CA1273884A - Process for the preparation of a herpes antigen, an agent suitable for this and a process for its preparation, and the use of this antigen - Google Patents
Process for the preparation of a herpes antigen, an agent suitable for this and a process for its preparation, and the use of this antigenInfo
- Publication number
- CA1273884A CA1273884A CA000433648A CA433648A CA1273884A CA 1273884 A CA1273884 A CA 1273884A CA 000433648 A CA000433648 A CA 000433648A CA 433648 A CA433648 A CA 433648A CA 1273884 A CA1273884 A CA 1273884A
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- deoxyribonucleic acid
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- dna
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- 238000000034 method Methods 0.000 title claims abstract description 21
- 238000002360 preparation method Methods 0.000 title claims abstract description 12
- XQFRJNBWHJMXHO-RRKCRQDMSA-N IDUR Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(I)=C1 XQFRJNBWHJMXHO-RRKCRQDMSA-N 0.000 title description 3
- 239000000427 antigen Substances 0.000 title description 3
- 102000036639 antigens Human genes 0.000 title description 3
- 108091007433 antigens Proteins 0.000 title description 3
- 239000003795 chemical substances by application Substances 0.000 title 1
- 108020004414 DNA Proteins 0.000 claims abstract description 35
- 102000053602 DNA Human genes 0.000 claims abstract description 34
- 230000000890 antigenic effect Effects 0.000 claims abstract description 15
- 241000700605 Viruses Species 0.000 claims abstract description 12
- 229960005486 vaccine Drugs 0.000 claims abstract description 8
- 239000013598 vector Substances 0.000 claims abstract description 8
- 229940039227 diagnostic agent Drugs 0.000 claims abstract 2
- 239000000032 diagnostic agent Substances 0.000 claims abstract 2
- 239000012634 fragment Substances 0.000 claims description 24
- 229920001184 polypeptide Polymers 0.000 claims description 13
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 12
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 11
- 239000013612 plasmid Substances 0.000 claims description 10
- 241000700584 Simplexvirus Species 0.000 claims description 5
- 241000588724 Escherichia coli Species 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims description 2
- 239000000126 substance Substances 0.000 claims 2
- 230000000694 effects Effects 0.000 claims 1
- 210000005253 yeast cell Anatomy 0.000 claims 1
- 108090000623 proteins and genes Proteins 0.000 abstract description 22
- 102000004169 proteins and genes Human genes 0.000 abstract description 17
- 241001529453 unidentified herpesvirus Species 0.000 abstract description 9
- 230000002068 genetic effect Effects 0.000 abstract description 3
- 210000004027 cell Anatomy 0.000 description 13
- 108020005202 Viral DNA Proteins 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 238000010367 cloning Methods 0.000 description 4
- 230000029087 digestion Effects 0.000 description 4
- 108090000288 Glycoproteins Proteins 0.000 description 3
- 102000003886 Glycoproteins Human genes 0.000 description 3
- 108020004999 messenger RNA Proteins 0.000 description 3
- 230000009466 transformation Effects 0.000 description 3
- 108020004635 Complementary DNA Proteins 0.000 description 2
- 108010042407 Endonucleases Proteins 0.000 description 2
- 102000004533 Endonucleases Human genes 0.000 description 2
- 101710091045 Envelope protein Proteins 0.000 description 2
- 101710188315 Protein X Proteins 0.000 description 2
- 102100021696 Syncytin-1 Human genes 0.000 description 2
- 239000011543 agarose gel Substances 0.000 description 2
- 229960000723 ampicillin Drugs 0.000 description 2
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 2
- 238000010804 cDNA synthesis Methods 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- MYWUZJCMWCOHBA-VIFPVBQESA-N methamphetamine Chemical compound CN[C@@H](C)CC1=CC=CC=C1 MYWUZJCMWCOHBA-VIFPVBQESA-N 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 241000701822 Bovine papillomavirus Species 0.000 description 1
- 108010054576 Deoxyribonuclease EcoRI Proteins 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 241001131785 Escherichia coli HB101 Species 0.000 description 1
- 108060002716 Exonuclease Proteins 0.000 description 1
- 101150096243 FENS gene Proteins 0.000 description 1
- 101800000342 Glycoprotein C Proteins 0.000 description 1
- 241000700721 Hepatitis B virus Species 0.000 description 1
- 241000700328 Herpes simplex virus (type 1 / strain F) Species 0.000 description 1
- 241000700588 Human alphaherpesvirus 1 Species 0.000 description 1
- 241000701074 Human alphaherpesvirus 2 Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 239000004098 Tetracycline Substances 0.000 description 1
- 108010003533 Viral Envelope Proteins Proteins 0.000 description 1
