JP2780961B2 - Herpes simplex virus protein and vaccine containing it - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [0001] TECHNICAL FIELD OF THE INVENTION 1.Field of the invention The present invention relates to a herpes simplex virus (HSV) glycoprotein.
Quality-related proteins, their production methods and
And new DNA sequences, plasmids
And microorganisms producing the protein (eukaryotes and
Methods for making and using both prokaryotes) and
Composition. [0002] The present invention relates to a method for preparing a viral DNA, plasmid D
DNA or DNA vectors such as bacteriophage DNA
Glycoprotein D (gD) or a part thereof
Use recombinant DNA technology to insert the coding DNA sequence.
That the vector can be transformed into a bacterium.
Only replicate the gD gene in a host or other single cell system
Have the ability to induce its expression
You. Introduce the obtained recombinant DNA molecule into host cells
And gD or a part thereof or a variant thereof by the host cell
Production of molecules becomes possible. The protein produced is
HSV type 1 (HSV-
Against both 1) and type 2 (HSV-2) infections
Modified for use as immunogens in certain vaccines. [0003] [Prior art] 2.BACKGROUND OF THE INVENTION 2.1.Recombinant DNA technology and gene expression Recombinant DNA technology uses a specific DNA sequence as a DNA vector
That can be inserted into a host cell and replicated in a host cell
This is for producing a recombinant DNA molecule. Generally, insert
DNA sequence is foreign to the recipient DNA vector
It is. That is, the inserted DNA sequence and the DNA vector
Are derived from organisms that do not exchange genetic information in nature.
Or the inserted DNA sequence is totally or
Is partially synthesized. Recently, recombinant DNA
Several general methods have been developed to enable the construction of molecules
Was. For example, U.S. Pat. No. 4,237,224 (Co
hen & Boyer), restriction enzymes and ligation
Using a method known as concatenation,
It describes the production of recombinant plasmids. That
Later, these recombinant plasmids were transformed into
It is introduced and replicated in spores. US Patent No. 4,23
General availability of the technology described in 7,224
Therefore, this patent is incorporated herein by reference.
You. [0004] Introduction of recombinant DNA molecules into unicellular organisms
Another method is rice by Collins and Hohn.
It is described in National Patent No. 4,304,863.
Is incorporated here as a reference. This method uses bacteria
Packaging / Transduction Using Ophage Vectors
It uses a system. How to use for construction
Nevertheless, the recombinant DNA molecule should be compatible with the host cell.
Yes, that is, it should be capable of autonomous replication in host cells
is there. The recombinant DNA molecule may also be
Markers that allow selection of transformed host cells
Should have function. In addition, proper duplication, transcription and
All of the translation signals are correctly placed on the plasmid
In some cases, the foreign gene is present in the transformed cell and its progeny.
Will be properly expressed in All viruses that infect eukaryotic cells
Herpes simplex virus is eukaryotic, as is characteristic of
Requires a host cell line and replicates its genome in it
Therefore, the viral gene is expressed and its progeny are produced. true
Signals and regulatory elements for DNA replication in nuclear cells
Element, gene expression and virus assembly are performed in prokaryotic cells
Different from the one. It occurs naturally only in eukaryotic cells.
Try to express the expressed gene in prokaryotic host cells
Is very important when [0006] These different gene signals and pro
The sexing process involves many levels of gene expression (eg,
DNA transcription and messenger RNA translation)
You. DNA transcription directs RNA polymerase binding
Promoters of DNA sequences that facilitate transcription.
Is dependent on the existence of Eukaryotic promoter
The DNA sequence differs from that of a prokaryotic promoter.
In addition, eukaryotic promoters and associated genes
Signals are those that are not recognized in prokaryotic systems.
Or cannot function. Similarly, messenger RNs in prokaryotes
Translation of A (mRNA) is performed in a suitable source different from eukaryotes.
It depends on the presence of a nuclear signal. M in prokaryotes
Efficient translation of RNA requires Shine Da on the mRNA.
Lugarno (Shine Dalgarno; SD) sequence
Requires a ribosome binding site called This array is
short nucleotide sequence of mRNA
A start codon encoding the amino-terminal methionine (AU
G). SD sequence is 16S rRNA
(Ribosomal RNA)
To form a double helix with rRNA,
MRNA by enabling the correct positioning of
Promotes binding to ribosomes. Gene expression
For a discussion on what to do, Roberts
and Lauer, Methodsin Enzym
org. 68: 473 (1979).
No. [0008] The appropriate signal is inserted and
Even after being attached, many factors are eukaryotic in prokaryotes
Complicates the expression of biological genes. Book of these factors
Immediate understanding of gender and mechanism of action
Missing. One such factor is Escherichia coli (Esche
(Richia coli) and other bacteria
The presence of a neutral protein hydrolysis system. This protein
Degradation systems are "abnormal", such as eukaryotic proteins.
That is, it seems to selectively destroy foreign proteins.
You. Therefore, eukaryotic proteins expressed in bacteria
By developing measures to protect the protein from hydrolysis.
A wide variety of uses will be possible. One strategy is true
The eukaryotic sequence is combined with the prokaryotic gene in phase.
In other words, by linking with the correct reading frame,
Protein products (prokaryotic amino acid sequence and exogenous
Tans that are hybrids with eukaryotic amino acid sequences
To construct a hybrid gene that can obtain
It is. [0009] The construction of hybrid genes has been described by many eukaryotes.
Biological proteins (eg, somatostatin, rat
Loinsulin, growth hormone and egg albumin-like
For the molecular cloning of the gene encoding
This was the method used. In addition, egg albumin and
And β-globin, a prokaryotic promoter
(Guarente)
Et al., Cell 20: 543 (1980)). Guar
The ent. et al. system uses a lac promoter containing an SD sequence.
At different distances before the ATG of the fusion gene.
And The molecular class of some eukaryotic genes
Roning and expression were achieved, but this was due to gD genetic
This has not been done for children. Also prokaryotic
Expression of foreign or eukaryotic genes in a host cell
It is not in a state of technology that can be done on a daily basis. 2.2.Vaccines There are three basic ways to protect and treat viral infections:
There is a way. That is, (1) elicit an active immune response
Vaccines, (2) chemotherapeutic agents that suppress viral replication,
And (3) drugs that induce the synthesis of interferon
is there. Use all three methods to prevent infection
But can be more effective when applied early in the infection
is there. Vaccination, or active immunization,
After starting, it usually has no effect. Vaccines generally destroy immunological properties
Prepared by detoxifying the virus without the need
Is done. Traditionally, this has been used for inactivated vaccines.
To inactivate the virus (ie, formaldehyde)
Will by treatment with various chemicals such as hide
"Killing" a live vaccine (attenuated vaccine)
Non-toxic or attenuated (modified) for
Achieved by selecting a virus. Attenuation
Can bring the virus to an unusual state (different animal hosts and
Cell culture), or
A large number of kina virus inoculants were passaged and lost their virulence
To show no or only a few symptoms
Obtained by selecting mutants that do not. Veterinary medicine
A small number of vaccines still used in the field of
Completely poison injected at sites where growth is of little consequence
It consists of a powerful infectious virus. This way
Is considered too dangerous for human use. Attenuated viruses used for live vaccines are:
It is generally an excellent immunogen. Because the attenuated virus
Because they grow in the host and elicit long-lasting immunity
It is. Attenuated vaccines contain all viral antigens (wi
Humoral for both surface and internal antigens
Of antibodies. However, the use of live vaccines
Have raised a number of issues, including virus
Weak attenuation, genetic instability of the virus, vaccine vaccine
By an external virus in the cell culture used to grow
Contamination, and finally, vaccine instability (eg,
Stability). Inactivated vaccine (non-proliferating "dead")
Alternatively, use an inactivated virus)
To avoid the difficulties associated with using
Do not grow in the immunized animal and are usually
Only antibodies against the components result. But the inactivation
The main difficulty with chins is that they supply the required amount of the relevant antigen.
To produce enough virus to produce Inactivated wa
Another difficulty with the use of Kuching is that the achieved immunity is short.
Often require additional immunity,
Is altered by chemical treatment of inactivation because it is immunogenic
Weakens or neutralizes viral infections
To induce less effective antibodies, and
Kuching often does not induce satisfactory levels of IgA
That is. [0014] Subunit vaccines are
With the decahedral virus capsid protein or coat
Viral peplomers (glycoprotein spikes)
Sometimes contain only necessary and appropriate immunogenic substances.
You. Subunit vaccines are highly purified
Isolate relevant subunits from the
Or by synthesizing related polypeptides.
Can be The main advantages of subunit vaccines are:
Elimination of viral-derived genetic material and host or dona
Elimination of interfering substances derived from However, at present
Subunit Wakuchi using these methods
The production of olefins is too expensive for widely used commercial applications. set
Recombinant DNA technology has many applications in the production of subunit vaccines.
To provide an immunogenic portion of the virus
Is the molecular class of the viral gene that encodes the protein.
Roning and expression in host cells are subunit
Producing enough immunogen for use with Kuching
Can be. [0015] Vaccines often contain various adjuvants.
Administered as an emulsion containing Adjuvant is free
Uses less antigen than when only the epigen is administered
And achieves high levels of sustained immunity at low doses
To help you. The mechanism of action of adjuvants is
Complex and not fully understood. However, that
It stimulates phagocytosis of the reticuloendothelial system, stimulates other activities, delays antigen
May include released and degraded. Adjuvant
For example, Freund's adjuvant (complete or non-adjuvant)
Complete), adjuvant 65 (falling oil, mannidomono)
Contains latet and aluminum monostearate
), And aluminum hydroxide and aluminum phosphate
Or there is a mineral gel like alum. Freund
Adjuvants are vaccines for humans and food animals
No longer used. Because it is non-metabolic
It contains mineral oil and may be a carcinogen.
You. However, mineral gels are widely used in commercial veterinary vaccines
It has been. 2.3.Herpesviruses Herpesviruses (Herpet viridae: Herper
toviridae) is a large DNA virus,
By establishing a potential infection that can last for the life of the host
Famous. After a primary infection that does not appear on the table, the virus
Some suspected stimuli, such as irradiation or immunosuppression
Remain stationary until reactivated by any of
Exist. The state of activity, the acute form of infection, is
Characterized by infection or lytic infection. That is,
Irus replicates, hijacks host cell machinery, and becomes infectious
Make mature virions and cause cell death
You. However, in the latent state, the virus is
Lurking in the passage. Infectious virus is produced during the incubation
Little evidence. [0018] Most primary infections occur in infancy and are superficial.
It does not appear. Infection can be due to mucosal virus inoculation or
Caused by irrus entering the skin from a ruptured epidermis
You. Therefore, the infectious virus is transmitted by close contact
Can be done. Once acquired, HSV is a lifelong body
And the patient is asymptomatic or
For example, lip sores (usually "febrile" or "cold
A disorder on the lips called "cold sores"
Harm), gingival stomatitis (mouth and gums covered with vesicles,
Ulcers), pharyngitis, tonsillitis, keratoconjunctivitis
(Progress to dendritic ulcer due to keratitis or inflammation of the cornea of the eye
And eventually leads to corneal scarring and blindness)
Many clinical cases such as mucocutaneous diseases including genital herpes
Vulnerable to recurrent attacks that cause symptoms. Rarely, H
SV infections include encephalitis, herpetic eczema, traumatic herpes, hepatitis and
May cause neonatal herpes. During active outbreaks, the infectious virus can
From or around the site, for example, in the case of lip rash
Can be isolated from saliva. These lesions
The moon tends to occur on the same part of the body of a particular individual
Overexposure to the pituitary or adrenal gland
Lemon, allergic reaction, or classically fever
Caused by many stimuli. Of these occurrences
While the patient is releasing the virus, others
Can transmit infectious viruses to During the latent period of the infection, the researchers suggested that the lesion
Virus is retained at sites other than those occurring after
Prove that. During this stationary phase, the virus
Localized in nodal neurons (in the case of facial lesions,
Usually associated with the trigeminal ganglion), isolated from normal disease
It is not possible. Most children rapidly get out of primary infection
Recovers, but sometimes disseminated, characterized by hepatitis
Neonatal herpes infection is to beat newborns
You. The most severe infections are more
Infants usually have a baby in the birth canal at birth from the infected mother
Acquired when you encounter herpes. Vulva in women and children
Vaginitis and penis infection caused by HSV-2
It was previously thought that HSV-1
Or HSV-2 can be the cause,
In addition, herpes sexually transmitted infections in women are associated with cervical cancer.
ing. Herpes infections are frequent in today's society.
ing. Currently, there is no cure for HSV infection.
Recent treatments use compounds that inhibit DNA synthesis
It is. Of course, these compounds are
Inhibits not only synthesis but also DNA synthesis in dividing cells
It is non-selective in that it controls. In addition, these compounds
The object is only effective during active infections, and
The effect of the period has not been demonstrated. Herpes virus is a eukaryotic virus.
Yes, size 80 × 10⁶~ 150 × 10⁶Dalton's
Includes a range of linear double-stranded DNA genomes. Each ui
Ruth particles are composed of icosahedral nucleocapsids and
Separated from host cell lipid bilayer during budding and budding
Has a membrane envelope formed by Virus
The envelope is partially present in the membrane, but the membrane envelope
Many virus-specific proteins protruding outward from the pump
It is a lipid bilayer containing dentin. During the primary infection, the membrane
Velocity is an efficient way for virus particles to enter cells
Is necessary for Viral tan outside the lipid bilayer
Antibodies that specifically bind to protein neutralize viral infection
Have the ability to Most for virus entry into cells
Important viral proteins are glycoproteins (sugar molecules are linked).
Protein molecules). Herpes simplex
Loose type-1 (HSV-1) and type-2 (HSV
V-2) are at least four antigenically distinct from each other
Make different glycoproteins. HSV-1 and HSV
Some of these proteins from -2
Has a tope (ie, antigenic or antibody binding site)
You. One example of such a protein is 49,000-5
It is a glycoprotein of 8,000 daltons and is called gD
It is. HSV-1 A polyvalent antiserum against gD is HSV-
Ability to neutralize both HSV-1 and HSV-2 infections
(Norrild, 1979, Current Top
ics in MiCrobiol. and Immu
nol. 19:67). [0023] [Problems to be solved by the invention] 3.Summary of the Invention Cloning of HSV glycoprotein gene in unicellular host organism
Methods and compositions for cleaning and expression are provided
Is done. In addition, these novel unicellular organisms are cultured to produce H
Method for producing SV gene product and gene product
A method for purification is described. Recombination D described here
The gD-related protein produced by NA technology is H
Vaccines to protect against SV-1 and HSV-2 infection
It can be formulated for use as an immunogen for a protein. The gD gene of HSV-1 (HSV-1 package)
Isolated from ton strains) were
By mapping mRNA, in the viral genome
Identified. Once the gene is in the specific segment of DNA
Once localized on the
And the amino acid sequence of the gD protein is deduced.
Was. The gD gene of HSV-2 (HSV-2G
Isolated from the HSV-1 genome).
By analogy with the position determination of the
Analysis identified in the viral genome. So
Followed by the isolated gD gene or gene fragment
(Type 1 or Type 2) in a plasmid vector
Recombination that acts as a biologically functional replication unit by inserting
A plasmid was prepared. These recombinant plasmids
Is used to transform the gD gene during the transformation of compatible host cells.
It was constructed to facilitate production and expression. In addition,
These plasmids are used to transform actively expressing the gD gene.
Provides one-step identification of a commuting microorganism. Finally, expressed
Gene product isolation method and vaccine formulation
Is described. [0026] [Means for Solving the Problems] 4.Description of the drawings The present invention will be described in detail with reference to the following detailed description of the present invention.
By reference to example embodiments and accompanying drawings
Will be more fully understood. FIG. 1a shows the HSV-1 genome.
Indicates the HSV-1 gD gene (hereinafter referred to as the gD-1 gene).
Indicates the position of a short unique area (Us)
You. FIG. 1b shows the lambda gtWES :: EcoR.
HSV-1 EcoRI-H fragment insert of IH
3 shows a restriction map. Show only restriction sites relevant to the discussion
I have. Restriction enzyme recognition sequences are indicated by the following abbreviations: Bam
HI (Ba4-Ba8); BglII (Bg2); Bs
tEII (Bs); EcoRI (RI); HindII
I (H3); KpnI (Kp); PvuII (Pv);
SacI (Sc); SalI (Sa); and XhoI
(Xh). FIG. 1c shows lambda gtW to pBR322.
ES: BamHI-8 to BamHI of EcoRI-H
Construction of pRWF6 including insertion of fragments up to -7
Is shown. FIG. 1d shows HSV-1 Sa of pSC30-4.
Figure 3 shows a restriction map of the cI DNA fragment insert. rod
The line (expanded area) is the gD-1 mRNA coding sequence.
Indicate localization and location. FIG. 2 shows the sequence for determining the gD-1 gene sequence.
Abbreviations and restriction maps are shown. The code area is indicated by a bar (enlarged area).
Area), and the non-coding region is indicated by a single line (thin line).
Is shown. Isolation, labeling and fragmentation of fragments
Indicates the restriction endonuclease cleavage site used for the next digestion.
It is. Horizontal arrows indicate regions of the sequenced DNA
Shown above the restriction map, the non-coding strand sequence was determined.
The template strand sequence was determined for those below the restriction map.
Indicates that it was done. FIG. 3 shows the nucleotide sequence of the gD-1 gene.
