Gly Arg Thr Leu Pro Arg Ala His Ala Ala Ala 783 810 10

ACG CCG TAC GCC ATC GAC CCC GCG CGG CCC TCG GCG GGC TCG CCG AGG CCC CGG

Thr Pro Tyr Ala Ile Asp Pro 'Ala Arg Pro Ser Ala Gly Ser Pro Arg Pro Arg. 837 864

CCC CGG CCC CGG CCC CGG CCC CGG OCG AAG CCC GAG CCC GCC CCG GCG ACG CCC

Pro Arg Pro Arg Pro Arg Pro Arg Pro List Pro Glu Pro Ala Pro Ala Thr Pro 15 891 918

GCG CCC CCC GAC CGC CTG CCC GAG CCG GCG ACG CGG GAC CAC GCC CCC GCG GGC

Ala Pro Pro Asp Arg Leu Pro Glu Pro Ala Thr Arg Asp His Ala Ala Gly Gly 945 972

20 OGC CCC ACG CCG OGA CCC CCG AGG OCC GAG ACG CCG CAC OGC CCC TTC GCC CCG

Arg Pro Thr Pro Arg Pro Pro Arg Pro Glu Thr Pro His Arg Pro Rie Ala Pro 999 1026

CCG GCC GTC GTG CCC AGC GGG TGG CCG CAG CCC GCG GAG CCG TTC CAG CCG CGG

Pro Ala Val Val Pro Ser Gly Trp Pro Gin Pro Ala Glu Pro Rie Gin Pro Arg 25 1053 1080

ACC CCC GCC GCG CCG GGC GTC TCG CGC CAC CGC TCG CTG ATC GTC GGC ACG GGC

Thr Pro Ala Ala Pro Gly Val Ser Arg His Arg Ser Val Ile Val Gly Thr Gly 1107 1134 ACC GCG ATG GGC GCG CTC CTG GTG GGC GTG TGC GTC TAC ATC TTC TTC CGC CTG 30 Thr Ala With Gly Ala Leu Leu Val Gly Val Cys Val Tyr Ile Rie Phe Arg Leu 1161 1188

AGG GGG GCG AAG GGG TAT CGC CTC CTG GGC GGT CCC GCG GAC GCC GAC GAG CTA

Arg Gly Ala Lys Gly Tyr Arg Leu Leu Gly Gly Pro Ala Asp Ala Asp Glu Leu 1215

35 AAA GCG CAG CCC GGT CCG TAG

List Ala Gin Pro Gly Pro DK 175114 B1

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28

SCHEME D BamHI-7 Fragment of PRV BanHI BstEII PvuII Kpnl Sall Kpnl Stu Dral BstElI Sphl Bsml BanHI

J_I_I_l-I_I_I_I_I_I_I_L

5 XXXXXXXXX 5050505050505050 63636363636363 IIIIIIIIIIIIIIIII

X - glycoprotein X (gX) 50 - glycoprotein 50 (gp50) 63 - glycoprotein 63 (gp63) ΊΟ I - glycoprotein I (gi) 15 20 25 30 35 SCHEME E. Construction of pPR28-4 and pPR28-l.

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B1 (a) BamHl-7 is degraded with BamHI and PvuII to form fragments 1 (1.5 kb) and 2 (4.9 kb).

5

BamHI PvuII

J_l XXXXXXXXXX 5050 fragment 1 10

PvuII Salall BamHI

(B) Fragments 1 and 2 are inserted separately between the BamHI and PvuII sites of pBR322 to form

20 BamHI Narl Narl Narl PvuII

* -1-1 _! _ I. I_ * XXXXXXXXXXXXXXXXXXXX 5050 pPR28-4

25 PvuII Sall Mae III BamHI

* -1-1_1_I_ * 50505050505050 pPR28-l 1 35

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30 DK 175114 B1 SCHEME F. Restriction enzyme cleavage sites used for pg50 sequence determination.

5

Ndel Narl PvuII Kpnl Sall Kpnl Sau3A Maelll J-1-1_I_I_I_I_l 505050505050505050505050505050505050505050505050505050 10 gp50 gene 15 20 25 30 35 SCHEME G. Construction of pPR28-4 Nar2.

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311 175114 B1 (a) pPR28-4 is digested with Narl to form fragment 3.