- 108010067390 Viral Proteins Proteins 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 1
- 229960005542 ethidium bromide Drugs 0.000 description 1
- 102000013165 exonuclease Human genes 0.000 description 1
- 108010092809 exonuclease Bal 31 Proteins 0.000 description 1
- 238000005188 flotation Methods 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 208000002672 hepatitis B Diseases 0.000 description 1
- 230000016784 immunoglobulin production Effects 0.000 description 1
- 238000009830 intercalation Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 238000011321 prophylaxis Methods 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- 101150118377 tet gene Proteins 0.000 description 1
- 229960002180 tetracycline Drugs 0.000 description 1
- 229930101283 tetracycline Natural products 0.000 description 1
- 235000019364 tetracycline Nutrition 0.000 description 1
- 150000003522 tetracyclines Chemical class 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 238000000844 transformation Methods 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 210000003501 vero cell Anatomy 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/16011—Herpesviridae
- C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
- C12N2710/16622—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Virology (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
ABSTRACT OF THE INVENTION
A process for the preparation of a recombinant deoxyribonucleic acid which contains the genetic code for an antigenic protein from herpes viruses is described. This combined DNA is obtained by bonding a corresponding genoma section of the virus to a so-called vector and introducing it into a cell which then produces the antigenic protein. The protein is isolated and used for the preparation of an antiserum, a vaccine or a diagnostic agent.
A process for the preparation of a recombinant deoxyribonucleic acid which contains the genetic code for an antigenic protein from herpes viruses is described. This combined DNA is obtained by bonding a corresponding genoma section of the virus to a so-called vector and introducing it into a cell which then produces the antigenic protein. The protein is isolated and used for the preparation of an antiserum, a vaccine or a diagnostic agent.
Description
~7388~
The invention relate~ to a recombinant deoxyribonucleic acid (DNA) which contains the genetic code for an antigenic protein from herpes viruse~, a cell which contains that recombinant deoxyribonucleic acid and produces this antigenic protein, the use of that recombinant deoxyribonucleic acid or this cell for the preparation of this protein and processes for their preparation, and to the use of this antigenic protein.
A protein which causes formation of antibodies against herpes viruses is of great interest for the preparation of vaccines for the prophylaxis and therapy of diseases caused by these viruses.
Investigations carried out recently give rise to the as~umption that certain types of tumor may be caused by herpes viruses (HV). A requirement of a vaccine, for example against herpes simplex virus type I (HSV-l) or type II (HSV-2) is therefore that this vaccine must be absolutely free from HSV-DNA
(deoxyribonucleic acid)~ or that the viral DNA must be inactivated, in order to exclude possible transformations.
The conventional preparation of a vaccine corresponding to these requirements would necessitate expensive purification steps.
Some glycoproteins which are integrated in the envelope of the virus (so-called envelope proteins) and to which it has been possible to allocate genetic loci on the HSV-DNA have so far been described as antigenic components of HSV.
There was therefore the ob~ect of preparing a herpes antigen which is free from the nucleic acid of herpes viruses and $8 suitable for th0 preparation of a vaccine against diseases caused by herpes viruses.
This ob~ect has now been achieved by isolating a genome section (DNA) from a herpes simplex virus and transferring this to a microorganism in which this DNA iS replicated and expressed.
Cloning of the gene section, which provides the code for the glycoprotein C (gC) is described in the following text. The process can also be carried out analogously for the other envelope proteins gA, gB, gD and gE, which have already been described, and for any other viral protein.
' . - .
i ~ 73 88
The invention relate~ to a recombinant deoxyribonucleic acid (DNA) which contains the genetic code for an antigenic protein from herpes viruse~, a cell which contains that recombinant deoxyribonucleic acid and produces this antigenic protein, the use of that recombinant deoxyribonucleic acid or this cell for the preparation of this protein and processes for their preparation, and to the use of this antigenic protein.