Sequence and deduced amino acid sequence of gD-1 protein
Is shown. Relevant restriction sites are indicated and gD-1
Numbered vertical arrow at the aminocode end of the
Is the gD-transfer to pJS413 in the following recombinant plasmid:
Indicate the ligation site at the end of the 1 amino code: pEH51, p
EH60, pEH62, pEH66, pEH71, pE
H73 and pEH73 (these are the remainder of the gD-1 sequence)
To its natural TAG); and pEH4-
2, pEH82, pEH3-25 and pEH90-N
Series (these are Cro / gD-1 / β-galactosidase)
Encoding a fusion protein). gD-1 carboxy
The numbered vertical arrows at the ends of the cord are
(1) The following recombinant plasmids: pEH4-2, pEH8
2. β-galactosidase of pJS413 in pEH3-25
The enzyme (z gene); or (2) the plasmid
g to pHK414 z gene in pEH90-N series
The D-1 carboxy code terminal ligation site is shown. FIG. 4 (not drawn to scale)
Part of gD-1 gene and E. coli expression vector pJ
Of recombinant plasmid pEH25 derived from S413
Show construction. The recombinant plasmid pEH25 is used for the expression vector.
Linked to a "leader" sequence (cro) derived from
About 87% of the gD-1 coding sequence
This leads to the production of HSV-1 gD-related proteins. FIG. 5 shows that Cro / gD-1 in pEH25
1 shows the DNA sequence and the deduced amino acid sequence of the junction. Figure
6 shows the competition present in the lysate of HSV-infected Hela cells.
Immunization of pEH25gD product by adding antigen
4 shows inhibition of sedimentation. Figure 7 (not drawn at a fixed ratio
Is the gD-1 expression plus from pEH25.
The construction of mid pEH4-2 is shown, in this plasmid:
205 nucleotides at the 3 'end of the gD-1 coding sequence
(69 amino acids) have been deleted and the β-galacto
Approximately 3,000 nucleonucleotides encoding the sidase protein
Replaced by a tide. This recombinant plasmid was gD-1
E. coli-related polypeptides (immediately
Cro and β-galactosidase) were fused
"Sandwich" proteins (ie fusion proteins)
Bring production. FIG. 8 (not drawn to scale)
Many gD-1 expression plasmids pEH50 + x (each
It contains the variable portion at the amino coding end of the gD gene)
The method for producing is described below. Figure 9 (drawn at a fixed ratio
No) is gD-1 fused to β-galactosidase.
Depending on the host cell in which the protein was transformed with pEH82.
GD-1 gene in pEH4-2 as expressed
Fig. 4 shows a method for reconstructing the amino acid end of a child. FIG. 10 (not drawn to scale)
Is gD derived from pEH3-25 and pHK414
1 shows the construction of the expression plasmid pEH90-10am.
You. The recombinant plasmid pEH90-10am is
Β in transformants of E. coli containing suppressor tRNA
-Fused or not fused to galactosidase
GD-1. FIG. 11a shows the HSV-2 genome.
GD gene of HSV-2 (hereinafter referred to as gD-2 gene).
BglII file in a short unique region (Us) with
Indicates the location of the fragment. B that defines the L fragment
The glII restriction site is indicated as Bg. FIG. 11b shows that p
Restriction map of HV1 BglII L fragment insert
Is shown. Of relevant restriction endonuclease recognition sites
Only show. Restriction enzyme recognition sites are indicated by the following abbreviations: Bam
HI (Ba); BglII (Bg); ClaI (C);
HindIII (H3); PvuII (Pv); Sal
I (Sa); SacI (Sc); SphI (Sp);
And XhoI (Xh). The bar (enlarged area) is gD
-2 indicates the location and location of the mRNA coding sequence. FIG. 11c shows HSV-2 Xh at pHV2.
Figure 3 shows a restriction map of the oI DNA fragment insert. Figure
12 shows the nucleotide sequence of gD-2 gene and gD
2 shows the predicted amino acid sequence of the protein. is related to
Restriction sites are indicated and the amino acid of the gD-2 gene
A numbered vertical arrow at the end of the
Rasmid pHV5 (all carboxy code ends of gD-2)
GD-2 to pJS413 in pHV6
Shows the joining site at the amino code end. BamHI site
Is the gD-2 to z gene of pHK414 in pHV6.
It is a linking site at the end of the carboxy code. FIG. 13 shows estimated amounts of gD-1 and gD-2.
4 shows a comparison of the noic acid sequences. Amino acid residues are represented by single letter code
I have. Amino acids homologous in gD-1 and gD-2
Noic acid residues are indicated by a dash in the gD-2 sequence. g
The D-2 sequence is one amino acid residue shorter than the gD-1 sequence
No. The "missing" amino acid in gD-2 is marked with an asterisk.
(*) Is attached. FIG. 14 (not drawn to scale)
Is derived from part of the gD-2 gene and pJS413
2 shows the construction of the constructed recombinant plasmid pHV5. Recombinant
Plasmid pHV5 is derived from the expression vector "Leader".
-"Of the gD-2 coding sequence linked to the sequence (cro).
HSV-2gD-related tans encoded by about 90%
This leads to the production of Parkin. FIG. 15 shows gD- derived from pHV5.
2 shows the construction of the expression plasmid pHV6.
259 nucleotides at the 3 'end of the gD-2 coding sequence.
The otide (86 amino acids) was deleted and the β-
Expression plasmid encoding the lactosidase protein
Approximately 3,000 additional nucleotides from pHK414
Is replaced. This recombinant plasmid, pHV6, contains gD-2
E. coli-related polypeptides (immediately
Cro and β-galactosidase) were fused
"Sandwich" protein (ie fusion protein)
Bring production. 5.Description of the invention The present invention relates to vaccine formulations using recombinant DNA technology.
HSV tamper that can be used as an immunogen in a plant
Producing polypeptides associated with proteins.
More particularly, it describes the production of gD-related proteins.
Multivalent antiserum against HSV-1gD is composed of HSV-1 and HSV.
Pure gD because of its ability to neutralize both V-2
The protein or an antigenically important part thereof is HSV-
Recipients from both primary 1 and HSV-2 infections
In subunit vaccines that can effectively protect
May be usable. The recombinant plasmids constructed as described herein
Rasmid is a stable gD that resists degradation in host cells.
Host cell production of proteins that are related polypeptides
Sprinkle. These plasmids are used for immunological antigen determination of gD.
GD-related proteins or fusion proteins containing
Allows you to produce in quantity. However, here
The described DNA composition is limited to the production of gD-related proteins.
Not defined, for polypeptides related to the HSV protein
Can be used for production. A variety of immunogens and vaccines
It will be readily understood that pharmaceutical formulations can be produced. The method of the present invention involves the following steps for illustrative purposes:
Can be divided. That is, (1) HSV glycoprotein
Identification and isolation of genes or gene fragments,
(2) Recombinant DNA molecule used for transformation of single cell organism
To produce a cloning expression vector
Insertion of a gene or gene fragment, (3)
Transformed single cell capable of replicating and expressing genes
(4) Identification and propagation of the gene product
And purification, and (5) its ability to elicit the production of neutralizing antibodies
Determination of the immunocompetence of a gene product by assessing its power. Light
For the purpose of confirmation, all methods are discussed with respect to the gD gene.
However, using the same technique as well, HSV sugar
Producing polypeptides related to proteins as well
Can be. 5.1.Identification of HSV glycoprotein gene
And isolation HSV glycoprotein (gD) is HSV type 1 or
It can be obtained from type 2 strains. gD gene (also
First, the HSV DNA is isolated.
Producing a DNA fragment of HSV,
And a fragment containing the glycoprotein gene sequence
Including setting. Prior to identification, the HSV DNA fragment
The vector is usually ligated into a cloning vector and the vector
The vector is used to transform host cells. This allows multiplexing
It is possible to generate copy number HSV DNA fragments
Supply for analysis and identification operations.
You. HSV DNA is (1) infected with virus
DNA from viruses purified from isolated eukaryotic cells
By direct isolation or (2) gD gene
Bacteriophage containing part of the viral genome containing
Alternatively, it can be obtained from a plasmid. HSV D
To make NA fragments, the DNA is
Cleavage by restriction enzymes at the position or in the presence of manganese
DNA is fragmented using DNase or DNase
A is physically crushed, for example, by sonication. That
Later, the linear DNA fragment is agarose or poly
Acrylamide gel electrophoresis, column chromatography
Standard technology, including but not limited to
Can be separated by Any restriction enzyme or combination of restriction enzymes
Using the HSV DNA fragment containing the gD sequence
Can be produced, provided that the enzyme is gD
It does not destroy the immunity of the offspring. For example, tongue
The antigenic site of protein can consist of about 7-14 amino acids
You. Thus, gD-sized proteins have many different
Can have antigenic sites, overlapping sequences, secondary structures, and
Acetylation, glycosylation, phosphorylation, etc.
Considering the aging process, there are probably thousands of antigen sites
I think that the. Therefore, many partial gD gene sequences are
Let's code the site. As a result, a large set of restriction enzymes
Can be used to create DNA fragments.
The DNA fragment is inserted into an appropriate vector.
GD-specific amino acids with different antigenic determinants
No acid sequences can be produced in unicellular organisms
U. Those containing the HSV DNA fragment
Transformation of host cells with recombinant DNA molecules
A large number of viral DNAs.
Rus DNA identifies a fragment containing the gD gene sequence
Can be analyzed to H to cloning vector
Insertion of the SV DNA restriction fragment is performed by cloning
Easy to reach if the vector has complementary cohesive ends
Is done. However, the phase used to fragment HSV DNA was
If there are no complementary restriction sites in the cloning vector
Both HSV DNA and cloning vector
The ends can be modified. Such modifications include
Digesting the DNA end of the main strand to blunt ends,
Repair single-stranded ends to allow blunt-end ligation
And are included. Also, a nucleotide sequence at the DNA end
Create desired ends by linking (linker)
The linker to be linked can have a restriction enzyme recognition sequence.
Specific chemically synthesized oligonucleotides encoding
May be included. Vector cleaved according to other methods
And HSV DNA fragment are homopolymerized
(Morrow, 1979,
Methods in Enzymology 68: 3
checking). Specific DNA fragment containing gD gene
Can be identified in a number of ways. First, ui
The sequence of the lus DNA fragment was determined (Maxam
& Gilbert, 1980, Methods in
Enzymology 65: 499), then estimated
Amino acid sequence compared to amino acid sequence of gD protein
The fragment containing the gD gene sequence is
It is possible to specify. Second, a file containing the gD gene
Fragments can be identified by mRNA selection. HSV
  DNA fragments are
Used to isolate complementary mRNA. Isolated
Analysis of in vitro translation products of isolated mRNA
And thus identify the gD gene sequence
The complementary HSV DNA fragment containing is identified. Most
Later, gD-specific mRNA was isolated from HSV infected cells.
Immobilized monoclonal to gD
Can be selected by allowing the antibody to adsorb. Radioactive
The labeled gD mRNA or cDNA is (adsorbed
Using selected mRNA as template
And can be synthesized. Then radioactively labeled
Using mRNA or cDNA as a probe, gD
Identifying an HSV DNA fragment containing the sequence
(Southern, 1975, j. Mol.
Biol. 98: 503; Alwine et al., 1979,
Methods in Enzymology 68: 2
20). Alternative methods for isolating the gD gene include, but are not limited to:
Not, but chemically synthesize the gene sequence itself
(Provided that the sequence is known)
) Or to the mRNA encoding the gD gene
Producing a cDNA (complementary DNA) is included. This
To achieve this, mRNA specific for gD
Isolate from Rus infected cells. Then, by standard technology
Converting the isolated mRNA to a cDNA copy, and
Insert directly into cloning vector. An isolated gene, cDNA or synthetic DN
Transforming host cells with a recombinant DNA molecule containing the A sequence
This allows the generation of high copy number genes.
You. Thus, transformants are grown and recombinant DNA molecules
From the transformant, and the isolated recombinant DNA
By recovering the inserted gene from
It can be obtained in microgram quantities. Also micro
Gram quantities of gD gene can be obtained from gD gene sources, for example,
If the herpes simplex virus or
Bacteria containing the viral gene in the recombinant plasmid
Alternatively, it can be obtained from phage. Virus or
Means that the plasmid DNA is used to confirm the location of the gene.
Isolated, fragmented, and sized in the same way
Fractionation by 5.2.HSV glycoprotein gene clone
Into the expression vector Once isolated, the gD gene or gene flag
Into the appropriate expression vector, i.e.
Into vectors containing elements necessary for transcription and translation of the gene
Insert (Roberts & Lauer, 197)
9, Methods in Enzymology, 6
8: 473). gD gene (or part of the gene)
For efficient expression of
Must be present in the reactor. RNA polymerase
Ze is usually linked to a promoter and
Of a group of genes and regulatory elements (called operons)
Start copying. The promoter has its "strength",
The ability to promote photography varies. Molecular cloning
For high level transcription, and thus high level relics
Use strong promoters to obtain gene expression
Is desirable. Depending on the host cell system used, many suitable
One of the various promoters can be used. An example
For example, when cloning into an E. coli host cell line,
From a bacterium, its bacteriophage or plasmid
An isolated promoter can be used (eg, la
c promoter). Additionally, recombinant DNA or other synthetic
Using an E. coli promoter made by DNA technology
Thus, transcription of the inserted gene may be promoted. Than
Specifically, P of coliphage lambda_RAnd P_Lpromotion
Mediates high-level transcription of adjacent DNA segments
You. In addition, the recA promoter from E. coli
This results in high levels of gene transcription of the fragment. Efficient Retention in Prokaryotic and Eukaryotic Cells
Specific initiation signals are also required for gene transcription and translation
Become. These transcription and translation initiation signals are
Specific messenger RNA and synthesized protein
The "strength" varies as measured by quality.
DNA expression vectors containing a promoter may further comprise a species.
Of various “strong” transcription and / or translation initiation signals
Any combination may be included. Thus, the host cell
The combination of SD-ATG that can be utilized by ribosomes
Used. Such combinations include the coliphage lam
From the cro or N genes of da or E. coli
S from tryptophan E, D, C, B or A gene
There are D-ATG combinations, but not limited to these. Change
Made by recombinant DNA or other synthetic techniques
Combinations of SD-ATG can also be used. [0052] Promoter and other regulatory elements in the vector
To ligate to a specific site in
Using the method described above to insert into a vector
it can. Thus, the gD gene (or part thereof)
Specific for promoters and regulatory elements of expression vectors
The gD gene sequence is ligated in place.
Correct translation reading frame for the ATG sequence
(Ie, in phase).
Alternatively, gD ATG or synthetic ATG may be used.
No. The resulting recombinant DNA molecule is then (vector
(Depending on the host cell system)
Transfection into a suitable host cell
You. Transformants should be of the appropriate genetic origin normally present in the vector.
Selected based on the expression of the offspring marker, for example, pBR3
22 resistance to ampicillin or tetracycline
Thymidine kinase in sexual or eukaryotic host systems
There is activity. Expression of such marker proteins
Indicates that the recombinant DNA molecule is intact and replicating.
Become a mark. Expression vector (normally containing marker function)
)) Include the following vectors or derivatives thereof:
: SV40 and adenovirus vector, yeast vector
Bacteriophage vectors (eg, lambda g
t-WES-Lambda B, Charon 28, Char
on 4A, lambda gt-1-lambda Bc, lambda gt
-1-Lambda B, M13mp7, M13mp8, M13
mp9) or a plasmid DNA vector (eg, p
BR322, pAC105, pVA51, pACY17
7, pKH47, pACYC184, pUB110, p
MB9, pBR325, ColE1, pSC101, p
BR313, pML21, RSF2124, pCR1
Or RP4). The expression vector used in the examples of the present invention is
U.S. Patent Application No. 449,187 (December 1, 1982)
3rd application) and refer to the teaching content
Here. In addition, host cell strains may be
Outside, those that suppress the action of the promoter are selected.
You. In this method, more than 95% of the vector promoter
Can be suppressed in non-induced cells. is there
The operon provides efficient transcription and translation of the inserted DNA.
In order to add certain inducers,
For example, the lac operon is lactose or IPTG
(I.e., isopropylthio-β-D-galactoside)
Induced by addition. Various other operons, such as
trp, pro, etc. are under different controls. trp operation
Ron is used when tryptophan is not present in the growth medium.
Induced and lambda P_LPromoter gives a sense of temperature
Lambda repressor protein (eg CI85)
7) induced by an increase in temperature in the host cell containing
You. Thus, the genetically engineered gD protein
Quality expression can be controlled. This means that
If the protein product of the gene is lethal to the host cell
It is important when: In such cases, the foreign gene is duplicated.
But not expressed during growth of transformants.
No. After the cells have reached the appropriate density in the growth medium,
The motor can be induced for protein production. The plasmid constructed as described above (immediately
Terminating at the native gD translation termination signal, and
AT provided by a gD gene or a synthetic sequence
Induces expression of gD-related proteins, starting with G
G) produced by cells transformed with
D-related proteins are hereinafter referred to as unfused gD proteins.
Or unfused gD-related protein. 5.3.Production of fusion proteins Maximize the level of gene expression by a particular transformant
For this purpose, the gene of interest must be
Desirable to link to genes encoding other proteins
Good. Functions that host cell proteins are inherently detectable
In the case of including, an additional advantage is obtained. Linked remains
Expression of the gene allows the fusion protein product (hereinafter referred to as gD fusion
Protein, which is a large molecule
Identification can be based on quantity and detectable function. example
Production of gD / β-galactosidase fusion protein
Is used for cloning and expression of gD in E. coli hosts.
It offers several advantages. First, Miller
Method (Experiments in Molecu)
lar Genetics, Cold Spring H
arbor Press, New York, N.W. Y.
1972, pp. 47-55 and 352-355)
Therefore, a colorimetric assay specific for β-galactosidase activity
Protein induced by vector using assay
It is possible to estimate the level of quality production (and therefore expression)
It works. Second, the production of the fusion protein
Simplify the identification and isolation of quality products. E. coli transformation
The unfused gD protein produced by the body is gD / β-
Smaller than the galactosidase fusion protein,
Analysis by SDS-polyacrylamide gel electrophoresis
Co-migrate with several other host proteins. I
However, the fusion protein produced has its unique
Due to the large molecular weight, SDS-polyacrylamide
Easy detection and detection by SDS PAGE
Can be identified. The present invention relates to a β-galactosidase fusion tan
It is not limited to production of parka. Eukaryote or prokaryote
Any gene derived from an organism can be replaced with the gD gene (or HSV tag).