5 Narl PvuII BanHI Narl

J_1-1 - L

soso xxxxxmxxx · (b) BamH1 linkers are added to the fragment and it is then treated with BamHI to form fragment 4.

BanHI PvuII BamHI

(I) 1-15050 (c) Fragment 4 is circularized with DNA ligase to form pPR28-4 Nar2.

BamHI PvuII

20 * _I_I __ * 5050 25 1 35 SCHEME H. Collection of the complete gp50 gene.

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321 175114 B1 (a) pPR28-4-Nar2 is digested with BamHI and PvuRII to form fragment 5 {160 bp).

5

Banttl PvuII

J-L

(B) pPR28-1 is digested with BamHi and PvuII to form fragment 6 (4.9 kb).

PvuII Maelll BanHI

15 J -! - L

50505050505050 (c) pPGX1 is digested with BamHi, treated with BAP and then ligated with fragments 5 and 6 to form PBGP50-23.

BamHi PvuII Maelll BamHi 25 * -1-1-1- '' 505050505050505050 1 35 Scheme I. Preparation of the plasmid pD50.

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331 175114 B1 (a) pBGP50-23 is digested with Maelll, provided with blunt ends with T4 DNA polymerase, added to EcoRI linkers 5g and digested with EcoRI, and then digested with BamHI to form fragment 7 (1.3 kb ).

BamHI PvuII EcoRI

(b) The plasmid pSV2dhfr is digested with BamHI and EcoRI to form fragment 8 (5.0 kb).

15 BamHI Hindi 11 PvuII EcoRI

I -1-1 -; - 1

1 1 1 R

ie SV40 AmpK

Ori 20 (c) Plasmid pD50 is prepared by ligating fragments 7 and 8.

BamHI Hindi 11 PvuII EcoRI 'PvuII

* _J_I_I_1_i_ *

III

ie SV40 AmpR 50505050505050

Ori dhfr = dihydrofolate reductase gene.

SV40 Ori = SV40 promoter and replication start.

β

Amp = Ampicillin resistance gene.

Scheme J. Preparation of the plasmid pDIE50.

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B1 (a) pD50 is digested with BamHI and treated with BAP to form fragment 9.

5

BamHI Hindi 11 PvuII EcoRI PvuII BamHI

I -J _! _! - 1 — l

III

i.e. SVAO AnrpR 5050505050505050 10 (b) Fragment 10 (760bp) containing the immediate early promoter of human cytomegalovirus (Towne) is isolated.

15

Sau3A SacI Sau3A

J_L__L

PPPPPPPPPPPPPPPP

(C) Fragments 9 and 10 are ligated to form the plasmid pDIE50.

Hindlll PvuII EcoRI Sau3A Salad Sau3A Sau3A

"* -1_I_I_I_I_I_I— *

III

ie SVAO Amp11 50505050505050PPPPPPPPP

Ori Scheme K. Preparation of plasmid pDIE50PA.

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(A) The plasmid pSVCOW7 is digested with PvuII and EcoRI to form fragment 11.

5 pSVC0W7

EcoRI PvuII PstI BamHI Hindlll PvuII

* _I _! _! _ I_I_t *

I I I

AAAAGOGOGGGGGGGGGGG ie SV40 Amp * 10 -.

Ori

fragment 11 EcoRI PvuII

J_L

AAAAG

(B) Fragment 11 is cloned into pUC9 to generate the plasmid pCOWT1.

EcoRI PvuII / Sma fusion BamHI Hall * I _! _ I_I_ *

2Q AAAAG

(c) pCOWT1 is digested with SalI, blunt-ended by T4 DNA polymerase, and EcoRI linkers are added and then digested with EcoRI to form fragment 12 (0.6 kb).

25,, ·

EcoRI BamHI EcoRI (at filled-in Sall location)

in - LI

AAAAG

(d) The plasmid pDIE50 is cleaved with Eco RI and the fragment 12 is cloned therein to form the plasmid pDIE50PA.

Hindlll PvuII EcoRI Banffi EcoRI Sau3A Sau3A

* 1_I_I_U_I i_ *

III

ie SV40 Amp * AAAAAG 5050505050PPPPPP

Ori A = Cattle growth hormone polyadenylation signal.

G = Genomic bovine growth hormone.

P = Immediate early promoter of human cytomegalovirus (Towne).

Scheme L. Preparation of plasmid pDIE50T.