A protein which causes formation of antibodies against herpes viruses is of great interest for the preparation of vaccines for the prophylaxis and therapy of diseases caused by these viruses.
Investigations carried out recently give rise to the as~umption that certain types of tumor may be caused by herpes viruses (HV). A requirement of a vaccine, for example against herpes simplex virus type I (HSV-l) or type II (HSV-2) is therefore that this vaccine must be absolutely free from HSV-DNA
(deoxyribonucleic acid)~ or that the viral DNA must be inactivated, in order to exclude possible transformations.
The conventional preparation of a vaccine corresponding to these requirements would necessitate expensive purification steps.
Some glycoproteins which are integrated in the envelope of the virus (so-called envelope proteins) and to which it has been possible to allocate genetic loci on the HSV-DNA have so far been described as antigenic components of HSV.
There was therefore the ob~ect of preparing a herpes antigen which is free from the nucleic acid of herpes viruses and $8 suitable for th0 preparation of a vaccine against diseases caused by herpes viruses.
This ob~ect has now been achieved by isolating a genome section (DNA) from a herpes simplex virus and transferring this to a microorganism in which this DNA iS replicated and expressed.
Cloning of the gene section, which provides the code for the glycoprotein C (gC) is described in the following text. The process can also be carried out analogously for the other envelope proteins gA, gB, gD and gE, which have already been described, and for any other viral protein.
' . - .
i ~ 73 88
- 2 -European Patent Application 0,013,828 published August 6, 1980 described a process for the preparation of a polypeptide with the antigenic properties of hepatitis B virus, in which a DNA sequence from the DNA of the virus which provides the code for an antigenic determinant of the virus is bonded to ~he DNA
of a so-called "cloning vehiclel~ or 'vector , and a host cell is invaded with this combined deoxyribonucleic acid 80 that it produces the polypeptide with the antigenic properties of hepatitis B viru~.
A process suitable for achieving the stated object is described in the following text. However, variants of this process can also be used for the purpose according to the invention, for example those with other vector/host cell combinations.
1. Obtaining the virus-specific nucleic acid 1.1 Choice of the HSV strain Infections with high infection doses lead in many virus systems to the formation of defective virus genomes. To avoid such artifacts, a cloned virus strain with which host cells, for example vero cells, were infected in low multiplicity was therefore used. We used HSV-1 strain F. The procedure would be the same if other strains were used (for example cos or McIntyre).
1.2 Isolstion of the HSV-DNA
To avoid the losses arising on purification of a virus, the viral DNA was obtained directly from the cell lysate.
Centrifugation in a high-resolution density gradient (KI, NaI) wa~ used for this; this centrifugation permits substantial removal of the cell DNA from the viral DNA, the cell DNA having a significantly different flotation density. The addition of intercalating dyestuffs, for example ethidium bromide, permitted direct visual monitoring of the separation result and the isolation of the viral DNA (Walbromers and Scheggat, Virology (1976) 74, 256-258).
35 2. Isolation of a gsnome section which provides the code for an antigenic protein from the virus envelope.
~' .: .
': ' .
~73~38~
HSVl-DNA was hydrolyzed with restriction endonucleases and the DNA fragments were separated electrophoretically in an agarose gel. The nuclease ~indIII is suitable for this, since, after digestion of the DNA with this enzyme, the complete information for gC is localized on a specific fragment (HindIII-L fragment). The HindIII-L fragment (DNA sections: 0.592 -0.647 about 8,200 bp, compare also Figure 1) was extracted from the agarose gel (J. Langridge et al., Analyt. Biochem. 103, 264-271 (1980). The DNA can also be isolated by methods such as tho3e described by H.O. Smith (Meth. Enzymol. 65, 371-380 (1980)).
of a so-called "cloning vehiclel~ or 'vector , and a host cell is invaded with this combined deoxyribonucleic acid 80 that it produces the polypeptide with the antigenic properties of hepatitis B viru~.
A process suitable for achieving the stated object is described in the following text. However, variants of this process can also be used for the purpose according to the invention, for example those with other vector/host cell combinations.