Reading frame (in
  phase) and ligated with β-galactosidase.
Similar advantages as the combined protein product can be obtained. So
Examples of galactokinase; trpD, E or
Pill; pili gene; and eukaryotic inheritance
Children, for example, thymidine kinase, β-globin, SV-
40T-antigen or Rous sarcoma virus transforming inheritance
Children can be mentioned, but are not limited to these. Construction of gene encoding fusion protein
To do this, two genes must be translated
Is maintained and not interrupted by the stop signal
As such, they need to be joined within their coding sequence.
Also, as described above, the host cell
If the strain suppresses the effect, the fusion protein
Produced only when responding to guidance. 5.4.Gene product identification Once a transformant containing the correct DNA construct has been identified
The recombinant DNA gD gene product immunoreactivity and
And antigenicity analysis is required. The host cell is naturally present
GD gene product in the same pattern as HSV-gD
In the absence of the ability to cosylate, the gD gene product is native
GD glycoprotein. Hence immunology
Analysis is particularly important for gD gene products. What
If so, the ultimate goal is to produce gD-related
The use of protein in vaccine preparations.
You. Analysis of immunoreactivity was based on gD glycotan of HSV infected cells.
Easiest to do with antiserum to protein
But the antigenicity in response to immunization with the gD gene product
Neutralizes antiserum titers and HSV infection in resulting test animals
Can be evaluated by measuring the ability of the antiserum.
it can. In analyzing the immunoreactivity, the total
Preparation of multivalent antibodies directed against ils or especially gD
A variety of antisera can be utilized, including products.
Recognizes only one antigenic site on gD protein molecule
By using a monoclonal antibody,
The isomerism is obtained. Many monoclonal antibodies against gD
And some of them are g of HSV-1 and HSV-2.
Not only specifically immunoprecipitates the D gene product,
It also neutralizes the infectivity of these viruses. Therefore, the protein described in the present invention
Identification is based on two requirements. First, the gD-related tamper
Protein should only be produced in response to promoter induction.
It is. Second, gD-related proteins are converted to HSV.
Against various polyclonal antibodies or gD
Should be immunoreactive with the monoclonal antibody;
Whether the protein is part of the gene sequence from the entire gene sequence
Or to produce a fusion protein
Resulting from two or more gene sequences
Should be immunoreactive. This reactivity is standard
Immunological techniques, such as immunoprecipitation, immunodiffusion, radiation
Sexual immunity competition, immunoelectrophoresis, or polyacrylamide
Separated by gel electrophoresis and then on nitrocellulose
Detected by immunological detection of proteins transferred to
obtain. 5.5.Purification of gene products gD gene (or any HSV glycoprotein gene)
Are grown in large volumes and the promoter
Proteins produced after induction are not
If the protein is secreted, the culture medium
Isolated from. Protein, ion exchange, affinity
Standard chromatograph containing tea or sizing resin
Fees, centrifugation, or protein
Isolation and purification by any other standard techniques for
Can be Certain fusion proteins are expressed in excess in cells.
Agglomerates form when produced or when produced in solution
Therefore, the present invention provides a method for isolating an aggregate-forming protein.
It is particularly useful for isolating the fusion protein produced in the above.
These fusion proteins are then used in vaccine formulations.
Can be. Purification of the fusion protein (hereinafter, aggregation
Product purification) is the disruption of cell culture by cell disruption.
Aggregate formation from protein mixtures such as lysates
Including extraction, separation and / or purification of proteins
is there. In addition, aggregates have strong hydrogen acceptors and strong hydrogen donors.
Solvents that are both in the body (or each solvent is one of these properties)
By adding a mixed solvent with
And then aggregate-forming proteins in a suitable buffer.
It can be diluted and settled. Suitable protein dissolution
The medium includes urea (about 4-8M), formamide (at least
Also about 80%, volume / volume basis) and guanidine hydrochloride
(About 4-8M), but not limited to these. Aggregate form
Solvent capable of solubilizing the synthesized protein, for example, SDS (D
Sodium decyl sulfate), 70% formic acid
Not suitable for use. Because it was solubilized
Insufficient subsequent sedimentation of aggregate forming proteins
Because I understand. Guanidine hydrochloride and other similar
The reagent is a denaturant, but experiments have shown that this denaturation is not possible.
Rather than reverse, the removal of denaturants (eg, by dialysis)
Or upon reconstitution upon dilution, immunological and / or
Or allows for the reconstitution of biologically active proteins
Turned out to be. One Embodiment of Fusion Protein Isolation Technique
Is outlined as follows (hereinafter referred to as the non-denaturing aggregate purification method).
U). Cell pellet using dry ice / methanol
Freeze quickly, weigh, and weigh 3-4 g of cells
At least 25 ml of buffer solution [e.g.
M Tris-HCl (trishydroxymethylamino meta
Hydrochloride), pH 8.0, 2 mM EDTA (ethylene
Diaminetetraacetic acid) and 200 mM NaCl].
Suspend. That is, the cells are about 100-
Suspended at an estimated concentration of 200 g cells / L. About 160g
/ L or less is preferred. Add the lysozye to this suspension
Was added at a final concentration of about 130 μg / ml and the resulting
Allow the mixture to stand at 4 ° C. for 20 minutes with occasional shaking
You. Nonionic surfactants used to solubilize membranes
Nonidet P40 (NP-40, She
11 registered trademark, polyoxyethylene (9) p-ter
t-octylphenol) at a final concentration of about 0.1%.
And then mix the solution. Next, the suspension is
tron crusher (Brinkman Instrument)
nts, Westbury, N.M. Y. ) Or equivalent
Grind for about 1 minute using a device. The suspension was centrifuged at 8,000 × g for 30 minutes.
Release and pellet in washing buffer [eg 2m
M Tris-HCl (pH 7.2), 1 mM EDTA,
150 mM NaCl and 2% Triton-X100
(Polyoxyethylene (9-10) p-tert-oct
Chilled phenol, nonionic surfactant)]
And crushed with a Polytron crusher
You. The centrifugation, washing and grinding steps remove the pellet.
To remove as much cell debris as possible
Can be repeated. The aggregate pellets can be prepared by adding a denaturing agent.
Can be further processed as follows (hereinafter, transformation
A method for purifying a soluble aggregate). Centrifuge the suspension and discard the supernatant
And remove the pellet to approximately 1/5 volume of 6M
Resuspend in anidine hydrochloride (distilled water). For example, 25
3 g of cells washed with ml of buffer are added at this stage
Should be resuspended in 5 ml of 6 M guanidine hydrochloride solution.
You. It is difficult to resuspend the pellet at this stage.
Sonication or homogenization
It will be necessary to get the liquid. Solution at 22 ° C for 20 minutes
Leave, then centrifuge at 8000 xg for 30 minutes
Except for the supernatant containing the fusion protein aggregate at this point
Get. The fusion protein is about four times the volume of aqueous buffer.
Precipitated from guanidine hydrochloride supernatant by addition of fur
Close. Use almost any aqueous buffer at this stage
Can be used in small amounts of nonionic surfactants [eg:
0.5% NP-40 or Triton X-100]
It would be helpful to add. 30 minutes at 4 ° C
And then centrifuge at 8,000 xg for 10 minutes
You. Discard supernatant and pellet (fusion protein precipitate
) In a suitable volume of a suitable buffer, such as phosphoric acid
Resuspend in buffer solution (PBS). Short-term sonication
Or a homogeneous suspension or homogenization
Will help you get a rally (what agglomerates
The forming protein is insoluble in water). 5.6.Production of unfused gD protein It is desirable to use fusion proteins in vaccine preparations.
I will. However, as explained earlier, non-fusion
The transformant producing gD protein is a gD fusion protein.
A smaller amount of the protein than the transformant producing the protein.
Produce the protein, which is
Even when the sequence is under the control of an inducible promoter
The same is true. Furthermore, it is produced by bacterial transformants.
The obtained non-fused gD protein is compared with the gD fusion protein.
All may be inferior in stability. In another embodiment of the present invention, a host cell
Transformants are co-aggregated for easy purification and fusion.
Produces large amounts of both unfused gD-related proteins
Can be operated as follows. According to this embodiment, g
The recombinant plasmid encoding the D fusion protein is g
D gene sequence and host cell protein gene sequence (eg
For example, the junction with β-galactosidase z gene sequence)
In the nonsense codon sequence, ie, amber (ambe)
r) (TAG), ocher (TAA)
Or chain like opal (TGA)
So that the terminator is located between the two gene sequences
Is changed to This chain terminator is a gD sequence
Translation reading frames for both gene and host cell gene sequences
Must be arranged in phase with the system. This
Such a change is a restriction site located between the two gene sequences
Cleavage of the plasmid at
Or a chain terminator such as an opal
DNA linker sequence to be inserted into the cleavage site of the plasmid
When linked, the chain terminator
Translation reading frame and arranged in phase
This is achieved by being done. These ambers, ochers or opers
Modified plasmid containing the appropriate tRNA suppressor.
When introduced into a host cell, the unfused gD-related protein
As well as the synthesis of both gD fusion proteins. A
Member, ocher or opal-modified plasmid
When used to transform presser cell lines,
Will produce an unfused gD-related protein. Amber, Ocher or Opal Modified Plate
Rasmid introduced into suppressor cell background
There are at least two ways to do this. That is,
(1) Transformants (ie, amber, ocher or o
Host cells transformed with pearl-modified plasmid
And a lysogenic form with the appropriate suppressor tRNA gene
Transfected phage (for example, φ80pSU3
That carry a different supF suppressor)
Can be. Or (2) amber, ocher or
Using an opal-modified plasmid, amber,
Contains ocher or opal suppressor tRNA
The cell line can be transformed. Of such strains
Examples include LE392 (amber mutations supE and s
upF suppressor), YMC (including supF)
And C600 (supE), but are not limited to these.
No. Various amber suppressors tRN in E. coli
As the A gene, supB, supC, supD, s
upE, supF, supG, supL, supM, s
upN, supO, supP, supU, supV
But not limited to these. Various okers in Escherichia coli
Presser tRNA genes include supB, supC,
supG, supL, supM, supN, supO,
supV, but is not limited to these. Various in E. coli
SupK is an opal suppressor tRNA gene of
But not limited to them (Backmann and Lo
w, 1980, Microbiological Re
views 44 (1): 1-56). Amber, Ocher or Opal Modified Plate
Appropriate suppressor transformed by Rasmid
The host cell containing the tRNA gene is a
Produces gD-related proteins as both fusion proteins
(Produced fused gD and non-fused gD
Protein ratio depends on degree of suppression in host cells
). Both proteins are immunoreactive with antisera against HSV
I do. The method for purifying non-denatured aggregates described in Section 5.5
Method is non-denaturingBothUsing an aggregate purification method)
Both the unfused gD and the gD fusion protein are co-purified.
It is. After solubilization, the unfused gD is based on size or charge.
Thus, it can be separated from the gD fusion protein. Consequent
In addition, large amounts of non-fusible
GD-related protein can be easily purified,
Formulated for use as 5.7.Production of vaccines It is an object of the present invention to provide HSV-
1 and / or vaccine for protecting against HSV-2 infection
GD-related proteins that can be used as immunogens for tin
Of HSV glycoprotein-related polypeptides such as
It is to be. Specially produced gD-related protein
With different HSV-1 and / or HSV-2 neutralizing antibodies
If immune response, the gD-related protein
Elicits an immune response that can neutralize related viruses in vivo
Would be expected. Genetically engineered immunity
Vaccines made from epidemics are made from attenuated viruses
Should be safer than regular vaccines. Because
The risk of infection of the recipient is completely eliminated.
You. Also, genetically engineered gD products are passively exempt.
It can be used to make antibodies useful for epidemiological treatment. GD / β-galactosidase fusion protein
While the quality product itself may be useful in vaccine formulations,
It may be necessary to remove the β-galactosidase moiety first.
In addition, rearrangement of the amino code end of the gD gene is
It may be important for the immunogenicity of the protein. When
That means that the amino terminus may contain additional important antigenic sites
That's because. The amino coding end of the gD gene is gD
A DNA sequence encoding the amino terminus of
Linking to an appropriate site in the gD coding region of the molecule
Therefore, it can be reconfigured. All recombinant DNA molecules
Since all necessary expression control elements are retained,
GD-related protein was reconstituted
By cells transformed with a DNA molecule containing the gene
Produced. Finally, the genetically engineered gD-
Related proteins are identified using standard protein isolation techniques.
Or by the aggregate purification method described herein.
Can be isolated and purified from the cells. After that, the final purified product is
Diluted to the appropriate concentration and use the appropriate vaccine adjuvant
Formulated and packaged for use.
You. Suitable adjuvants include surfactants, such as
For example, hexadecylamine, octadecylamine, lysole
Cytin, dimethyldioctadecyl ammonium bromide,
N, N-dioctadecyl-N'-N-bis (2-hydro
Xyethyl-propanediamine), methoxyhexadeci
Luglycerol and pluronic polyols; poly
Anions such as pyran, dextran sulfate, poly
IC, polyacrylic acid, carbopol; peptides, eg
For example, muramyl dipeptide, dimethylglycine, tuft
Shin; oil emulsion; and alum
But not limited to these. Finally, the protein product
Even if it is held in liposomes for vaccine preparation,
Complexes with polysaccharides and other polymers may be formed. The genetically engineered D described herein
NA molecules offer great flexibility in vaccine production.
For example, any part of the gD gene sequence or gD gene
Recombination involving multiple copies of (or part of) in tandem
GD- produced by a transformant containing a DNA molecule
Vaccines can be formulated using related proteins
Wear. The gD gene sequence (or a portion thereof) can be
The fusion protein is linked to the gene encoding the
The quality product may be used in the production of a multivalent vaccine. Furthermore,
gD gene sequence (or a part thereof) can be combined with any combination
In other HSV glycoprotein sequences (or parts thereof)
It could be linked. Finally, the vaccine immunogen
The gD sequence may be reconstituted to enhance sex. An example
For example, a protein product may provide a specific epitope to the immune system.
(Eg, the normally unexposed gD antigenic site is
Alter the gene sequence (as presented to the immune system). Ah
Alternatively, gD fragments encoding the immunosuppressive portion of the protein
Regions of the gene sequence can be deleted. [0076] BEST MODE FOR CARRYING OUT THE INVENTION 6.Example: HSV-1gD According to the method of the present invention, the Us portion of the HSV-1 genome
The DNA fragment (see FIG. 1a) was transferred to the vector pBR3
22 each having a different HSV-1 genome
To make a recombinant plasmid containing the fragment
Therefore, the gD-1 gene was localized and identified. gD-
Plasmids containing one gene can be obtained by the following three techniques (not necessarily
(Not in the order shown). Sand
(1) DNA sequencing, (2) gD-1 specific mR
NA hybridization experiments and (3) recombination
In vivo of mRNA hybridizing to plasmid
tro translation. A plasmid containing the gD-1 gene once
Once identified, DNA sequencing will
Confirm the position of the gD-1 gene end in the plasmid.
With this, the gene was characterized. Code array
GD-1 gene lacking the first 156 nucleotides
DNA fragment from the identified plasmid
Isolated. This DNA fragment is converted into a DNA vector
Linked to pJS413, cloned the gene in E. coli
The pEH25 used for training and expression
did. Gene products isolated from induced transformants
Is a monoclonal antibody against gD-1 and HSV
GD- by immunoprecipitation with a multivalent antibody against
1 as a unique protein. Finally, the gD-1 gene fragment was
PEH2 by restriction endonuclease cleavage at specific sites
5 to thereby terminate the gD-1 coding sequence.
Row (TAG) was deleted. A part of the gD-1 gene and
And plasmid promoters and regulatory elements (eg, S
This DNA fragment containing D-ATG) was
Ligated into a vector containing the lactosidase coding sequence
Was. The obtained plasmid pEH4-2 was prepared using croSD-
Lac promoter and β-ga of plasmid with ATG
GD-1 code flanked by lactosidase gene
Sequence and contains the
To express the fusion protein. Next, this fusion tamper
Protein is isolated from host cell lysates and used as an immunogen.
Formulated for use. In addition, the gD-1 gene
The resulting gD-1 protein was reconstituted with the ends of the minocode.
Quality formulated for injection into animals and preliminary clinical trials
did. The details of each step of the construction are described in the following sections.
You. GD-1 related protein produced here
And fusion proteins are not glycosylated and
Different from the naturally occurring HSV-1 gD glycoprotein
It is. However, the gD-1 related protein is H
Immunization with antibodies against SV-1 and HSV-2 gD
It has reactivity. 6.1.General procedures used for plasmid construction The next segment is used for DNA isolation, enzymatic reactions and ligation reactions.
Describes the general procedures and materials used. 6.1.1.Plasmid DNA isolation The host cell bacterium Escherichia (Escherichia)
E. coli) (Gautier & Bon)
eward, 1980, Molec. Gen. Gene
t. 178: 375), and grown in M-9 broth.
Large amounts (micrograms) of cultured E. coli transformants
Plasmid DNA was isolated (Miller, E.
xperiments in Molecular G
enetics, Cold Spring Harbo
r Press, New York, N.W. Y. , 197
2, p. 431). Plasmids are from Guerry et al.
Method (1973, J. Bacteriol. 116: 10).
63) Isolate from late-log phase cells using a modification of 63).
did. 6.1.2.Conditions for restriction enzyme digestion Restriction enzymes used in the present invention, unless otherwise indicated,
New England, Beverly, MA
  Biolads, Inc. It is obtained from
Enzyme units were 1.0 μg in 50 μl total reaction mixture.
Required to digest lambda DNA for 15 minutes at 37 ° C
Defined as a quantity. Complete digestion with all restriction enzymes is as follows:
Under the following conditions. 1 μg of each DNA is 0.5 unit of enzyme
At 37 ° C for 60 minutes in 20 μl of buffer.