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36 DK 175114 B1 (a) Plasmid pDIE50 is digested with SalI and Eco RI to form a 5.0 kb fragment.

5

Salad Sau3A Sau3A HindiII PvuII EcoRI

J-1-1_I_I_L

III

5050505050PPPPPPPP ie SV40 AmpR

10 and a 0.7 kb fragment.

EcoRI Sau3A Salad

J_I_L

505050505050 (b) The 0.7 kb fragment is digested with Sau3AI and a 0.5 kb SalI / Sau3AI fragment is isolated.

SauSA Salad

J_L

(C) The 5.0 kb EcoRI / SalI fragment, the 0.5 kb SalI / Sau3Al fragment and the condensed oligonucleotides (see text) are ligated to generate the plasmid pDIE50T.

25

Sau3A Salad Sau3A Sau3A Hindi II PvuII EcoRI

* _! _ I_I _: _ I_I_I _! _ *

I I I

T505050505050505050PPPPPPPP ie SV40 Amp * 30 r

Ori T = stop codon.

35

Claims (11)

A recombinant DNA molecule containing a DNA sequence encoding a polypeptide exhibiting pseudorabies virus (PRV) glycoprotein gp50 immunogenicity, wherein said DNA sequence is operably linked to an expression control sequence, wherein the DNA sequence that encodes the polypeptide is selected from the sequence encoding gp50 and which is ATG CTC CTC GCA GCG CIA TTG GCG GCG CTG GTC GCC CGG ACG ACG CTC GGT GCG A host cell transformed with a recombinant DNA-35 molecule according to claim 1. DK 175114 B1 2. ACG TAC AAC TTC CGC GCG TGC TGG ACC GAC GAC AGC TTC AAG CGG GGC GTG GAC GTG ATG CGA TTC CTG ACG CCG TTC TAC CAG CAG CCC CCG CAC CGC GAG GTG GTG AAC TAC TGG TAC CGC AAG AAC GGC CGG ACG CTC CCG CGG GCC CAC GCC GCC GCC ACG CCG TAC GCC ATC GAC CCC GCG CGG CCC TCG GCG GGC TCG CCG AGG CCC CGG CCC CGG CCC CGG CCC CGG CCC CGG CCG AAG CCC GAG CCC GCC CCG GCG ACG CCG 25 GCG CCC CCC GAC CGC CTG CCC GAG CCG GCG ACG CGG GAC CAC GCC GCC GGG GGC CGC CCC ACG CCG CGA CCC CCG AGG CCC GAG ACG CCG CAC CGC CCC TTC GCC CCG CCG GCC GTC GTG CCC AGC GGG TGG CCG CAG CCG CCG TTC CAG CCG CGG ACC CCC GCC GCG CCG GGC GTC TCG CGC CAC CGC TCG GTG ATC GTC GGC ACG GGC ACC GCG ATG GGC GCG CTC CTG GTG GGC CTG TGC GTC TAC ATC TTC TTC CGC CTG 30 AGG GGG GCG AAG GGG TAT CGC CTC CTG GGC GGT GCC GCG GAC GCC GAC GAG CTA AAA GCG CAG CCC GGT CCG TAG, and fragments thereof encoding polypeptides exhibiting PRV antigenicity. The host cell according to claim 2, which is derived from bacteria, fungi, plants or animals. The host cell of claim 3, which is E. coli. The host cell of claim 3, which is a yeast cell. 5 The host cell of claim 3, which is a Chinese hamster ovary cell (CHO cell). A method of producing a polypeptide exhibiting PRV-gp50 antigenicity, comprising: (a) producing a recombinant DNA molecule, wherein the molecule contains a DNA sequence encoding a polypeptide exhibiting PRV-gp50 antigenicity, wherein said DNA sequence is operably linked to an expression control sequence, (b) transforming a suitable host cell with said recombinant DNA molecule, (c) culturing