1. Obtaining the virus-specific nucleic acid 1.1 Choice of the HSV strain Infections with high infection doses lead in many virus systems to the formation of defective virus genomes. To avoid such artifacts, a cloned virus strain with which host cells, for example vero cells, were infected in low multiplicity was therefore used. We used HSV-1 strain F. The procedure would be the same if other strains were used (for example cos or McIntyre).
1.2 Isolstion of the HSV-DNA
To avoid the losses arising on purification of a virus, the viral DNA was obtained directly from the cell lysate.
Centrifugation in a high-resolution density gradient (KI, NaI) wa~ used for this; this centrifugation permits substantial removal of the cell DNA from the viral DNA, the cell DNA having a significantly different flotation density. The addition of intercalating dyestuffs, for example ethidium bromide, permitted direct visual monitoring of the separation result and the isolation of the viral DNA (Walbromers and Scheggat, Virology (1976) 74, 256-258).
35 2. Isolation of a gsnome section which provides the code for an antigenic protein from the virus envelope.
~' .: .
': ' .
~73~38~
HSVl-DNA was hydrolyzed with restriction endonucleases and the DNA fragments were separated electrophoretically in an agarose gel. The nuclease ~indIII is suitable for this, since, after digestion of the DNA with this enzyme, the complete information for gC is localized on a specific fragment (HindIII-L fragment). The HindIII-L fragment (DNA sections: 0.592 -0.647 about 8,200 bp, compare also Figure 1) was extracted from the agarose gel (J. Langridge et al., Analyt. Biochem. 103, 264-271 (1980). The DNA can also be isolated by methods such as tho3e described by H.O. Smith (Meth. Enzymol. 65, 371-380 (1980)).
3. Preparation of a plasmid which has the HSV-DNA HindIII-L section, and transformation of bacteria ~ith this recomb~nant plasmid.
The HindIII-L fraqment was ligated (F. Bolivar and K.
Backman, Meth. Enzymol. 65, 245 (1980) (Fig. 1)) in the tetracycline-gene (Tet-gene) of the plasmid pBR 322 (F. Bolivar et al., Gene 2, 95 (1977)) and transformed into Escherichia coli strain HB 101.
In addition to plasmid pBR322, it is also possible to use other suitable vectors, such as, for example, pBR327, pBR328 or pBR329 (L. Covarrubias and F. Bolivar, Gene 17, 79 - 89 (1982)) or pUC7, pUC8 or pUC 5 (J. Messing, Recombinant DNA, A.G.
Walton, ed. 143-153 (1982), Elsevier Scientific Publishing Comp., Amsterdam) or the plasmid pUR 222 (U. R~ther et al., Nucl. Acids Res. 9, 4087 - 4098 (1981)).
Besides the strain HB 101, other strains of E. coli, such as C600 (B. Bachmann, Bacteriol. Rev. 36, 525 - 557 (1972)), RRl, SF8 or SX1592 are transformable by similar methods to that used by ourselves ~S.L. Peacock et al., Biochim. Biophys. Acta 655, 243-250, (1981); and M.G.M. Brown et al., FENS Microbiol.
Lett. 5, 219 - 222 (1979)).
For example, the following vectors could be used in transformation of eucaryotic cellss bovine papilloma virus DNA
35 (N., Sarver et al., Nol. Cell. Biol. 1, 486 to 496 (1981); SV40 (J.T. Elder et al., Ann. Rev. Genetics 15, 295 - 340 (1981)) or :
. ~i - : , ' . : -1;~73~4 chimeric plasmids, such as pSG ~R.N. Sendai-Goldin et al., Mol.
Cell. Biol. I, 743-752 (1981)).
The HindIII-L fraqment was ligated (F. Bolivar and K.
Backman, Meth. Enzymol. 65, 245 (1980) (Fig. 1)) in the tetracycline-gene (Tet-gene) of the plasmid pBR 322 (F. Bolivar et al., Gene 2, 95 (1977)) and transformed into Escherichia coli strain HB 101.