Incubated. Partial digestion depends on the conditions used for complete digestion.
This was done by making the following changes. 1 μg of D
NA for 15 minutes at 37 ° C with 0.1 units of enzyme
Was added. 0.1% sodium dodecyl sulfate (SD
The reaction was stopped by the addition of S). Thus, DN
Reaction conditions so that an average of one cleavage per A molecule occurs
Was adjusted. 6.1.3.Restriction enzyme buffer The buffer used for SacI, SacII or SmaI digestion
Fur was 6.6 mM Tris-HCl (pH 7.4),
6.6 mM MgCl₂And 6.6 mM β-ME
(Β-mercaptoethanol).
BglII, BstEII, EcoRI, HindII
Used for I, NruI, PstI or PvuII digestion
The buffer was 60 mM NaCl, 6.6 mM Tris-
HCl (pH 7.4), 6.6 mM MgCl₂and
From 6.6 mM β-mercaptoethanol (β-ME)
It consisted. BamHI, SalI or XbaI digestion
The buffer used for 150 mM Tris-HCl (pH
7.4), 6.6 mM MgCl₂And 6.6 mM
It consisted of β-ME. 2 or more restriction endonu
Low if the creatase reaction is performed on DNA.
Reaction in a salt buffer
It should be noted that before doing. Two enzymes are the same
If you need a buffer, run the reaction simultaneously
Is also good. 6.1.4.DNA modification Bal 31 nuclease is a highly specific single-stranded enzyme.
Dodeoxyribonuclease activity and advanced exonuclease
It is a multifunctional enzyme containing creatase activity and has a double-stranded DN
A (dsDNA) at both the 3'- and 5'-ends.
Sometimes decomposes. Nuclease Bal 31 (New
England Biolabs, Inc. , Beve
rly, Ma. ) Is composed of denatured bovine thymus DNA (6
50 μg / ml) to 1.0 μg of acid-soluble nucleotid
As the amount required to release the drug at 30 ° C for 1 minute.
Is defined. The reaction buffer used for Bal 31 digestion was
20 mM Tris HCl (pH 8.0), 600 mM
NaCl, 12m MgCl₂, 12 mM CaC
l₂Consisting of, 1.0 mM EDTA and DNA
Met. Incubation was performed at 30 ° C. Ba
The l31 digestion converts 30 μg of DNA to 0.5 units of Ba.
l for 1, 2, 4, 6, 8 and 10 minutes with 31
Performed by incubation. Add EDTA
To 50 mM or heat inactivation of Bal 31
(For example, at 65 ° C. for 10 minutes) to stop the reaction.
Was. S1 nuclease can be RNA or denatured DN.
A (ie, single-stranded DNA) is decomposed into mononucleotides
However, double-stranded DNA and DNA / RNA hybrids
Does not decompose (under appropriate conditions). S1 nuclease (B
oehringer Mannheim, Indian
apolis, Ind. ) Is 1 μg of denatured
Necessary for acid solubilization of thymus DNA at 37 ° C for 30 minutes
It is defined as the amount of enzyme that does. S1 nuclease
The reaction buffer used was 30 mM sodium acetate
(PH 4.6), 250 mM NaCl, 1 mM Zn
SO₄And 5% glycerol.
S1 digestion involves adding 2,000 units of enzyme to 0.1 μg of DNA.
And for 30 minutes at 45 ° C with 20 μg RNA
Performed. Exonuclease VII (Exo VI
I) degrades single-stranded DNA (ssDNA). Ex
o The mechanism of action of VII is that of forward exonuclease
It seems to be. Exo VII (Bethesda
Research Laboratories, Roc
kville, Md. 1) is a linear unit as a substrate
Denaturation [³H] -SV40 DNA at 37 ° C., 30
Enzyme that yields 1 nmol of nucleotide monomer per minute
Is defined as the amount of Used for Exo VII
The reaction buffer was 10 mM Tris-HCl (pH 7.
9), 100 mM NaCl, 10 mM β-ME and
And 8 mM EDTA. Exo V
II digestion requires 0.1 μg of D in a 250 μl reaction volume.
1 unit at 45 ° C. with 4 units of Exo VII per NA
Time went. 6.1.5.DNA polymerase reaction DNA polymerase catalyzes stepwise elongation of DNA strands
You. Chain growth is in the 5 'to 3' direction (i.e.,
Addition of the reotide occurs at the 3 'end) and
The sequence is determined by the sequence of the template. If not, enter
The coming nucleotide must be complementary to its template
Not form a correct base pair with the template strand.
Because it must be. Known DNA polymerases are strands
Synthesis cannot be started. Therefore, for DNA synthesis
Is the free 3'-hydroxy which is annealed to the template strand
A primer having a terminus is required. Chain elongation
3 'of the primer to the 5'-phosphate of the coming nucleotide
-Proceeds by nucleophilic attack of the hydroxy terminus. new
The strands are base-paired and antiparallel to the template strand.
You. As a result of the DNA polymerase reaction,
The type strand is "repaired" or becomes a double-stranded DNA molecule
Is converted. The second important feature of DNA polymerase is
"Proof-readin
g) "function. Related to DNA polymerase I
The 3'-5 'exonuclease activity is base-paired.
Works at both ends and both single-stranded and double-stranded DNA
Is decomposed in the 3 'to 5' direction to produce a mononucleotide.
To achieve. Thus, incorrectly added nucleotides
Removal before continuing, improving the fidelity of the enzyme.
To increase. DNA polymerase I is a double-stranded DNA
Also has specific 5'-3 'exonuclease activity
Cage (5'-mononucleotide and oligonucleotide
This can excise mismatched regions of DNA.
Can be. The method of Klenow (Klenow et al., 1
971, Eur. J. Biochem. 22: 371)
Hydrolysis of DNA polymerase I by two
Gives a fragment, the big and the small.
Large fragment, Klenow fragment
(76,000 daltons) have 3'-
Retains 5 'exonuclease activity, while retaining small
The fragment retains 5'-3 'exonuclease activity
I do. DNA polymerase I or DNA polymerase
I—one unit of the large fragment (New England)
  Biolabs, Inc. , Beverly, M
a. ) Is 10 nmole of deoxyribonucleotide
Is converted to an acid-insoluble form at 37 ° C for 30 minutes.
Is defined. The assay conditions for both enzymes are the same, but DN
The reaction mixture for A polymerase I was 6.6 mM M
gCl₂1.0 mM β-ME, 2 mM dAT
Rimmer, 33 μM dATP, 33 μM³H-dTT
67 mM KPO in buffer consisting of P and enzyme
₄(PH 7.5), while the Klenow fragment
Reaction mixture is 40 mM KPO₄(PH 7.5)
Was included. In the present application, single-stranded DNA or restriction
In order to repair the cohesive ends resulting from enzymatic cleavage,
When using a remerase large (Klenow) fragment
In that case, the following procedure was used. Restriction enzyme reaction at 68 ° C for 10
Stopped by heating for minutes. Same reaction mixture
First, each deoxynucleoside triphosphate was added to a final concentration of 50 μM.
1 microgram of DNA in the reaction mixture.
10-100 units of DNA polymerase Kleno
w fragment was added. In other words, the single-stranded end
Excess DNA Polymerase to ensure complete repair
ZeKlenow fragment was used. 6.1.6.Gel fragmentation of DNA fragments
Made After restriction or nuclease treatment, various sizes
DNA fragments from agarose or polyacrylic acid
Low voltage (using agarose gel)
About 2 volts / cm on polyacrylamide gel
Gel electrophoresis at 10 volts / cm)
Release, stain with ethidium bromide, and
(Southern, 1979, Me
things in Enzymology 68:15
2). Recovering a specific DNA fragment from a gel
Excise the appropriate band from the gel and dialyze
The DNA was electroeluted into the tube. Next, the DNA was
Isolated on E-cellulose or ethanol precipitated
And resuspended in a suitable buffer (Smi
th, 1980, Methods in Enzymo
logic 65: 371). 6.1.7.Ligation of DNA All ligation reactions were performed using T4 DNA ligase.
(New England Biolabs, In
c. , Beverly, Ma. ). T4 DNA rigger
One unit of the enzyme contains 20 μl of ligase buffer and
0.12 μM (300 μg / ml) 5′-DNA end
Of bacteriophage lambda DNA at different concentrations
50% ligation of the dIII fragment at 16 ° C. for 30 minutes
Defined as the amount required to bring. DNA ligation was performed using 60 mM Tris-HCl.
(PH 7.8), 10 mM MgCl₂, 20 mM
Otreitol (DTT), 1.0 mM ATP and
Riga consisting of a DNA concentration ranging from 15 to 50 μg / ml
Performed in lysis buffer. The ligation reaction was performed with 10 μl of the reaction.
Use about 300 units of T4 DNA ligase per equivalent volume
And incubated at room temperature for 4 to 24 hours. 6.2.gD-1 gene localization and
Isolation The proteins identified by the HSV-1 × HSV-2 recombinant
Analysis of the protein shows that the gD-1 gene
0.9 to 0.945 of the genome map unit in the Us region
(Ruyechan et al., 19
79, J.A. Virol. 29: 667). FIG. 1a and
See FIG. 1b. The Us region is fragmented with a restriction enzyme.
Insert these fragments into the cloning vector
Thus, various recombinant plasmids were prepared. Each plasmid
Contained a specific portion of the Us region. Next, in the Us area
These recombinant plasmids are used to map the gD-1 gene to
Rasmid was analyzed. Map of gD-1 in HSV-1
The location has recently been described by Lee et al. (1982, J. Virol.
43:41). 6.2.1.Identification of HSV-1 Us region
Recombinant DNA plasmid containing a part Vectors pBR322 and HSV-1 (Patton strain)
Several types of recombination using various fragments of the Us region of
A plasmid was prepared. Of these plasmids
One, pRWF6 is found to contain the entire gD-1 gene
did. pRWF6 is described below. HSV-1 of the recombinant plasmid pRWF6
The insert (FIG. 1c) is lambda gtWES: EcoRI-H
(Umene & E) obtained from the Us region of the clone.
nguist, 1981, Gene 13: 251).
Lambda gtWES: EcoRI-H clone to EcmR
I digested completely. Lambda gtWES: EcoRI-
EcoRI-H fragment of H clone, about 15-1
6 kb (kilobase) includes the entire UsV region of HSV-1
I have. The plasmids pBR322 and HSV-1
EcoRI-H fragment (as isolated above)
Were each completely digested with BamHI. Obtained pBR
322 4.4 kb fragment and HSV-1 6.4
kb fragment and a 1: 1 ratio
Ligation was performed to obtain pRWF6. 6.2.2.Position of mRNA coding sequence unique to gD-1
Confirmation Locates coding sequence unique to gD-1 in pRWF6
And reselect the viral DNA insert for selection.
And subcloned into pBR322. Subclone
Denatured viral DNA restriction fragment of pSC30-4
(FIG. 1d and description below) was immobilized on nitrocellulose.
Cytoplasmic RNA extract of HSV-1 infected cells
MRNA from (complementary base pair hybridization
To isolate). Two mRNA species
(3.0 kb and 1.7 kb) and pSC30-4
I hybridized. In vitro of these mRNAs
o 3.0 kb or 1.7 kb mRNA by translation
Encodes a gD-1 protein. This
The details of this procedure are described below and are shown in FIG. Plasmid pRWF6 was transformed into E. coli transformant.
And digested with the restriction enzyme SacI. this
Gave three fragments. That is, all pBR
322 vector and part of the HSV-1 DNA sequence
6.2 kb fragment, 2.9 kb HSV-1
DNA fragment and 1.7 kb HSV-1D
NA fragment. The 2.9 kb HSV-1 DNA fragment
In order to subclone the plasmid (see FIG. 1d),
BR322 to PvuII (to linearize pBR322)
And a SacI linker (New E
ngland Biolabs, Inc. , Bever
ly, Ma. ) Using T4 DNA ligase in the presence of
Connected. Therefore, the unique Pvu of pBR322
II site was converted to a unique SacI site [Sac
I (Pvu II^(-))]. Modified with SacI enzyme
After cutting the decorated pBR322 vector, the vector is
2.9 kb HSV-1 SacI DNA fragment
And ligation was performed to obtain pSC30-4. This recombinant plastic
Escherichia coli was transformed using the sumid. Transformants
Screening and micro-lysate technology (mini-lys
ate technology; Clewell & H
elinski, 1970, Biochem. 9:44
28) selected by restriction mapping. The subclone pSC30-4 was replaced with the mRNA
Used for hybridization selection as follows
(Ricciardo et al., 1979, Proc. Nat.
l. Acad. Sci. , U.S. S. A. 76: 492
7). Plasmid pSC30-4 was transformed from E. coli transformant.
And 200 μg of pSC30-4 DNA was isolated from Sac
HSV-1 DNA insert was cut out by digestion with I.
Cut the plasmid into chloroform / phenol (1: 1)
And ethanol precipitated. 2 m of sediment
resuspend in 0.3 M NaOH for 10 minutes at room temperature
The DNA was denatured by incubation. Then this suspension
The product was treated with 4.4 ml of distilled water, 0.2 ml of 3M HCl,
0.2 ml of 1 M Tris-HCl (pH 7.5) and
3 ml of 20 × SSC (SSC is 0.15 M NaC
1, 0.015 M sodium citrate)
More neutralized. PH measured by indicator paper is between 6 and 8
Met. Denatured and neutralized DNA is distilled water, then
25mm Nitrose previously immersed in 6 × SSC
Lulose filter (Schleicher and
Schuell, Keene, N.M. H. A) through the vacuum
Filtered. After vacuum filtration, remove the nitrocellulose filter
Wash with 6 × SSC, dry and bake at 80 ° C. for 2 hours
Done. Use a half nitrocellulose filter
Used in the hybridization method. About this below
Briefly stated. The nitrocellulose containing the viral DNA was
Cut into small pieces and incubate with total cytoplasmic RNA
did. The RNA was prepared from HSV-infected V prepared as follows.
It was isolated from the lysate of ero cells. Ve
10 plaque forming units (pfu) per cell
50 μg to stain and prevent DNA replication
/ Ml cytarabine (β-cytosine arabinoside)
Incubate at 37 ° C for 7 hours in Dulbecco's medium
Was. Cells were treated with 0.15 M NaCl, 1.5 mM MgCl
₂And 0.01 M Tris-HCl (pH 7.9)
Dissolved in 0.65% NP-40. 3000 × g, 2 minutes
The nuclei were sedimented by centrifugation between
EDTA (pH 7.9) and 0.5% SDS final
The concentration was adjusted. This solution is chloroform / phenol
Extract twice with (1: 1) and once with chloroform
did. Ethanol precipitation of cytoplasmic RNA (Vero
One confluent roller bottle of vesicles has about 1.5 mg RN
A) and was partially dried. RNA precipitation
400 μl of the deposit (about 0.4-0.8 mg of RNA)
Hybridization buffer (50% formami
, 0.4 M NaCl, 40 mM PIPES, pH
6.4, dissolved in 1 mM EDTA and 1% SDS)
And add to chopped nitrocellulose filter
Was. Hybridization at 55 ° C in a shaking water bath
Performed for 3 hours. Next, filter pieces were placed at 60 ° C. in 0.5% S
Wash 10 times with SSC containing DS, then SS at 60 ° C
Washed twice with 1 ml of 2 mM EDTA in C. last
Then, use 2 mM EDTA, pH 8.0 for the filter piece.
And washed at 60 ° C. for 5 minutes. Remove the solution and filter
-The pieces were thoroughly dried with a cotton swab. Hive
The re-mRNA is synthesized using formamide and heat.
Eluted from nitrocellulose pieces as follows. 120 μl
95% formamide / 10 mM PIPES (pH
6.5) was added to the filter and the ink was incubated at 65 ° C for 5 minutes.
Was added. Transfer eluate to microcentrifuge tube and keep on ice
did. The second 120 μl of elution buffer was
Add to the lulose pieces and incubate again at 65 ° C for 5 minutes.
I did it. Transfer the eluate to a second microfuge tube and place on ice
Held. Fill a 720 μl aliquot of sterile distilled water
And then stirred. Each microcentrifuge containing eluate
Approximately 360 μl was transferred to the tube. Eluted RNA in microcentrifuge tubes
20 μl of 5 M NaCl, 5 μg of a suitable carrier
-(Eg, rabbit liver tRNA) and 1 ml of anhydrous
Tanol was added. The mixture is incubated at -70 ° C for 1 hour.
Incubate or centrifuge tubes on dry ice / E
Soaking the RNA in a slurry of tOH for 20 minutes
Settled. Precipitated RNA is pelleted in a microcentrifuge
(12,000 × g for 10 minutes) and settle one tube
The precipitate was washed with 1 ml of buffer (0.5 M NaCl, 10
mM Tris-HCl (pH 7.9) and 0.5% S
DS) to dissolve the RNA. Aliquot
Combine and dissolve to dissolve all precipitated RNA
Was added to the same type of tube. Polyadenylated RNA
[Poly (A) RNA] is replaced with oligo (dT) cellulose
(BethesdaResearch Laborat
ories, Inc. Rockville, Md. )
From dissolved RNA by chromatography using HPLC
did. Poly (A) RNA is used as an elution buffer in 10
mM Tris HCl (pH 7.9) and 0.1% SDS
Elute from the oligo (dT) cellulose using
(A) Precipitate the RNA with ethanol as described above.
Was. Hybridization with pSC30-4 DNA
MRNAs, 3.0 kb and 1.7
The kb mRNA was isolated. Rabbit net
Erythroid cell-free system (³⁵(Including S-methionine)
Was translated in vitro using (Pelha
m & Jackson, 1976, Eur. J. Bi
ochem. 67: 247). In vitro translation extraction
Effluent was a monoclonal antibody directed against gD-1.
Body 4S (provided by M. Zweig of the National Institutes of Health)
Gift) and S as described previously.