said host cell, and (d) collecting said polypeptide, wherein the DNA sequence is selected from the gp50 sequence which is 20 AJC CIC CTC GCA CCG CIA TIC GCC GCG CTG CTC GCC CGC ACG ACG CTC GCT GCG GAC GIG GAC GCC CIG CCC GCG CCG ACC TTC CCC CCG CCC GCG TAC CCC TAC ACC GAG TCG TGC CAG CTG ACG CTG ACG ACG GTC CCC TCG CCC TTC CTC GGC CCC GCG GAC GTC TAC CAC ACG CGC CCG CTG GAG GAC CCG TGC GCG GIG CTG GCG CTG ATC ICC GAC CCG CAG GTG GAC CGG CIG CIG AAC GAG GCG CTG GCC CAC CGG CGG CCC 25 ACG TAC CGC GCC CAC GTG GCC TGG TAC CGC AIC GCG GAC GGG TGC GCA CAC CTG CIG TAC TIT ATC GAG TAC GCC GAC TGC GAC CCC AGG CAG CTC ΤΓΓ GGG CGC TGC OGG CGC CGC ACC ACG CCG AIC TGG TGG ACC CCG ICC GAC TAC AIG TTC CCC ACG GAG GAC GAG CIG GGG CIG CTC A1G GTG GCC CCG GGG CGG TTC AAC GAG GGC CAG TAC GGG CGC CTG GIG TCC GTC GAC GGC GTG AAC AIC CTC ACC GAC TTC AIG 30 GTG GCG GTC CCC GAG GGG CAA GAG TGC CCG TIC GCC CGC GIG GAC CAG CAC CGC ACG TAC AAG TTC GGC GCG TGC TGG AGC GAC GAC AGC TTC AAG CGG GGC GTG GAC GTG ATG CGA TTC CTG ACG CCG TTC TAC CAG CAG CC CAC CGG GAG GTG GTG AAC TAC TGG TAC CGC AAG AAC GGC CGG ACG CIC CCG CGG GCC CAC GCC GCC GCC ACG CCG TAC GCC AIC GAC CCC GCG CGG TCG GCG GGC TCG CCG ACG CCC CGG CCC CGG CCC OGG CCC CCG CCG AND AAG CCC GAG CCC GCC CCG GCG ACG CCC GCG CCC CCC GAC OGC CTG CCC GAG CCG GCG ACG OGG GAC CAC GCC GCC GGG GGC 35 DK 175114 B1 CGC CCC ACG CCG CGA CKCO ^ AGGCCCGAGACGCCGCACOGCCCCTTCGCCCCG 5 CCG GCC CTC GIG CCC AGC GGG TGG CCG CAG CCC CCC GAG CCG TTC CAG CCG GGG ACCCCCGCCGCCCCGGGCCrCTCGCGCCACCGCTCGGTGATCCTCGGCACGGGC ACC GCC AIC GCC GCC CTC CIG CTC CGC CTC ICC CTC TAC ATC TTC TTC CGC CTG AGG GGG GCG AAG GGCIATOGCCTCCICGGCGGTCCCGCGGACGCCGAC GAG CIA AAA GCG CAG CCC GGT CCG TAG, 10, and fragments thereof, which encode polypeptides exhibiting pseudorabies virus antigenicity. The method of claim 7, wherein the host cell is selected from bacteria, fungi, plant cells and animal cells. The method of claim 7, wherein the host cell 15 is E. coli. The method of claim 7, wherein the host cell is yeast. 10 GAC GTC GAC GCC GTG CCC GCG CCC ACC TTC CCC CCG CCC GCG TAC CCG TAC ACC GAG TCG TGG CAG CTG ACG CTG ACG ACG GTC CCC TCG CCC TTC GTC CGC CCC GCG GAC GTC TAC CAC ACG CGC CCG CTG GAG GAC CCG TGC GCG GTG GTG GCG CTG ATC TCC GAC CCG CAG GTG GAC CGG CTG CTG AAC GAG GCG GTG GCC CAC CGG CGG CCC ACG TAC CGC GCC CAC GTG GCC TGG TAC CGC ATC GCG GAC GGG TGC GCA CAC CTG 15 CTG TAC ITT ATC GAG TAC GCC GAC TGC GAC CCC AGG CAG GTC ΤΠ GGG CGC TGC CGG CGC CGC ACC ACG CCG ATG TGG TGG ACC CCG TCC GCG GAC TAC ATG TTC CCC ACG GAG GAC GAG CTG GGG CTG CTC ATG GTG GCC CCG GGG CGG TTC AAC GAG GGC CAG TAC CGG CGC CTG GTG TCC GTC GAC GGC GTG AAC ATC CTC ACC GAC TTC ATG CTG GCG CTC CCC GAG GGG CAA GAG TGC CCG TTC GCC CGC GTG GAC CAG CAC CGC The method of claim 7, wherein the host cell is CHO.