In addition to plasmid pBR322, it is also possible to use other suitable vectors, such as, for example, pBR327, pBR328 or pBR329 (L. Covarrubias and F. Bolivar, Gene 17, 79 - 89 (1982)) or pUC7, pUC8 or pUC 5 (J. Messing, Recombinant DNA, A.G.
Walton, ed. 143-153 (1982), Elsevier Scientific Publishing Comp., Amsterdam) or the plasmid pUR 222 (U. R~ther et al., Nucl. Acids Res. 9, 4087 - 4098 (1981)).
Besides the strain HB 101, other strains of E. coli, such as C600 (B. Bachmann, Bacteriol. Rev. 36, 525 - 557 (1972)), RRl, SF8 or SX1592 are transformable by similar methods to that used by ourselves ~S.L. Peacock et al., Biochim. Biophys. Acta 655, 243-250, (1981); and M.G.M. Brown et al., FENS Microbiol.
Lett. 5, 219 - 222 (1979)).
For example, the following vectors could be used in transformation of eucaryotic cellss bovine papilloma virus DNA
35 (N., Sarver et al., Nol. Cell. Biol. 1, 486 to 496 (1981); SV40 (J.T. Elder et al., Ann. Rev. Genetics 15, 295 - 340 (1981)) or :
. ~i - : , ' . : -1;~73~4 chimeric plasmids, such as pSG ~R.N. Sendai-Goldin et al., Mol.
Cell. Biol. I, 743-752 (1981)).
4. Selection of suitable clones The cells transformed with recombinant plasmids were streaked out on Petri dishes with L broth as a source of nutrients, with addition of Na ampicillin (50 mg/liters) and were incubated at 37C for 1 day. Clones which proved to be resistant to ampicillin and sensitive to tetracycline were hybridized against radioactive HSV-1 HindIII-L fragment (E. Southern, ~eth.
Enzymol. 65, 152 - 176 (1980)).
Positive clones were bred in 5.0 ml portions each of liquid culture and the DNA was isolated (H.M. Goodman and R.J.
MacDonald, Meth. Enzymol. 65, 75 - 90 (1980); and D.S. Holmes and M. Quigley, Anal. Biochem. 114, 193 - 197 (1981)).
Clones which had integrated the HindIII-L fragment into the Hind III position of pBR322 were unambiguously identified by digestion of the recombined plasmids with the endonucleases Hind III, ~am HI, Sal I, Pvu I, Pvu II and Mst II and comparison with the restriction card of HSV-I strain COS. Orientation of the L-fragment and the gC-mRNA in pNB HSV 9 is shown in Figure 1.
Enzymol. 65, 152 - 176 (1980)).
Positive clones were bred in 5.0 ml portions each of liquid culture and the DNA was isolated (H.M. Goodman and R.J.
MacDonald, Meth. Enzymol. 65, 75 - 90 (1980); and D.S. Holmes and M. Quigley, Anal. Biochem. 114, 193 - 197 (1981)).
Clones which had integrated the HindIII-L fragment into the Hind III position of pBR322 were unambiguously identified by digestion of the recombined plasmids with the endonucleases Hind III, ~am HI, Sal I, Pvu I, Pvu II and Mst II and comparison with the restriction card of HSV-I strain COS. Orientation of the L-fragment and the gC-mRNA in pNB HSV 9 is shown in Figure 1.
5. Shortening of the HindIII-L fragment and subcloning of the fragment.
gC is a late (r) protein, the mRNA of which is localized on the right section of the HindIII-L fragment (Fink et al., J.
Virol. in pres~, 1983). Since gC-mRNA, which provides the code of the peptide part of gC, is probably not spliced, the genomic information can be expressed in procaryotes. It is worth endeavoring to remove the unnecessary DNA portions of the HindIII-L fragment for the synthesis of gC. This can be achieved, inter alia, by digestion of the DNA with suitable endonucleases. ~y digestion of the Hind III-L fragment upstream from the translation start with the exonuclease Bal 31 up to the translation start and bonding of the shortened L-fragment behind an efficient promoter (such as, for example, a Trp-, Lac- or Tac-promoter), a high rate of ~ynthesis of the gC-polypeptide can be achieved in procaryotic systems (Figure 2).
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,- ~ , .