Prepared for DS-PAGE. Cell-free translation system
Of the immunoprecipitated protein product of
Of course, these selected mRNAs are 50,000 daltons.
Demonstrates Specificity of gD-1 Specific Protein
Was done. According to the size of the gD-1 protein, the smallest
The mRNA coding sequence will be approximately 1.6 kb. It
Therefore, consider the mRNA leader sequence and poly (A) tail.
Considering that the larger (3.0 kb) mRNA is gD-
It was expected to encode one polypeptide. 6.2.3.Characterization of gD-1 mRNA
Set of the 3.0 kb mRNA sequence contained in pSC30-4
Map locations and further characterize mRNA
What is required is S1 mapping, ie, single-strand specific nuclei.
Complementation using ase S1 and exonuclease VII
Of a DNA probe that hybridizes with a specific mRNA sequence
By mapping regions (Berk & Shar
p, 1978, Proc. Natl. Acad. Sc
i. , U.S. S. A. 75: 1274) and gD-
By sequencing the DNA of one gene coding region
(Maxam & Gilbert, 1980, Me
things in Enzymology, 65:49
9) Performed. With the S1 mapping technique, 3.0 kb
And the 1.7 kb mRNA species are both spliced.
Not (ie, including intervening sequences or introns)
Not), and they are common with different 5 'ends
It was found to have a 3 'end. 3.0 kb mRNA
Of the 1068 nucleotides, including the coding region at the 5 'end of
The DNA sequence is determined and the open end closest to the 5 'end is determined.
By positioning the start codon (ATG), translation
Coding frame was estimated. The principle of the S1 mapping technique is that RNA and release
Radioactively labeled single-stranded DNA (ssDNA) probe (eg
For example, those obtained from pSC30-4)
Is formed. RNA spliced
If the mRNA is mature, the intron will be
Will form a loop. The enzyme nuclease S1 is an RNA
Of radiolabeled DNA not protected by duplex formation with
Digest all ssDNA regions. On the other hand, Exonucle
Aase VII digests terminal-only ssDNA, thus
Will not digest the ssDNA loop. (C with RNA
These nucleases (by hybridization)
By comparing the size of DNA that is not sensitive to
Can detect spliced mRNA transcripts
You. In the present invention, a 3.0 kb mRNA
Radiolabeled DNA used to position the 5 'end
Is its BstEII5 'end before cutting with BamHI.
At the end (using the polynucleotide kinase enzyme,
(By the method of Maxam & Gilbert)³²P
3.8 kb BamHI-8 / of pRWF6 labeled with
It was a BstEII fragment. 3.0 kb mR
Radiolabel D used to position the 3 'end of NA
NA has its HindIII before cutting with SalI.
At the 3 'end (Klenow of DNA polymerase
Using fragments, Maxam & Gilber
by the method of t)³²Of P-labeled pSC30-4
With a 4.7 kb HindIII / SalI fragment
there were. Cytoplasmic mRNA is present in the presence of cytarabine as described above.
HSV-1 infected Vero cells grown for 7 hours in the presence
Isolated. The S1 mapping technique used is Berk &
  A modification of Sharp's method (Watson et al., 198
1, J. Virol. 37: 431). gD-
1 The 5 'end of the mRNA is the HindI of pSC30-4.
Maps near the II site, while the 3 'end is about
Mapped 2.8 kb downstream (FIG. 1d). Finally, HSV-1D of pSC30-4
NA is sequenced using the method of Maxam and Gilbert
Decided. FIG. 2 shows only the HSV-1 gD gene coding region.
1 shows a sequencing strategy for Both coding and non-coding strands
Was determined. FIG. 3 shows the yield of the HSV-1 gD gene.
1 shows the DNA sequence obtained. This DNA sequence corresponds to position 241
394 open codons from ATG
It was clear that it contained a coding frame. This part
Position is presumed to be the initiator of the gD-1 gene.
This putative gD-1 gene was transformed into the expression vector pJS41.
3 by cloning
(See Section 6.3). 6.3.Cloning of gD-1 gene and
Expression The gD-1 coding sequence is placed under the control of the lac promoter.
Was cut. For this purpose, the starting sequence ATG and the amino
First 156 nucleobases at the C-terminus (5'-terminus of the gene)
Part of the putative gD-1 gene lacking tide (hereinafter gD-
DNA cloning expression vector
pEH25 was prepared by linking to pJS413 (see FIG. 4).
See). The partial gD-1 gene has a protein coding sequence
Open readie correct for the starting ATG of the vector
It was inserted so that it might become a frame. As a result,
The translation of the mRNA is determined by the start sequence of the vector (ATG).
Initiated, the gD-1 gene (its own starting ATG and
And the first 156 nucleotides of the gD-1 coding sequence
Through to the natural termination signal of gD-1
Good. PEH25 plasmid containing the gD-1 gene
Was used to transform E. coli host strains,
Transcription of DNA from the lac operon depends on the promoter.
It is suppressed unless otherwise induced. Primary transformant
Drug resistance (the vector carries the ampicillin resistance gene)
For further analysis of resistant clones.
Was. The structure of the obtained recombinant plasmid pEH25 is
Confirmed by restriction analysis and DNA sequencing. pEH
Induction of 25 transformants resulted in 46,000 daltons
Is produced, which is an anti-blood against HSV.
By monoclonal antibody to gD
Even immunoprecipitated. These steps are detailed in the following sections.
I will describe it. 6.3.1.Expression vector pJS413 The expression vector pJS413 (FIG. 4)^r(Β-
Lactamase) gene, lac promoter (Pla
c), lac and cro ribosome binding sites (SD
^LacZAnd SD^croIs SD for all drawings
^ZAnd SD^CRO), Cro 69
Chain Initiated ATG with Nucleotides and Modified β-Gala
The custosidase gene (lac iz gene, hereinafter z
-Described as a gene). p
Between the cro-start ATG of JS413 and the z-gene (immediately
BglII, SmaI or Bam of pJS413
Correct reading frame in HI cloning site)
When the gene is inserted in the
The expression of the protein is enabled. However, this embodiment
Has deleted the z-gene and the partial gD-1 gene
With the cro-initiated ATG of pJS413 and cro-nuclei
Otide was ligated in phase. PJS4 for inserting gD-1 gene
13 (see FIG. 4), pJS413 was converted to Sm
aI (generating blunt ends) and SacI (SacI
Digestion with 3 'cohesive ends). Promoter, S
4.7 including D sequence, starting ATG and partial cro sequence
kb fragment was isolated by gel electrophoresis
Was. 1.8 kb fragment containing the 5 'end of the z-gene
Account was deleted. 6.3.2.pJS41 of gD-1 gene
Insertion into 3 After mapping the gD-1 gene in pSC30-4
(Section 6.2.3), carboxy-co of gD-1 gene
2.2 including the last 1026 bp (base pairs) of the card end
kb DNA fragment with PvuII (blunt ends
And SacI (resulting in SacI 3 'cohesive ends)
Selective from pSC30-4 by digestion with
(FIG. 4). 2.2 kb PvuII / SacIpSC
30-4 fragment and 4.7 kb SmaI / S
The acIpJS413 fragment was converted to T4 in a 1: 1 ratio.
  Ligation was performed using DNA ligase (FIG. 4). Obtained
E. coli NF1829 using recombinant plasmid
The quality has changed. E. coli NF1829 strain is lac repressed.
LacI for sir overproduction^QF'-la with mutation
K-12 MC1000 derivative carrying c episome
It is. Β-gala present on F'-lac episome
The lacz-gene encoding custosidase is Tn5
(Transposon) Inactivated by insertion.
Therefore, in the NF1829 strain, the pJS413 plasmid
Lac promoter to obtain expression of the inserted gene
Must be guided. 6.3.3.Form expressing gD-1 gene
Transformant identification A plasmid isolated from an ampicillin-resistant E. coli transformant
Sumid was further purified by pJS4 by restriction enzyme mapping.
13 at the junction between the vector 13 and the gD-1 gene insert
Analyzed by NA sequencing. Plasmid pEH2
5 (FIG. 4) is correct along the Cro-gD-1 junction.
Having a nucleotide sequence (shown in FIG. 5) and gD-
Its ability to express one related polypeptide was examined. l
The ac promoter in NF1829 (lac repressor
Because it is transcriptionally inactive (due to overproduction of sir)
gD-1 protein is 1 mM IPTG or 1-10
After inducing the promoter with any of mM Lactose
Could only be detected. Clones transformed with pEH25
For the IPTG-specific induction of gD-1 related proteins
When tested, 23 amino acids of cro protein
(Encoded in pJS413) and gD-1
342 amino acids of the protein (i.e., the first 52
46,000 daltons consisting of amino acids)
Protein production was confirmed. This protein
Shows that all rabbit antisera against HSV-1 (DAKO C
chemicals, Inc. , Hicksville,
N. Y. ) And HSV-1 specific for gD
Noclonal antibodies (1S, 4S, 55S and 57S)
(Showalter et al., 1981, Infectio.
n and Immunity, 34: 684)
Immunoprecipitated. The monoclonal antibodies 1S, 4S, 55S and
And 57S recognize many different sites on the gD-1 molecule
(Eisenberg et al., 1982, J. Viro)
l. 41: 478). What should be noted is the monochroma
Antibody 4S demonstrates the infectivity of HSV-1 and HSV-2
Capable of neutralizing and produced by both viruses
Ability to immunoprecipitate produced gD protein
That is. Protein produced by pEH25
Is immunoprecipitated by the 4S antibody, indicating that pEH2
5 gD-1-related proteins are HSV-1 and HSV-2
The antigenic determinant shared by both gD proteins
Has been demonstrated. Furthermore, the gD of pEH25
-1 related protein is a 1S that neutralizes only HSV-1
Immunoprecipitation was also performed with a monoclonal antibody. Also,
pEH25gD-1 related proteins are HSV-1 and H
55S and 5 do not neutralize any infectivity of SV-2
Immunoprecipitated by 7S monoclonal antibody. Each license
Plasmid sediment was analyzed by SPS-PAGE. All steps
Will be described in detail below. All pEH25 transformants were L-brominated.
An aliquot of the overnight culture at 37 ° C. in
Dilute 20-fold with -9 broth and 90 minutes at 37 ° C
Proliferated by shaking (Miller, Ex.
peripherals inMolecular Gen
etics, Cold Spring Harbor
Rless, New York, N.W. Y. , 197
2). These cultures for expression of gD-1 protein
For the product assay, 1 mM IPTG and 25 μCi
/ Ml³⁵S-methionine was added to the culture (control
Is³⁵Labeled with S-methionine but no induction
T). After 60 minutes at 37 ° C., the culture is centrifuged
Let's go. Lyse cells and release cell contents
Each pellet of cells with an equal volume of lysis buffer IP.
-3 (20 mM Tris-HCl (pH 8.1), 100
mM NaCl, 1 mM EDTA, 1% NP-40,
Resuspend in 1% deoxycholic acid and 0.1% SPS)
It became cloudy and was immediately frozen in liquid nitrogen and sonicated. cell
Lysate was centrifuged at 5,000 xg for 5 minutes at 4 ° C,
The supernatant was divided into aliquots. Control serum (non-immune or
Indicates pre-immune serum or test antiserum (for HSV-1)
Polyvalent antiserum or monoclonal antiserum against gD)
Was added to each aliquot, followed by ink at 4 ° C. for 60 minutes.
Was added. (The amount of antiserum added is based on a known amount of antigen.
And testing serial dilutions of the antiserum
Determined by calibrating the value. ) The immune complex was washed with Pansorbin (Pansorb).
in) (Staphylococcus aureus)
Protein A, Calbiochem-Behring
  Corp .. , LaJolla, Cal. )
Centrifuge (unless otherwise noted)
(Centrifugation was performed at 5,000 xg at 4 ° C for 5 minutes.)
Was recovered. The pelleted immunoprecipitate was transferred to IP-2.
(Same as IP-3, but to eliminate non-specific adsorption
Add 20 mg / ml bovine serum albumin BSA.
Washed with IP-3, washed twice with IP-3, and
P-1 [20 mM Tris HCl (pH 8.1), 10
0 mM NaCl, 1 mM EDTA and 1% NP-4
0]. Transfer this suspension to a new tube
And centrifuge it to pellet the SDS-polyamide.
Cryamide gel sample buffer (Laemml
i, 1970, Nature 227: 680).
Suspended. After heating at 95 ° C for 3 minutes, trace the sample
Centrifuge with a centrifuge at 12,000 xg for 2 minutes.
Soluble components were removed. Remove the supernatant, and remove the SDS
Loaded on acrylamide gel (10%). Electrophoresis
Later, the protein is stained with Coomassie Blue dye and
Treated with sodium citrate for fluorography
(Chamberlain, 1979, An)
al. Biochem. 98: 132). SDS Polya
Results of acrylamide gel electrophoresis (SDS-PAGE)
Is 46,000 daltons produced from pEH25 after induction.
Ruton's gD-1 related protein binds to HSV-1
Whole rabbit antiserum and monoclonal antibody 1S, 4
Clearly immunoprecipitated by S, 55S and 57S
It was clearly shown. Finally, the large intestine transformed with pEH25
Induced gD-1-related proteins expressed from fungal NF1829
Effect of HSV-1-infected Hela cells on immunoprecipitation of proteins
Competition experiments were performed to determine the effect of the lysate (Figure
6). Control (uninfected) and HSV-1 infected Hel
a cells (infection as described above for Vero cells)
A serial dilution was prepared from the lysate. Mo
Noclonal 55S ascites (gD-1 specific monoclonal)
5 μl aliquot of a 100-fold dilution of
Add each aliquot of serial dilutions of the la cell lysate
Was. After incubation at 4 ° C. for 30 minutes, the induced p
From lysates of EH25 transformants³⁵S-methioni
Of labeled protein to HeLa cell lysate
And incubated for an additional 60 minutes at 4 ° C. Exemption
The epidemic complex was recovered by immunoprecipitation, and the
Analyzed by S-PAGE and fluorography
Was. Gel specific immunoprecipitated protein bands
And radioactivity for scintillation counting
Therefore, it was measured. FIG. 6 shows the results of these experiments. each
The radioactivity of the sample was determined by immunoprecipitation of pEH25.
It represents the protein product,
And plotted. Circles are serially diluted control (uninfected)
Represents Hela cell lysate. Squares represent serially diluted H
3 shows SV-1 infected Hela cell lysates. This is Ming
Clearly, HSV-1 protein is 55S monoclonal
Release from pEH25 for formation of immune complexes with antibodies
It shows that it competes with the radiolabeled protein. 6.4.Cro / gD-1 / β-galacto
PEH4-2 resulting in the production of a sidase fusion protein
Production of The gD-1 gene was isolated from pEH25 and its termination signal
Nulls were deleted. To this end, the gD-1 gene sequence
DNA fragment containing the gD-1 termination sequence TAG
Cleavage at the over restriction site. Next, DNA nuclease
TAG by forward digestion of the ends with Bal 31
Was removed. Then, the gD-1 gene fragment
Is a fusion protein: Cro / gD-1 / β-galacto
It was inserted into pJS413 so as to encode for a sidase.
This procedure is described below and shown in FIG. The plasmid pEH25 was completely digested with NruI.
Digest and progressively digest with Bal 31 nuclease
Was. Next, the obtained DNAs of various lengths were digested with the restriction enzyme Ps
Cleavage at tI produces a wide range of fragments, many of which
Or lacked the gD-1 stop codon, but most gD-1
-1 gene sequence. Appropriate DNA fragmentation
(1.5-1.9 kb) was separated by gel electrophoresis.
Released and eluted as previously described. Vector pJS4
13 was completely digested with PstI and SmaI,
A 5.5 kb DNA fragment was isolated (FIG. 7).
pEH25 fragment and pJS413 fragment
Were ligated at a ratio of 1: 1 to obtain the form of E. coli NF1829.
Used for quality conversion. Ampicillin resistant colonies are indicated cold
Assay β-galactosidase activity on a top plate.
And the production of the fusion protein was investigated (Mil
ler, Experiments in Molecu
lar Genetics, Cold Spring
Harbor Press, New York, N.M.
Y. , 1972). Positive colonies were induced with IPTG.
SDS-PAGE analysis of all lysates of selected transformants
Thus, the Cro / gD-1 / β-galactosidase fusion tag
The presence of protein was tested. Cro / gD-1 /
These expressing β-galactosidase fusion proteins
160,000 dalton fusion protein
High quality producers were isolated. Included in this clone
The resulting plasmid was named pEH4-2 (FIG. 7). pE
Produced by E. coli clones transformed with H4-2
The resulting fusion protein is inducible by IPTG
And cross-exempt with both HSV-1 and HSV-2
It was found to be epidemiologically reactive. pEH4-2 Plasmi
Isolates by restriction mapping and DNA sequencing
analyzed. pEH4-2 is the BamHI part of pJS413
No digits are included (see FIG. 8). The plasmid was named pEH3-25.
Other transformed E. coli isolates were transformed with pEH4-2.
Less amount of Cro / gD-1 / β-galact than the recombinant
A sidase fusion protein was produced. pEH3-25 type
Transformants are described later in the text (see Section 6.5).
See). 6.4.1.pEH4-2 fusion protein is anti-HSV-
Immune reaction with 1 serum Fusions produced from E. coli transformed with pEH4-2
Synthetic protein is characteristic of rabbit antiserum against HSV-1.
Interacted differently (data not shown). pEH4
-2 and pEH25 IPTG-inducible proteins
Separated by DS-PAGE and transferred to nitrocellulose
(Ie, a protein “blot” was performed).