73~
5 _ In order to be able to induce antibody production, not all the protein qC is necessarily required, but only a part of the protein carrying the antigenic determinants. Cloned DNA
fragments in which degradation has been effected in the coded region of the gC with exonucleases, such as Bal 31, or sub-fragments of the HindIII-L fragment, for example the Eco-Hind I-L fragment, can be used for the production of such polypeptides.
Bonding of the Eco-Hind I -L fragment in the Eco RI
position behind the lac W 5 promoter in pUC 8 permits synthesis of a fusion protein with the first NH2-terminating amino acids of ~-galactosidase and most of the gC-polypeptide (Figure 3).
Cloning and expression of antigenic glycoproteins of HSV
other than gC can be carried out analogously to the procedures listed here.
If the corresponding mRNAs are spliced, the complementary DNA (cDNA) can be used for expression of the glycoproteins, in which case small cloned DNA fragments, such as the Eco-Hind I-L fragment, to which only the gCmRNA of HSV-l bonds, can be used for selecting the corresponding mRNAs from a lysate of cells infected with HSV. However, cloning of the genomic DNA is also conceivable in such cases, since the corresponding gene product formed in procaryotes can, in spite of the absence of splicing mechanisms here, be a protein which carries some or all of the antigenic determinants. Linking of DNA fragments from different genes of only one or of two different types of herpes viruses and expression of such a hydrbid protein is even possible.
Expression in eucaryotic cells (yeast, animal cell cultures) is also possible, especially if glycosilation of the protein is desirable.
Because of its antigenic properties, such a protein is suitable for isolating antisera and/or vaccines, if necessary using ad~uvants and conventional auxiliaries.
It can also be used for diagnostic purposes, for example for the detection of antibodies against herpes viruses.
Antisera against such a protein can themselves be used for detection of herpes viruses.
:
:' , '
gC is a late (r) protein, the mRNA of which is localized on the right section of the HindIII-L fragment (Fink et al., J.
Virol. in pres~, 1983). Since gC-mRNA, which provides the code of the peptide part of gC, is probably not spliced, the genomic information can be expressed in procaryotes. It is worth endeavoring to remove the unnecessary DNA portions of the HindIII-L fragment for the synthesis of gC. This can be achieved, inter alia, by digestion of the DNA with suitable endonucleases. ~y digestion of the Hind III-L fragment upstream from the translation start with the exonuclease Bal 31 up to the translation start and bonding of the shortened L-fragment behind an efficient promoter (such as, for example, a Trp-, Lac- or Tac-promoter), a high rate of ~ynthesis of the gC-polypeptide can be achieved in procaryotic systems (Figure 2).
- .
,- ~ , .
73~
5 _ In order to be able to induce antibody production, not all the protein qC is necessarily required, but only a part of the protein carrying the antigenic determinants. Cloned DNA
fragments in which degradation has been effected in the coded region of the gC with exonucleases, such as Bal 31, or sub-fragments of the HindIII-L fragment, for example the Eco-Hind I-L fragment, can be used for the production of such polypeptides.
Bonding of the Eco-Hind I -L fragment in the Eco RI
position behind the lac W 5 promoter in pUC 8 permits synthesis of a fusion protein with the first NH2-terminating amino acids of ~-galactosidase and most of the gC-polypeptide (Figure 3).
Cloning and expression of antigenic glycoproteins of HSV
other than gC can be carried out analogously to the procedures listed here.
If the corresponding mRNAs are spliced, the complementary DNA (cDNA) can be used for expression of the glycoproteins, in which case small cloned DNA fragments, such as the Eco-Hind I-L fragment, to which only the gCmRNA of HSV-l bonds, can be used for selecting the corresponding mRNAs from a lysate of cells infected with HSV. However, cloning of the genomic DNA is also conceivable in such cases, since the corresponding gene product formed in procaryotes can, in spite of the absence of splicing mechanisms here, be a protein which carries some or all of the antigenic determinants. Linking of DNA fragments from different genes of only one or of two different types of herpes viruses and expression of such a hydrbid protein is even possible.
Expression in eucaryotic cells (yeast, animal cell cultures) is also possible, especially if glycosilation of the protein is desirable.
Because of its antigenic properties, such a protein is suitable for isolating antisera and/or vaccines, if necessary using ad~uvants and conventional auxiliaries.
It can also be used for diagnostic purposes, for example for the detection of antibodies against herpes viruses.
Antisera against such a protein can themselves be used for detection of herpes viruses.
:
:' , '
Claims (10)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A process for the preparation of a polypeptide with an antigenic determinant of the herpes simplex virus type I, which comprises isolating from the deoxyribonucleic acid of this virus the HindIII-L fragment (genome section 0.592 to 0.647) which provides the code for such a polypeptide, bonding the HindIII-L fragment (genome section 0.592 to 0.647) enzymatically to the deoxyribonucleic acid of a vector, introducing this combined deoxyribonucleic acid into an E.
coli or yeast cell in which the combined deoxyribonucleic acid effects production of this polypeptide, and isolating this polypeptide.
coli or yeast cell in which the combined deoxyribonucleic acid effects production of this polypeptide, and isolating this polypeptide.
2. A process as claimed in claim 1, in which part of the HindIII-L fragment is isolated from the deoxyribonucleic acid of the herpes simplex virus.
3. A process as claimed in claim 1, or claim 2, in which the vector is the plasmid pBR322.
4. A process as claimed in claim 1, in which the combined deoxyribonucleic acid consists of the genome section 0.592 to 0.647 and the deoxyribonucleic acid section of the plasmid pBR322.
5. A process as claimed in claim 1, wherein the combined deoxyribonucleic acid is part of the genome section 0.592 to 0.647 and of a section of the deoxyribonucleic acid of a vector.
6. An HSVI gC polypeptide whenever obtained according to a process as claimed in claim 1 or by an obvious chemical equivalent thereof.
7. An HSVI gC polypeptide whenever obtained according to a process as claimed in any one of claims 2, 4 or 5, or by an obvious chemical equivalent thereof.
8. The genome section 0.592 to 0.647 of herpes simplex virus I.
9. A process as claimed in claim 1, which further comprises isolating an antiserum using the polypeptide.
10. A process as claimed in claim 1, which further comprises preparing a vaccine or a diagnostic agent using the polypeptide.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DEP3228501.9 | 1982-07-30 | ||
| DE19823228501 DE3228501A1 (en) | 1982-07-30 | 1982-07-30 | METHOD FOR PRODUCING A HERPES ANTIGENT, MEANS THAT ARE SUITABLE FOR ITS PRODUCTION, AND METHOD FOR THE PRODUCTION THEREOF AND THE USE OF THIS ANTIQUE |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1273884A true CA1273884A (en) | 1990-09-11 |
Family
ID=6169723
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000433648A Expired - Lifetime CA1273884A (en) | 1982-07-30 | 1983-07-29 | Process for the preparation of a herpes antigen, an agent suitable for this and a process for its preparation, and the use of this antigen |
Country Status (7)
| Country | Link |
|---|---|
| EP (1) | EP0100521B1 (en) |
| JP (1) | JPS5948095A (en) |
| AT (1) | ATE56044T1 (en) |
| AU (1) | AU577516B2 (en) |
| CA (1) | CA1273884A (en) |
| DE (2) | DE3228501A1 (en) |
| IL (1) | IL69374A0 (en) |
Families Citing this family (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| PT77014B (en) * | 1982-07-20 | 1986-01-24 | Molecular Genetics Inc | Production of herpes simplex viral proteins |
| US4818694A (en) * | 1982-07-20 | 1989-04-04 | American Cyanamid Company | Production of herpes simplex viral protein |
| US7264817B1 (en) | 1983-08-30 | 2007-09-04 | Genentech, Inc. | Immunogenic composition based on a truncated derivative of a membrane bound protein and process for making it |