Uninduced pEH4-2 and pEH25 transformation
Body lysates were processed in the same procedure as controls. next,
Nitrocellulose with rabbit antiserum to HSV-1
Processing, followed by rabbit immunoglobulin as a probe
Against¹²⁵Treated with I-labeled goat antiserum (Towb
in et al., 1979, Proc. Natl. Acad. S
ci. , U.S. S. A. 76: 4350). Autoradiogram of protein blot
Is a 160,000 dalton fusion from pEH4-2
Rabbit antiserum against HSV-1 and immune response
Responding, and pEH25 at 46,000 Daltons
GD-1 related protein derived from HSV-1
Demonstrated immunoreactivity with heron antiserum. 6.4.2.Anti-blood against pEH4-2 fusion protein
Qing immunoprecipitates HSV-1 and HSV-2 proteins
Let The antiserum to the pEH4-2 fusion protein was HSV
-1gD and HSV-2gD are immunoreactive.
Thus, the pEH4-2 fusion protein is gD-specific.
It has been demonstrated to contain foreign antigenic determinants. This is pE
Immunoprecipitation with antiserum against H4-2 fusion protein
HSV-1 protein and HSV-2 protein
Indicated by quality SDS-PAGE analysis. The following considerations
Please refer to. Rabbit against pEH4-2 fusion protein
A goat antiserum was prepared as follows. Transformation with pEH4-2
The transformed E. coli clone is grown to mid-log phase and
Induction was performed using mM IPTG. Four hours after induction,
Bacteria are sedimented by centrifugation and SDS-PAG
Lyse in E sample buffer and separate SDS-
Placed on polyacrylamide gel. After electrophoresis, outside
Lanes are stained with Coomassie Blue dye for protein
Visualized. Next, a 160,000 dalton fusion tongue
The protein band was cut out of the gel. Gel slices with liquid nitrogen
In water, crushed, and resuspended in PBS. Equal amount
Freund's complete adjuvant was added. Well mixed
Later, the solution was added to two New Zealand rabbits (01
8 and 019). 25-5 for each rabbit
0 μg of protein was injected. 28 days later, incomplete flow
With the same amount of fusion protein suspended in India adjuvant
Rabbits were boosted. More than once every 10 days
Booster immunizations were performed. 55 days after the first injection
Blood was collected and serum was collected and subjected to immunoprecipitation analysis. The immunoprecipitation was performed as follows. Confluent culture
HSV was added to the cells at 10 pfu / cell (10 plaque forming cells).
Position / cell) and after infection³⁵1 with S-methionine
Labeled for 6 hours. [For GBK (Georgia Bovine Kidney) cells
HeLa cells were infected, while HeLa cells were infected with HSV-1.
-2. HSV-1 or Vero cells
HSV-2 was infected. ]³⁵S-Metio
Lysates of HSV-infected cells labeled with nin
l of pre-immune rabbit serum to pEH4-2 fusion protein
Rabbit antiserum (eg, 018 or 019 antiserum)
Qing) or with 1 μl of monoclonal antibody 4S
Incubated. Immune complexes described in Section 6.3.3
Collected using pansorbin as a solid and immunoprecipitated
Separation of radiolabeled proteins by SDS-PAGE
And subjected to fluorography. SDS-PA
The GE results were as follows: (1) GBK cells infected with HSV-1
52,000 dal produced from vesicles or Vero cells
Tons of gD protein was obtained from pEH4-2 transformants.
Antiserum to the produced fusion protein, and 4
Being immunoreactive with the S monoclonal antibody, (2)
HeLa cells infected with HSV-2 or Vero cells
50,000 dalton gD protein produced from vesicles
The quality is determined by the antiserum against the pEH4-2 fusion protein and
And immunoreactive with 4S monoclonal antibody
did. HSV-2 when compared to gD from HSV-1
Apparent low molecular weight of gD of origin is consistent with published observations
(Ruechan et al., 1979, J. Viro)
l. 29: 677). The fluorography result is H
G produced by SV-1 or HSV-2 infected cells
D protein was produced from pEH4-2 transformants
Immunoreactive with antiserum to fusion protein
Was shown. 6.4.3.Antiserum to pEH4-2
Is HSV-1 and HSV-2 infection in vitro
Neutralize Rabbit antiserum to pEH4-2 fusion protein was
It was also analyzed for its ability to neutralize the infectivity of the virus.
Was. Viral neutralization is a measure of the potential for infected cells in tissue culture.
Tested by reduction of Lark number (Showalter
Et al., 1981, Infection and Immu.
ny 34: 684). For this, 50-100p
fu HSV-1 or HSV-2 was added to the pre-immune serum (vs.
), Immune antiserum (018 or 019) or Monoc
Active serum complement with dilution of Lonal Antibody 4S
Pre-incubate in the presence or absence of (C ')
Was. Preparation of HSV-1 or HSV-2 in Vero cells
Infected things. After 3-4 days, count HSV plaques
Then, the effect of antiserum on the reduction of plaque number was examined.
The results shown in Table 1 indicate that the antiserum obtained from each rabbit
  Neutralizes HSV-1 and HSV-2 in vitro
It indicates that you have the ability. Neutralization is in the absence of active complement
However, it has been proven that complement significantly increases neutralizing activity.
Was. Neutralization is better for HSV-1 than for HSV-2
The layers were effective. [0128] [Table 1] ¹Serum 018 and 019 from rabbit were heat-inactivated 5%
Sexualized complement (30 min at 56 ° C, -C ') or active morpho
On Vero cell monolayers in the presence of Cit complement (+ C ')
Tested for irus neutralization. The number is 5 plaques
The antibody titer is shown as the reciprocal of the serum dilution rate reduced by 0%.
You.² In these tests, GBK cells were used instead of Vero cells.
Vesicles were used. The results shown are for HSV-1 and HSV
PEH4- as a subunit vaccine against -2
This shows the availability of two fusion proteins. 6.4.4.gD-1 residue in pEH4-2
Reconstruction of genes Using plasmids pJS413 and pRWF6, g
The D-1 gene was reconstructed (FIG. 8). Thus, pRW
F6 is digested with HindIII and Bal31 and
A blunt-ended fragment of random size was generated. This
After digesting these fragments with SacI, 2.2-2.2
2.4 kb blunted / isolated SacI fragment
did. These fragments have different lengths of gD
-1 gene (see FIG. 8).
See). The gD-1 fragment was added to pJS413.
It was cloned. For this purpose, pJS413 was converted to Sm
aI (bringing blunt ends) and SacI (SacI
Digestion with 3 'cohesive ends). 4.7 kb S
The maI / SacI pJS413 fragment was blunt-ended.
End / SacI pRWF6 fragment (2.2-
2.4 kb) at a 1: 1 ratio and E. coli NF18
It was used to transform 29 strains. This is Aminoco
GD-1 gene randomly deleted at the
A population of clones transformed with the containing plasmid
(FIG. 8). pEH50 + x (where x is 1 to
24), for a total of 24 plasmids
(About 7 kb each) is a mapping of restriction enzyme recognition site.
And analyzed it. PvuII site in the gD-1 gene
(19 of 24 transformants)
Contains the longest portion of the amino-coded terminus of gD-1
Clones were identified. In the complete gD-1 gene sequence, gD
The distance between the starting ATG and the internal PvuII site is 156 salts
It is a base pair. Therefore, the 19 pEH50 + x plasmids
Were digested with BglII and PvuII.
The resulting fragments were separated by gel electrophoresis and Bg
The size of the II / PvuII fragment was determined. 1
BglII / PvuII fragment smaller than 60 base pairs
And identified 15 pEH50 + x plasmids with
Was. That is, the end point of Bal31 digestion shown in FIG.
One was somewhere between the initiation ATG and the PvuII site.
By analyzing these 15 sequences, the pJS413 plasmid
The linking site to was determined exactly. Seven plasmids, pE
H51, pEH60, pEH62, pEH66, pEH
71, pEH73 and pEH74 are those of pJS413
croATG translational reading frame and inph
ase-linked gD-1 sequence (see FIG. 3).
See the number of pEHs corresponding to these junctions.
And vertical arrows). In fact, pEH51 is g
D-1 All but the first 6 amino acids at the amino terminus
Code. PEH51, pEH60, pEH62 and
And pEH71 transformants were induced by IPTG.
Or do not do³⁵Labeled with S-methionine and lysed
The digest was treated with monoclonal antibody 55S. Immunoprecipitation
The yield was analyzed by SDS-PAGE as described above. pE
H51, pEH60, pEH62 or pEH71
Each of the transformants was subjected to monoclonal 55S.
46,000 dalton inducible tamper
Produced (data not shown). Finally, a recombinant encoding the fusion protein
Plasmid is the gD-1 plasmid present in pEH4-2.
To reconstruct the aminocoding end of the gene sequence, pE
It was prepared using the amino-code end of H51 (see FIG. 9).
See). Plasmid pEH4-2 was replaced with PstI and Sac
II and digested with partial gD-1 / β-galactosidase
A 6.2 kb fragment containing the gene was isolated. Plastic
Smid pEH51 was completely digested with PstI and SacI
Partially digested with I. 1.25 kb P of pEH51
The stI / SacII fragment was converted to pEH4-2-6.
2 kb PstI / SacII fragment and 1: 1
Ligation was performed at a ratio to produce pEH82. Transformants
As described above, the β-galactosidase activity
And identified. Large carrying pEH51 plasmid
The Enterobacteriaceae transformant was named Escherichia coli WW51 strain and was named pEH8
E. coli strain WW82
It is named. We have developed HSV gD-1 fusion protein.
Can be produced in large quantities in E. coli.
Was. The fusion protein is probably the structure of the fusion protein.
Fusing for growth itself or in dense, insoluble inclusion bodies
Very stable for protein isolation. we
Extracts the fusion protein from E. coli and immunizes animals.
It can be used for sensitization. Obtained blood
Qing performed HSV-1 and HSV-2 in vitro.
Both plaque formations can be neutralized. 6.5.Cro / gD-1 and Cro /
Produces gD-1 / β-galactosidase fusion protein
Preparation of pEH90-10amLE392 In a suitable host cell, both fused and unfused gD
-1 recombinant protein capable of producing related proteins
Rasmid pEH90-10am was constructed (see FIG. 10).
See). The pEH90-10am plasmid contains Cr
o / gD-1 / β-galactosidase fusion protein
Derived from coding pEH3-25 (see section 6.4)
Derived from a series of recombinant plasmids pEH-90-N
(See FIGS. 3 and 10). pEH
The -90-N series includes the cro, gD-1 and z-genes.
Translation reading frames match (in pha
pEH4-2 (Section 6.4) in that
Although similar, the pEH-90-N line is similar to the gD-1 gene.
Additional unique restriction endonuclei at the z-gene junction
Contains a rease recognition sequence. One transformant of the pEH-90-N series
A recombinant, designated pEH-90-10, isolated from
The plasmid was further modified as follows. (1)
pEH-90-10 was obtained by converting the gD-1 gene sequence and the z-gene
(2) Next, pEH-90-
10 is a DNA linker containing the amber chain termination sequence TAG.
Ligation was performed in the presence of the Kerr sequence. The obtained recombinant plus
Mid pEH-90-10am is composed of gD-1 gene and z-
A gene that matches both translational reading frames of a gene
It contains the member chain termination sequence TAG. The pEH90-10am was transformed into E. coli NF182.
9, when used for the transformation of ampicillin-resistant transformants.
The body does not synthesize any detectable fusion protein
Was. However, the pEH-90-10am transformant
Lysogenic trait containing the gene for suppressor tRNA
When infected with the introduced phage φ80 SuIII
Indicates that the induced transformant is Cro / gD-1 / β-Ga
A lactosidase fusion protein was produced. This lysogen
It is named pEH90-10am SuIII. Also, the pEH90-10am plasmid was
Using two amber suppressor mutations (SupE and
And SupF) were transformed into E. coli LE392.
Was. pEH90-10amLE392 transformants were induced
Under both induced and uninduced conditions, Cro / gD-1 / β-gal
Cuctosidase fusion protein was produced (LE392 cell
The vesicle contains NF1 which leads to overproduction of the lac repressor.
LacI of 829^QNo mutation). pEH90-1
Protein produced by 0amLE392 transformant
Quality SDS-PAGE analysis revealed that the fusion protein (approximately
160,000 daltons) and unfused gD-1 related
Both proteins (about 38,000 daltons) are produced
It was confirmed that. Both proteins are rabbit anti-HS
Immunoreacts with V-1 serum. pEH90-10amLE
The 392 transformant harbors both proteins in approximately equimolar amounts.
Probably produces two proteins listed in Section 5.5
Co-purified when isolated by a purified undenatured coaggregate purification method.
You. The details of this procedure are described in the following sections. 6.5.1.Production of pEH90-N series As previously described in Section 6.4, the pEH3-25 trait
The transformant is a Cro / gD-1 / β-galactosidase fusion
Produces protein, but its production is pEH4-2
Less than transgenic. pEH3-25 recombinant plasmid
Is pEH4-2 except that it has a gD-1 transmembrane sequence.
(See FIG. 3). Transmembrane sequence forms host cell
Involved in inefficient expression of fusion protein by transformant
Maybe you are. The expression vector pHK414 (FIG. 10)
It is a JS413 derivative. In fact, pHK414 is pJS
413, including all elements but crossing the cro-z junction
Has a unique cloning site. p
The cloning site of HK414 was BglII, Hin
dIII, SmaI and BamHI. The z-gene is
If croATG matches the reading frame
Therefore, the intact plasmid is β-galactosidase.
Do not dominate the production of However, an appropriate length (3n +
2) The DNA fragment of
Inserted into either of the z-genes
Reading frame readjusted to croATG
And the inserted DNA sequence is croATG or
Is the z-gene and the termination signal in phase
(Eg: TGA, TAA or TAG)
Under the conditions, the β-galactosidase fusion protein is
Cell transformants. The pEH90-N series was constructed as follows.
(See FIG. 10). First, pEH3-25 with BamHI
Digestion, then carboxy-terminal gD-1 transmembrane
The cut plasmid was Bal31
Processed. After Bal31 digestion, pEH3-25 was
Cleavage with PstI, PstI cohesive end, amp^rHeredity
Child amino-coding end, lac promoter and translation
Regulatory elements, all or part of the transmembrane sequence
GD-1 gene, and blunt ends (BamH
I^(-)1.7-1.9 kb characterized by
Population of DNA fragments for agarose gel electrophoresis
Therefore, it was isolated. The expression plasmid pEK414 first contains B
Cleavage with glII, the BglII cohesive end was cut with DNA polymer.
Repair was performed using the Klenow fragment of the lase.
Next, the linear blunt-ended pHK414 was digested with PstI.
To blunt ends (BglII^(-1)), Z gene, a
mp^rThe carboxy coding end of the gene, and PstI
5.5 kb DNA fragment characterized by cohesive ends
Fragment was isolated by agarose gel electrophoresis. The 1.7-1.9 kb P of pEH3-25
stI / BamHI^(-1)DNA fragments and pH
5.5 kb BglII of K414^(-1)/ PstI
The DNA fragments were mixed at a 1: 1 molar ratio with T4 DNA
Ligation was performed using ligase. Named pEH90-N
The resulting plasmid population was grown on indicated agar plates.
E. coli NF1829. Transformation of 24 Producing Fusion Protein
Next recombinant plasmid derived from 10 of the body
To D through the gD-1 / z-gene junction (see FIG. 3).
Analyzed by NA sequencing. That is, pEH90-
2, pEH90-3, pEH90-4, pEH90-
5, pEH90-6, pEH90-7, pEH90-
9, pEH90-10, pEH90-11 and pEH
90-12. All membranes except pEH90-12
Includes all or part of the penetrating sequence. pEH90-
4 and pEH90-5 contain almost half the transmembrane sequence.
No. Except for pEH90-12, these plasmids
Is the high level of Cr provided by pEH4-2
o / gD-1 / β-galactosidase fusion protein
Led to production. Plasmid for the next step in this procedure
PEH90-10 was selected (pEH90-10 type
The gD-1 fusion protein produced by the transformant
The same as that produced by the pEH4-2 transformant
is there. However, it was produced by the pEH4-2 transformant.
The last four amino acids of gD-1 are pEH90-10
Lost from fusion proteins produced by transformants
Is). 6.5.2.Preparation of pEH90-10am gD-1 sequence and z-inheritance of pEH90-10 plasmid
To introduce an amber chain termination sequence between offspring,
The procedure was used (see FIG. 10). Hi the pEH90-10
ndIII and then BamHI to give gD
That is present at the junction between the -1 gene and the z-gene
Plasmid at a unique restriction endonuclease site.
Cut (see below). [0147] Embedded image7.3 kb HindIII / BamHI
Isolate truncated plasmid by agarose gel electrophoresis
And HindIII (using T4 DNA ligase)
/ BglII cohesive end and internal XbaI site and
Next DN characterized by the member sequence (TAG)
Linked with A linker. [0149] Embedded image Each complementary strand of the DNA linker is represented by Chow
Et al., 1981, Nucleic Acids Res9.
(12): According to the solid-phase method reported in 2807-2817
Was synthesized. Linker was ligated to the cut plasmid
Thereafter, the obtained recombinant plasmid is digested with XbaI.
Then, a linear 7.3 kb DNA fragment was agarose
Isolated by Sgel electrophoresis and T4 DNA ligase
And recircularization (this is actually
GD-, as indicated by the presence of a single XbaI site
Between the HindIII and BamHI sites at the 1 / z junction
PEH90-10 plus containing linked linker sequence
Mid). As a result of the ligation, the DNA linker
The amber sequences of gD-1 and z-genes
Reading frame matched (see below). [0151] Embedded image The obtained plasmid was pEH90-10a.
m. 6.5.3.pEH90-10am transformant Transformation of pEH90-10am into E. coli NF1829
It was used for. Ampicillin resistance when induced by IPTG
Transformants synthesize fusion proteins at detectable levels
Did not (red on MacConkey indicated agar plate)
No colonies). Next, amber suppressor was added to the transformant.
Lysogenic transduction plasmid harboring the
Page 80 SuIII. Guided by IPTG
The lysogenized transformant thus obtained was subjected to MacConkey-indicated cold.