| NZ209308A (en) * | 1983-08-30 | 1991-08-27 | Genentech Inc | Vaccine against hsv involving a truncated membrane-free derivative of a membrane-bound protein |
| NZ209307A (en) * | 1983-08-30 | 1990-07-26 | Genentech Inc | Diagnostic product: antigenic peptide produced by recombinant dna techniques |
| JPS6051120A (en) * | 1983-08-31 | 1985-03-22 | Chemo Sero Therapeut Res Inst | Herpes simplex subunit vaccine |
| US4642333A (en) * | 1983-09-16 | 1987-02-10 | Stanley Person | Immunologically reactive non-glycosylated amino acid chains of glycoprotein B of herpes virus types 1 and 2 |
| US5171568A (en) * | 1984-04-06 | 1992-12-15 | Chiron Corporation | Recombinant herpes simplex gb-gd vaccine |
| DE3587991T2 (en) * | 1984-04-06 | 1995-07-20 | Chiron Corp | RECOMBINANT HERPES SIMPLEX GB-GD VACCINE. |
| JPS615786A (en) * | 1984-06-19 | 1986-01-11 | Chemo Sero Therapeut Res Inst | Recombinant dna having integrated herpes simplex virus, transformant animal cell and production of herpes simplex virus protein |
| DE3582200D1 (en) * | 1984-07-20 | 1991-04-25 | Chemo Sero Therapeut Res Inst | RECOMBINANT DNA, CONTAINING A HERPES SIMPLEX VIRUS GENE OR A FRAGMENT THEREOF, YEAR TRANSFORMED WITH THIS RECOMBINANT DNA AND METHOD FOR PRODUCING HERPES SIMPLEX VIRUS PROTEINS. |
| JPS6153226A (en) * | 1984-08-24 | 1986-03-17 | Chemo Sero Therapeut Res Inst | Purification of simple herpes subunit vaccine |
| US4659568A (en) * | 1985-02-27 | 1987-04-21 | American Cyanamid Company | Process for solubilization, purification and characterization of protein from insoluble protein aggregates or complexes and compositions of matter therefrom |
| DE3510734A1 (en) * | 1985-03-25 | 1986-09-25 | Behringwerke Ag, 3550 Marburg | METHOD FOR PRODUCING A HERPES SIMPLEX VIRUS TYPE 2-SPECIFIC ANTIQUE, MEANS THAT ARE SUITABLE FOR THIS, AND USE OF THIS ANTIQUE |
| JPH0668B2 (en) * | 1985-08-30 | 1994-01-05 | 財団法人化学及血清療法研究所 | Recombinant plasmid with herpes simplex virus gene |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| IL63224A (en) * | 1980-07-17 | 1985-05-31 | Scripps Clinic Res | Synthetic peptide specific antigenic determinant and method of manufacturing antigenic materials therefrom |
| AU1678783A (en) * | 1982-07-20 | 1984-01-26 | Molecular Genetics, Inc. | Production of herpes simplex viral proteins |
| PT77014B (en) * | 1982-07-20 | 1986-01-24 | Molecular Genetics Inc | Production of herpes simplex viral proteins |
| NZ209308A (en) * | 1983-08-30 | 1991-08-27 | Genentech Inc | Vaccine against hsv involving a truncated membrane-free derivative of a membrane-bound protein |
-
1982
- 1982-07-30 DE DE19823228501 patent/DE3228501A1/en not_active Withdrawn
-
1983
- 1983-07-27 DE DE8383107373T patent/DE3381843D1/en not_active Expired - Fee Related
- 1983-07-27 EP EP83107373A patent/EP0100521B1/en not_active Expired - Lifetime
- 1983-07-27 AT AT83107373T patent/ATE56044T1/en not_active IP Right Cessation
- 1983-07-29 JP JP58137894A patent/JPS5948095A/en active Pending
- 1983-07-29 IL IL69374A patent/IL69374A0/en unknown
- 1983-07-29 CA CA000433648A patent/CA1273884A/en not_active Expired - Lifetime
- 1983-07-29 AU AU17452/83A patent/AU577516B2/en not_active Ceased
Also Published As
| Publication number | Publication date |
|---|---|
| EP0100521B1 (en) | 1990-08-29 |
| EP0100521A3 (en) | 1985-12-04 |
| IL69374A0 (en) | 1983-11-30 |
| AU577516B2 (en) | 1988-09-29 |
| DE3381843D1 (en) | 1990-10-04 |
| ATE56044T1 (en) | 1990-09-15 |
| JPS5948095A (en) | 1984-03-19 |
| DE3228501A1 (en) | 1984-02-02 |
| EP0100521A2 (en) | 1984-02-15 |
| AU1745283A (en) | 1984-02-02 |
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