A red colony forms on the top plate, which is the fusion protein
Indicates quality production. These lysates were converted to pEH90-10a
It was named mSuIII. Further, the pEH90-10am plasmid
Are two amber suppressors (supE and su
pF) but with the lacI of NF1829^QWith mutation
Does not oversynthesize the lac repressor
LE392 was used to transform. These traits
The transformant was named pEH90-10amLE392.
It is a fusion with trying to induce with IPTG
The protein was produced. 6.5.4.pEH90-10amLE3
Analysis of protein produced by 92 transformants Fusion proteins (about 160,000 daltons) and 3
8,000 daltons of protein, in approximately equimolar amounts,
Produced from EH90-10amLE392 transformant
Was. Each protein was combined with a rabbit antiserum against HSV-1.
I found an immune response. For this, cell tamper
Is isolated by the mini-aggregate method (described below)
Separated by DS-PAGE and transferred to nitrocellulose
And then treated with rabbit antiserum to HSV-1.
To rabbit immunoglobulin as a probe
¹²⁵Treated with I-labeled goat antiserum (Towbin
Et al., 1979, Proc. Natl. Acad. Sc
i. , U.S. S. A. 76: 4350). Host cell aggregates for screening assays
For the isolation, the following mini-aggregate method was employed. (1) Fresh L containing 100 μg / ml ampicillin
Duplicate culture tubes containing 5 ml of uria broth
To inoculate 200 μl of cells from overnight culture
And grown at 37 ° C. for 90 minutes. One of each duplicate is 1
Induction was performed by the addition of mM IPTG (final concentration). Next
The inoculum was grown for another 5 hours at 37 ° C. with stirring.
I let you. (2) In a 3 ml aliquot of the culture
Cells to be centrifuged in a microcentrifuge (12,000 × g) for 2 hours.
Centrifuged for 10 minutes to pellet. (Note: All microcentrifuge tubes
The volume is 1.5 ml. Therefore, first 1.5 ml
The recoat was pelletized and the supernatant was removed. Same
Add another 1.5 ml aliquot to the microcentrifuge tube and add the same
Pelletized in a centrifuge tube. ) (3) Cell pellet containing 50 μl of 25% sucrose
Suspend in 50 mM Tris-HCl, pH 8, dry eye
By placing the centrifuge tube in a water / ethanol bath.
The suspension was frozen. (4) Thaw the frozen suspension and add 0.25M
10 mg / ml lysozyme in squirrel-HCl, pH 8
Add 50 μl and suspend the suspension on ice for 10-15 minutes
For a while. (5) After incubation for 10 to 15 minutes, 400
μl of TET buffer (100 mM Tris-HCl
pH 8, 50 mM EDTA, 2% Triton X-10
0; Triton X-100 is a nonionic surfactant polio
Xylene (9,10) p-tert-octylfe
Is added to the suspension), gently mix the suspension, and add ice
And incubated for 5 minutes. Next, 500 μl of 2
× RIPA (2 × RIPA is 20 mM Tris-HCl,
pH 7.4, 300 mM NaCl, 2% deoxycord
Sodium luate, 2% NP-40, 0.2% SPS
) And mix the suspension gently, on ice for 5 minutes
Incubated. (6) Next, the cells in the suspension were
Sonicate using a probe for 10 seconds, and remove the suspension
valley SS34 rotor with 20,000 rpm (4
(7,800 × g) and clarified by centrifugation for 30 minutes. (7) Decant supernatant and include coaggregate protein
The pellet was resuspended in 50 μl of distilled water,
l of 2x sample buffer (2x sample buffer
Is 10% SDS, 40 mM Tris-HCl, pH6.
8, 0% of 2% β-ME and bromophenol blue.
02 volume saturated solution) and mix well
Was. Boil mixture for 5 minutes and aliquot 25 μl aliquot
On a 10% SDS-polyacrylamide gel for electrophoresis
I applied. The proteins were separated by SDS-PAGE.
Later, they were transferred to nitrocellulose (ie,
A protein "blot" was performed). Next, the nitrocell
Loin is treated with rabbit antiserum to HSV-1,
Next, for rabbit immunoglobulin as a probe
¹²⁵Treated with I-labeled goat antiserum (Towbin,
1979, supra). Autoradiography of protein blots
Grams show two bands immunoreactive with anti-HSV-1 antibody
It clearly indicated what was done. That is, 160,000 da
Ruton fusion protein (Cro / gD-1 / β-gala)
Csidase), and 38,000 dalton gD-
1 related protein (Cro / gD-1) or unfused g
D protein (not fused to β-galactosidase)
It is. Therefore, to isolate proteins
When using the isolation method, the non-fusion protein
Co-purified with quality. Then, non-fusion gD (Cro
/ GD-1) and gD fusion protein (Cro / gD-1)
/ Β-galactosidase) are both anti-HSV serum and immunoreactive.
Respond. 7. Example:HSV-2gD Comparison of HSV-1 genome map with that of HSV-2
did. The location of the gD coding sequence in the HSV-2 genome is
Analogous from gD position and size in HSV-1 genome
It was decided. BglII in the UsV region of HSV-2
The L fragment was inserted into pBR322 and the recombinant plasmid
Smid pHV1 was prepared. gD contained in pHV1
-2 coding sequence is identical to the hybridization probe.
Of gD-1 DNA obtained from pSC30-4
By performing hybridization using
The position was determined. pHV1 containing gD-2 coding sequence
Is then subcloned into pBR322.
Thus, a recombinant plasmid pHV2 was prepared. Within pHV2
The DNA sequence of the gD-2 gene of
The sequence of -2 was compared to that of gD-1. D of gD-2
The NA sequence is one codon shorter than the gD-1 DNA sequence
However, there is considerable homology between these two sequences. The gD-2 gene is controlled by the lac promoter.
The first 12 of the protein coding sequence are
A portion of the gD-2 gene sequence lacking 0 nucleotides is
Isolated from HV2. This DNA fragment is
small encoding the c promoter and translational regulatory elements
The DNA fragment was ligated. The obtained recombinant plastic
Smid pHV5 was obtained from host cell transformants by gD-2
A series of proteins was produced. Finally, restriction endonucleases at specific sites
The gD-2 gene fragment is converted to pHV
5 to thereby terminate the gD-2 coding sequence.
The stop sequence (TAG) was deleted. This DNA fragment
Shows the part of the gD-2 gene and the promoter of the expression plasmid.
Beta-galactosidase
Ligated into the vector pHK414 containing the enzyme coding sequence
Was. The obtained recombinant plasmid pHV6 was prepared using croSD
-Plasmid lac promoter having ATG and β-ga
GD-2 coding sequence flanked by lactosidase genes
In the derived E. coli transformant
The fusion protein was expressed. pHV6 fusion protein
Is isolated from host cell lysates and used as an immunogen
Formulated to See below for details on each construction process.
This is explained in the section below. 7.1.General procedures used for plasmid preparation
order Unless otherwise specified, DNA isolation, enzymatic reactions and
The general description of the knot reaction described in Section 6 and its subsections
The method was utilized in the cloning of gD-2. 7.1.1.Restriction enzyme buffer The buffer used for SmaI digestion was 6.6 mM Tris-
HCl (pH 7.4), 6.6 mM MgCl₂and
Consists of 6.6 mM β-ME. BglII, ClaI, EcoRI or
The buffer used for PstI digestion was 60 mM NaC
1, 6.6 mM Tris-HCl (pH 7.4), 6.6
mM MgCl₂And 6.6 mM β-ME.
BamHI, SalI or XhoI digestion
The fur was 150 mM NaCl, 6.6 mM Tris-HC.
1 (pH 7.4), 6.6 mM MgCl₂And 6.
Consists of 6 mM β-ME. When two or more restriction endonuclease reactions are
When performed on NA, the reaction in a low salt buffer
Note that the reaction is performed before the reaction in a high salt buffer.
Should. Two enzymes require the same buffer
In such a case, the reactions may be performed simultaneously. 7.2.Localization and isolation of gD-2 gene As mentioned earlier, the genome of HSV-1 and HSV-2
Approximately the gD in the HSV-2 genome
The position and size were determined. The gD-2 gene is used for 8.
Us contained within a 5 kb BglII L fragment
Between 0.9 and 0.945 genomic map units in the region
Was pinged. The BglII L fragment was converted to HSV
-2 excised from DNA and pBR3 for further analysis
22. 7.2.1.pHV containing gD-2 gene
Construction of 1 HSV-2 (G strain) genomic DNA digested with BglII
And an approximately 8.5 kb DNA flag containing the gD-2 gene.
Mentions were isolated by agarose gel electrophoresis. Step
Rasmid pBR322 was completely digested with BamHI and obtained.
4.4 kb pBR322 DNA and HSV-2
Of the 8.5 kb BglII L DNA fragment
Anneal (BamHI cohesive end and BglII
The cohesive ends are complementary) and ligated in a 1: 1 ratio
pHV1 was obtained (FIG. 11b). 7.2.2.gD-2 specific mRNA coding
Check the position of the array The gD-2 mRNA coding sequence is a hybridase
GD obtained from pSC-30-4 as a probe
-1 Hybridization using a part of DNA
(Southern, 1975, J. Mol. Bio.
l. 98: 503) and localized within pHV1
(About the protocol used for Southern transfer
Is described in Maniatis et al., 1982, Molecula.
r Cloning, A Laboratory Ma
nual, Cold Spring Harbor L
laboratory, pp. See 382-389
When). Thus, pHV1 was cut with XhoI,
Agarose gel electrophoresis of the obtained DNA fragment
Separated. Next, the DNA fragments in the gel
Denatured in alkali, neutralized, and
Moved to Luther. Then, D attached to the filter
NA fragment³²High for P-labeled gD-1 probe
It was bridged. [0168]³²The P-labeled gD-1 probe is as follows:
Produced. pSC30-4 (FIGS. 1d and 4) was converted to Pv
digestion with uII and the first 52 codons of gD-1 (15
500 bp DNA fragment containing 6 nucleotides)
Were isolated by polyacrylamide gel electrophoresis.
Was. Release gD-1 DNA by nick translation
Radioactively labeled (BRL Kit, Bethesda R
essearch Laboratories, In
c. , Gaithersburg, MD). Nick Tiger
Translation of double-stranded DNA into high radioactivity
It is a preferred method for n vitro labeling. this
The method is based on several activities of E. coli DNA polymerase I.
The two are used. That is, 5'-3 'exo
Nuclease activity and 5'-3 'polymerase activity
You. In nick translation, this enzyme is
Binds to the nick on one strand of the single-stranded DNA. Then before
5'-3 'exonuclease ahead of the proceeding polymerase
ASE activity hydrolyzes nucleotides in nicked strands
Understand. Thus, the nick is moved along the chain.
Translated). The rate of hydrolysis is equal to the rate of polymerization
And there is no net synthesis. However, this exchange reaction is in progress
The radioactive deoxynucleotides present in the reaction mixture
Dotriphosphate is incorporated into DNA (Kelly et al., 1).
970, J.A. Biol. Chem. 245: 39). So
After,³²P-labeled gD-1 double-stranded DNA was
Modified for use as probes (Maniatis
Et al., Molecular Cloning, A Lab.
operative Manual, ColdSpring
  Harbor Laboratories, pp. 1
09-112). By autoradiography, pHV1
About 2.3 kb of XhoI / XhoI DNA fragment
Must be complementary to the radioactive gD-1 probe.
And therefore contains the gD-2 coding sequence
Was out. Next, further characterize the gD-2 coding sequence
XhoI / XhoI DNA flag of pHV1
Was subcloned into pBR322. That
First, pHV1 was digested with XhoI and the gD-2 coding system was digested.
The 2.3 kb DNA fragment containing the sequence was
(The cohesive end generated by SalI is
(Complementary to the cohesive end generated).
Annealed linear pBR322 in a 1: 1 ratio
Ligation was performed to create pHV2 (FIG. 11c). 7.2.3.gD-2 mRNA coding sequence
Column characterization The HSV-DNA of pHV2 is Maxam and Gilber
t method (1980, Methods in Enzy)
(Molology, 65: 499).
Was. FIG. 12 shows the obtained DNV of the HSV-2 gD gene.
A sequence is shown. This DNA sequence has 393 codons (gD
-1 DNA codon less than the DNA coding sequence).
Including Pun reading frame, ATG of 267th
It was growing. This site is the initiator of the gD-2 gene.
The estimated gD-2
The gene sequence was transformed into the expression vectors pJS413 and pHK4.
13 (see below)
I confirmed that Predicted amino acids of gD-2 protein
The sequence is the predicted amino acid sequence of the gD-1 protein.
(FIG. 13). The two sequences are about 82% homologous
It seems that the non-homologous region exists in the following three regions.
You. That is, leader sequence, transmembrane region, and putatively
Is the anchor area. gD-2 protein is gD-1
One amino acid residue shorter than the protein. 7.3.Cloning of gD-2 gene
And expression The gD-2 gene was placed under the control of the lac promoter.
First, the first amino-code end (5 'end of the gene)
The part of the putative gene sequence that lacks 120 nucleotides is p
Isolated from HV2. pHV2 DNA fragment
Lac promoter, translation regulatory element, croATG
And small pJS4 containing 69 nucleotides of cro
13 DNA fragment (about 200 bp)
Was. The resulting recombinant plasmid pHV5 (FIG. 14)
including all but the first 40 codons of the gD-2 sequence
I have. In fact, pHV5 is actually a mature gD-2 protein.
All but 15 amino acids. Through
Usually, the first 25 amino acid residues of gD-2 (signal sequence)
Column) is where the native gD-2 protein is processed
When it is cut. The construction of pHV5 is described below. 7.3.1.Construction of pHV5 The recombinant plasmid pHV2 was digested with ClaI (FIG. 14).
), And the resulting cohesive ends were replaced with Kle of DNA polymerase.
Repaired with now fragment. Next, blunt-ended
The linear pHV2 was cut with EcoRI. amp^rgene
And 120 nucleotides of the gD-2 coding sequence
5.4 kb ClaI, including all^(-)/ EcoR
I DNA fragments were subjected to agarose gel electrophoresis.
Therefore, it was isolated. The expression vector pJS413 (section 6.3.1) was used.
Section) was cut with SmaI and EcoRI. lac
Promoter, SD^z, SD^cro, CroATG and
200 bp encoding the 69 nucleotides of
(0.2 kb) fragment
Electrophoresis. 5.4 kb ClaI
^(-)/ EcoRI pHV2 DNA fragment
0.2 kb EcoRI / SmaI pJS413 D
The NA fragment was used at a 1: 1 molar ratio with T4 DNA
Ligation was carried out using the enzyme. The resulting recombinant plasmid p
HV5 was used for transformation of E. coli NF1829 strain. 7.3.2.Form expressing gD-2 gene
Transformant identification Ampicillin-resistant plasmids isolated from E. coli transformants
Rasmids are obtained by restriction endonuclease mapping.
Analyzing and further expressing a gD-2-related polypeptide
I checked its ability. In NF1829, (l
lac promoter (for overproduction of ac repressor)
Since the protein is transcriptionally inactive, the gD-2-related protein
The protein is 1 mM IPTG or 1-10 mM lactose
Can only be detected after inducing the promoter with
Would. The recombinant plasmid designated pHV5
The transformed clones are inducible gD-2-related tags.
It was found to produce protein. Inductive tamper
The protein is immunoprecipitated with rabbit antiserum against HSV-2.
It turned out to be. 7.4.Cro / gD-2 / β-galactosidase fusion
Production of pHV6 for protein production Translation reading frames to create fusion proteins
GD-2 so that the system is not interrupted by the termination signal.
The rows were ligated to the bacterial host gene sequence. for that reason
The deletion of the gD-2 termination signal (TAG)
The gD-2 coding sequence of pHV5 was cleaved. lac
Motor, translational regulatory element and Cro / gD-2 sequence
The pHV5 DNA fragment containing
PHK414 DNA fragment.
The resulting recombinant plasmid pHV6 (FIG. 15)
/ GD-2 / β-galactosidase fusion protein
Is loaded. 7.4.1.Construction of pHV6 Plasmid pHV5 digested with PstI and BamHI
(See FIG. 15). amp^rAmino code end of gene
Part of the end, lac promoter, SD^zSD^cro, C
1.7 encoding roATG and cro / gD-2
5 kb PstI / BamHI pHV5 DNA fragment
Fragment was isolated by agarose gel electrophoresis. The expression vector pHK414 was replaced with PstI and B
Digested with amHI. z gene and amp^rGenetic
5.54 kb encoding part of the carboxy code end
BamHI / PstI pHK414 DNA Flag
Mentions were isolated by agarose gel electrophoresis.
1.75 kb PstI / BamHI pHV5 DN
A fragment and 5.54 kb BamHI / PstI
  Anneal pHK414 DNA fragment
And ligated in a 1: 1 molar ratio and E. coli NF1
829. Instructions β-gala on agar plate
By assaying for custosidase activity,
The fusion protein production ability of the non-resistant colonies was examined. Positive
Colonies show all of the transformants induced by IPTG.
SDS-PAGE analysis of the lysate revealed Cro / gD
-Examine the existence of β-galactosidase fusion protein
Tested. 160,000 dalton fusion protein high
The level producer is isolated and the plasmid obtained from this transformant is
The rasmid was named pHV6. E. coli transformed with pHV6
The fusion protein produced from the loan was
And HSV-1 and HSV-2.
Were found to be cross-immunoreactive with both. Produced
The fusion protein was composed of 265 gD-2 proteins.
Contains amino acid residues. 7.4.2.Antiserum against pHV6 fusion protein
Immunoprecipitation analysis The pHV6 fusion protein is the denatured protein described in Section 6.4.2.
Purified using the protocol, three rabbits (R15
9, R160, R161) to make antiserum
Used as E. coli clone transformed with pHV6
Is grown to mid-log phase and 1 mM IPTG
Induced by Four hours after induction, the bacteria are centrifuged.
Pellet by separation, SDS-PAGE sample
Lysis using buffer and separate SDS-poly
Placed on acrylamide gel. After electrophoresis,
Stained with Coomassie Blue dye to allow protein
Visualized. Next, a 160,000 dalton fusion tamper
The dentin band was cut out of the gel. Gel section with liquid nitrogen
And suspended in PBS after crushing. Next
An equal volume of Freund's complete adjuvant was added. Ten
After mixing, the solution was added to three New Zealand
Subcutaneous injection into herons (R159, R160, R161)
Was. Inject 100-200 μg of protein into each rabbit
did. 28 days later, during incomplete Freund's adjuvant
Boost rabbits with the same amount of turbid fusion protein
Was given. Animals receive a total of 5 boosts every 10 days.
I got it. 48 days after the first injection (ie two additional immunizations)
The serum collected after the post-infection) was used for immunoprecipitation analysis. The immunoprecipitation was performed as follows. Increase congestion
The grown cells were infected with HSV at 10 pfu / cell
(Infect Vero cells with HSV-1 while Hela
Cells were infected with HSV-2). After infection³⁵S-me
Cells were labeled with thionine for 16 hours.³⁵S-methioni
-Labeled HSV-1 infected Vero cells or HSV-2
The lysate of infected Hela cells was used for pre-immune rabbit serum or
Is a rabbit antiserum against the pHV6 fusion protein (immediately
R159, R160 or R161 antiserum) 10μ
l. Described in Section 6.3.3
The immune complex was recovered using pansorbin as
The radiolabeled immunoprecipitated protein obtained was purified by SDS-P
Separated by AGE and subjected to fluorography. SDS
-According to PAGE results, HSV-2 infected Hela cells
50,000 dalton gD tan produced by vesicles
The protein is contained in the fusion protein produced from the transformant.
Immunoreacted with the corresponding antiserum. R159 and R161
The antiserum is specific for gD-2, while R160
The antiserum was isolated from gD-1 (HSV-1 infected Vero cells).
52,000 dalton gD protein produced)
And gD-2 are both immunoprecipitated. Therefore, R16
0 is “type-common”. By pEH4-2
Induced against the loaded gD-1 fusion protein
018 (Section 6.4.2) is also type-common
It should be noted. 7.4.3.Herpes simplex virus in
in vitro neutralization Rabbit antiserum as described above against pHV6 fusion protein
Its ability to neutralize HSV infection in vitro
Used to find out. For that, Plaque Asses
Virus neutralization experiments using A were performed as described above. Collection
Each dish (35 mm) of dense cells contains a control (pre-immune serum) or
Is the test antiserum dilution (the following antisera were tested: 018,
R159, R160, R161)
Was infected with 50 pfu of HSV (Vero
Cells are infected with HSV-1 while Hela cells are infected with H
SV-2). After 3 days, fix cells and stain
And counted the plaques. Table 2 shows two such experiments.
The result is shown. Neutralization reduces plaque count by 50%
It is expressed as the required serum dilution. From Table 2, p
Antiserum against HV6 fusion protein (R159, R1
60, R161) is HSV-2 infected in vitro
Can be neutralized while the pEH4-2 fusion protein
Antiserum (018) is HSV-1 infected in vitro
It is clear that can be neutralized. 018 and R160
Type (based on immunoprecipitation data in section 7.4.2)
-Common but 018 (anti-gD-1 fusion protein)
Is more effective at neutralizing HSV-1 in vitro
And R160 (anti-gD-2 fusion protein) has i
more effective in neutralizing HSV-2 infection in vitro
is there. [0182] [Table 2][0183]¹The figures show blood that reduced plaque count by 50%
The antibody titer is represented as the reciprocal of the dilution ratio. Assay is serum
Performed in the presence of complement. 7.4.4.Antiserum against pHV6 fusion protein
Immunofluorescence analysis The immunofluorescence assay was performed as follows. Herpes on confluent cells
Virus at 0.1 pfu / cell at 37 ° C for 20 hours
(Vero cells were infected with HSV-1 while
Hela cells were infected with HSV-2). Cell to P
Wash with BS, then use acetone: methanol (1: 1)
And fixed at room temperature for 90 seconds. Remove fixative and remove cells
Was air dried. Then diluted 1:20 in PBS
Each rabbit antiserum (018, R159, R160 and R
161) Separate 20 μl aliquots to the marked locations
Individual drops were added to the cells on the plate. 37 ° C, 3
After the 0 minute incubation, wash the cells with PBS and allow
Then it was dried. Next, a fluorescein-conjugated swine anti-rabbit
A 20 μl aliquot of protein serum was added to the marked location.
Was. Wash cells after 30 minutes incubation at 37 ° C
Dried naturally, fixed with glycerol,
And observed under UV light. The presence of fluorescence is a rabbit
Antiserum is immunoreactive with HSV-infected cells
You. The results are shown in Table 3. [0184] [Table 3] [0185]¹Antiserum was added at a 1:20 dilution.² ++ is the strongest cell staining ++ is moderate cell staining + Is positive but weak staining 8.Example: Vaccination 8.1. Using gD-1 fusion protein in vaccine formulation
WasProtection from in vivo HSV-2 infection The following preparations were prepared for 3 groups of 10 Balb C mice per group.
The material was injected intraperitoneally (IP). Group 1 (Non-Wakuchi
Group received saline, and group 2
Natural HSV-1gD in Complete Freund's Adjuvant
Received 3 μg of protein, and group 3
PEH4-2 fusion protein in saline (described in Section 5.5)
30 μm isolated by the denatured aggregate purification method described above)
g received. All groups received 4 IP injections
Excision, administration of each injection at 2-week intervals, and
Plasmids are 1 μg gD (group 2) or 10 μg fusion
Contained protein. After the fourth injection, mice were collected retro-orbitally.
Bloody, previously described in Sections 6.4.3 and 7.4.2
In vitro (complementation) using the plaque assay
Serum in the absence of the body) for herpes virus neutralization.
Tested. One week after the fourth inoculation, 10L
D₅₀Mice were challenged with HSV-2 strain 186
I did it. HSV-2 is suspended in 0.5 ml and injected intraperitoneally.
Fired. Survival of challenged mice indicates protection.
Table 4 shows the results. [0187] [Table 4] 8.2.GD-2 fusion in vaccine formulation
From in vivo HSV-2 infection using proteins
Defense of The following preparations were vaccinated with 4 groups of 10 mice per group:
Seeded. Group 1 (control) is pNB9-1 bovine growth host
Receiving Lumon / β-galactosidase fusion protein
Group 2 is pEH4-2 Cro / gD-1 / β
-Receiving galactosidase fusion protein (Section 6.4)
In group 3, pEH82 reconstructed Cro / gD-
1 / β-galactosidase fusion protein (Section 6.5
Group 4 is pHV6Cro.
/ GD-2 / β-galactosidase fusion protein (primary
(See section 7.4). The fusion protein may be derived transformants.
Is disrupted with lysozyme / surfactant, and
Lett (non-denaturing coagulation as described in Section 5.5)
Various purification of Escherichia coli transformants
Isolated. The immunization schedule is as follows:
Was. 0.2 ml in Balb C mice (6-7 weeks old)
Non-denatured fusion protein in saline
μg was inoculated intraperitoneally. 75-100 μg fusion to mouse
Intraperitoneal re-inoculation of protein three times or more every two weeks (additional
(Immunization) (4 inoculations in total). After the fourth injection,
Blood was collected from the mice retro-orbitally, at sections 6.4.3 and
Using the plaque assay described previously in section 7.4.2
And herpes wis in vitro (in the absence of complement)
The sera were tested for ruth neutralization. One week after the fourth inoculation, 1
7LD₅₀Of mice with HSV-2 strain 186
I ranged. HSV-2 was suspended in 0.5 ml,
Injection. Survival of challenged mice shows protection
You. Table 5 shows the results. [0191] [Table 5][0192]¹Fusion protein suspended in physiological saline
Was inoculated with IP.² HSV-1 or HSV-2 neutralization in the absence of complement
And tested serum. Numbers reduce plaque count by 50%
The antibody titer is expressed as the reciprocal of the serum dilution rate. 8.8.Comparison of vaccine formulations pEH4-2 and pEH90-10amLE392
Fusion protein preparations produced from
Mice with a mixture of different adjuvants
did. The obtained antiserum was used for HSV-1 infected cells in culture.
Its ability to immunoprecipitate proteins from
Was evaluated. The non-denaturing technique described in Section 8.2
PEH4-2 and pEH90-10amLE39
The fusion protein was isolated from two transformants. Aggregates
Resuspend (form a slurry) by sonication and
And solubilized by treatment in hot alkali. 0.5MNa
OH was added to the aggregate pellet at a final concentration of 50 mM.
Was. Agglomerates can be removed by heating at 65 ° C for 15 minutes.
Solubilized. Next, the solution is cooled and tris-HCl (pH
7.5) was added (final concentration: 100 mM Tris-
HCl). Fusion protein preparation (slurry or heat
Alkaline preparation) mixed with the following adjuvant before inoculation
did. (1) L121 (Hunter et al., 1981,
J. of Immunol. 127 (3): 1244-
1250; BASF Wyandotte Corpo
ration, Wyandotte, Mich. )pull
Ronic polyol (2.5 mg per animal
(2) Aluminum hydroxide gel (Rehe
is Chemical Company, Berke
ley Heights, N.M. J. ) 0.2% final concentration
Every time. Using these preparations, the following schedule
Mice (CD-1 strain) were immunized according to Each ma
Mice receive a primary injection of 100 μg of fusion protein,
After 22 days, 50 μg of the fusion protein was re-injected. fusion
Suspend protein in a total volume of 0.2 ml and intramuscularly into thigh
Injected. 7 days after the last injection, from the retro-orbital of the mouse
Blood was collected. The collected serum was subjected to the following immunoprecipitation.
And analyzed.³⁵S-methionine labeled HSV-1 infected V
The lysate of ero cells was added to 5 μl of mouse serum (anti-pE
H4-2 or anti-pEH90-10amLE392),
Pre-immune serum (negative control) or monoclonal 4S (positive)
Sex control) for 2 hours at 4 ° C.
l rabbit anti-mouse serum (Dako, 30 minutes at 4 ° C)
Incubated. The immune complex is described in section 6.3.3.
Collect with pansorbin as described, immunoprecipitate
Radiolabeled protein was analyzed by SDS-PAGE.
Released and subjected to fluorography. Fluorography
From the results of the above, the alkali with L121 adjuvant added
Solubilized fusion proteins (pEH4-2 and pEH90-1)
0amLE392 both) immunized with all mice
Was found to have a strong immune response. Hydroxylation
Alkali solubilized pEH4 administered with aluminum
A weaker immune response was observed with the -2 fusion protein
Was. Other mice were exempt from this test.
It did not appear to show epidemics. 9.Microbial deposit All base pair sizes given for nucleotides are approximate
Should be understood as a value and used for explanation.
is there. In addition, many modifications of the invention shown above and
Changes are made without departing from the spirit and scope of the
It is clear that this can be done. Specific embodiments described
Is only an example, and the present invention is defined by the appended claims.
It is stipulated only. The next strain of E. coli carrying the indicated plasmid was
American Type Culture Collection (Am
erican Type Culture Colle
ction: ATCC) (Rockville, M
d. ) And was assigned the following accession numbers: The next strain of E. coli carrying the indicated plasmid was Agricultural
Natural Research Culture Collection (Agr
icultural Research Cultur
e Collection: NRRL) (Peori
a, Ill. ) And given the next accession number
Was. The present invention is not limited in its scope by the deposited microorganism.
What is claimed is that the deposited embodiments may
This is because the embodiment is exemplified. In fact, here
In addition to what has been described, various modifications of the invention are described above.
And the accompanying drawings will be apparent to those skilled in the art. like this
Modifications are also included in the claims.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 (a) HSV-1 genome, (b) HSV-1 EcoRI of lambda gtWES :: EcoRI-H
Figure 3 shows the restriction map of the -H fragment insert, (c) pR
(C) shows construction of WF6 and (d) H of pSC30-4
FIG. 3 shows a restriction map of the SV-1 SacI DNA fragment insert. FIG. 2 shows a gD-1 gene sequence sequencing strategy and a restriction map. FIG. 3. Nucleotide sequence of gD-1 gene and gD
FIG. 2 is a diagram showing a deduced amino acid sequence of -1 protein. FIG. 4. Recombinant plasmid pEH2 derived from a part of gD-1 gene and E. coli expression vector pJS413
FIG. 5 is a diagram showing the construction of Example 5; FIG. 5. DN of the Cro / gD-1 junction in pEH25.
FIG. 3 is a view showing an A sequence and a deduced amino acid sequence. FIG. 6 shows the inhibition of immunoprecipitation of the pEH25gD product by adding a competing antigen present in the lysate of HSV-infected Hela cells. FIG. 7 shows the construction of a gD-1 expression plasmid pEH4-2 derived from pEH25. FIG. 8. Many gD-1 expression plasmids pEH50 + x
FIG. 4 is a view showing a method of manufacturing the device. FIG. 9 is a diagram showing a method for reconstructing the amino coding end of the gD-1 gene in pEH4-2. FIG. 10 shows the construction of a gD-1 expression plasmid pEH90-10am derived from pEH3-25 and pHK414. FIG. 11 shows (a) HSV-2 genome and (b) pH
Figure 6 shows a restriction map of the BglII L fragment insert of V1 and (c) the HSV-2 XhoI of pHV2.
FIG. 3 shows a restriction map of a DNA fragment insert. FIG. 12. Nucleotide sequence of gD-2 gene and g
It is a figure showing the deduced amino acid sequence of D-2 protein. FIG. 13 is a diagram showing a comparison between deduced amino acid sequences of gD-1 and gD-2. FIG. 14 shows the construction of a recombinant plasmid pHV5 derived from a part of the gD-2 gene and pJS413. FIG. 15 shows the construction of a gD-2 expression plasmid pHV6 derived from pHV5.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI // C12N 1/21 C12N 15/00 ZNAA (C12N 15/09 ZNA C12R 1:92) (C12P 21/02 C12R 1:91) Microorganism accession number NRLL B-15451 Microorganism accession number NRRL B-15471 (72) Inventor Lynn William Enquist United States 55331 Excelsior Zambra Drive 5691 Minnesota

Claims (1)

(57) [Claims] The following amino acid sequence: Herpes simplex virus type 1 (HSV-1)
A non-glycosylated polypeptide of a gD glycoprotein; a partial sequence of an HSV-1 polypeptide having at least one immunological antigenic determinant of said gD glycoprotein; the following amino acid sequence: Herpes simplex virus type 2 (HSV-2)
A herpes simplex virus gD glycoprotein selected from the group consisting of: a non-glycosylated polypeptide of a gD glycoprotein; and a partial sequence of an HSV-2 polypeptide having at least one immunological antigenic determinant of said gD glycoprotein. Non-glycosylated polypeptide. 2. E. coli having ATCC accession number 39,159, A
E. coli having TCC accession number 39,160, NRRL
Produced by E. coli having the accession number B-15449, E. coli having the NRRL accession number B-15450, E. coli having the NRRL accession number B-15451, and E. coli having the NRRL accession number B-15471. A non-glycosylated polypeptide of a herpes simplex virus gD glycoprotein according to claim 1. 3. The following amino acid sequence: Herpes simplex virus type 1 (HSV-1)
A non-glycosylated polypeptide of a gD glycoprotein of the following; a partial sequence of an HSV-1 polypeptide having at least one immunological antigenic determinant of said gD glycoprotein; the following amino acid sequence: Herpes simplex virus type 2 (HSV-2)
A herpes simplex virus gD glycoprotein selected from the group consisting of: a non-glycosylated polypeptide of a gD glycoprotein; and a partial sequence of an HSV-2 polypeptide having at least one immunological antigenic determinant of said gD glycoprotein. A vaccine preparation for protection against herpes simplex virus infection, comprising a non-glycosylated polypeptide. 4. E. coli having ATCC accession number 39,159, A
E. coli having TCC accession number 39,160, NRRL
Produced by E. coli having the accession number B-15449, E. coli having the NRRL accession number B-15450, E. coli having the NRRL accession number B-15451, and E. coli having the NRRL accession number B-15471. 4. The vaccine formulation of claim 3, comprising a non-glycosylated polypeptide of herpes simplex virus gD glycoprotein. 5. The following amino acid sequence: Herpes simplex virus type 1 (HSV-1)
A non-glycosylated polypeptide of a gD glycoprotein; a partial sequence of an HSV-1 polypeptide having at least one immunological antigenic determinant of said gD glycoprotein; the following amino acid sequence: Herpes simplex virus type 2 (HSV-2)
A herpes simplex virus gD glycoprotein selected from the group consisting of: a non-glycosylated polypeptide of a gD glycoprotein; and a partial sequence of an HSV-2 polypeptide having at least one immunological antigenic determinant of said gD glycoprotein. A method for producing a non-glycosylated polypeptide, comprising: a) the following DNA sequence: A DNA sequence encoding a polypeptide having the amino acid sequence of a gD glycoprotein of HSV-1 consisting of: HSV-1DN encoding a polypeptide having at least one immunological antigenic determinant of the gD glycoprotein
A partial sequence of A; the following DNA sequence: A DNA sequence encoding a polypeptide having the amino acid sequence of an HSV-2 gD glycoprotein; and an HSV-2DN encoding a polypeptide having at least one immunological antigenic determinant of the gD glycoprotein.
A subsequence of A comprising a DNA sequence selected from the group consisting of: culturing a unicellular organism capable of expressing said DNA sequence or subsequence to produce said polypeptide; and b) Isolating the peptide. 6. The method according to claim 5, wherein the unicellular organism carries a recombinant vector containing the DNA sequence or a partial sequence thereof. 7. The unicellular organism is ATCC Accession No. 39,159.
E. coli having ATCC accession number 39,160, E. coli having NRRL accession number B-15449, E. coli having NRRL accession number B-15450, E. coli having NRRL accession number B-15451,
7. The method of claim 6, wherein the E. coli strain is selected from the group consisting of E. coli having NRRL accession number B-15471.