CA1341644C - Recombinant dna molecules and their method of production - Google Patents
Recombinant dna molecules and their method of productionInfo
- Publication number
- CA1341644C CA1341644C CA342497A CA342497A CA1341644C CA 1341644 C CA1341644 C CA 1341644C CA 342497 A CA342497 A CA 342497A CA 342497 A CA342497 A CA 342497A CA 1341644 C CA1341644 C CA 1341644C
- Authority
- CA
- Canada
- Prior art keywords
- hepatitis
- hbv
- antigen
- recombinant dna
- polypeptide
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Abstract
Recombinant DNA molecules and hosts transformed with them which produce polypeptides displayihg HBV anti-genicity and genes coding therefor and methods of making and using these molecules, hosts, genes and polypeptides. The recombinant DNA molecules of this invention are characterized by structural genes that code for at least one polypeptide displaying HBV antigenicity. In appropriate hosts these recombinant DNA molecules permit the production and identification of genes and polypeptides characteristic of HBV antigenicity and their use in compositions and methods for detecting HBV virus infections in humans and stimulating the production of antibodies against this infection.
Description
11111 , -, \ -' ' RECOMBINANT DNA MOLECULES
AND THEIR METHOD OF PRODUCTION
TECHNICAL FIELD OF INVENTION
This invention relates to recombinant DNA
molecules and their method of production. This invention relates more particularly to recombinant DNA molecules expressed in appropriate host organisms. The recombinant DNA molecules disclosed herein are characterized by the DNA
that codes for polypeptides with the specificity of hepatitis B viral antigens. As will be appreciated from the disclosure to follow, the recombinant DNA molecules may ( be used in the detection of hepatitis B virus in man and the stimulation of antibodies in humans against this virus:
BACKGROUND ART
, The virus that causes hepatitis B or serum - - hepatitis appears to infect only man. Hepatitis B virus ("HBV") infection in humans is widespread. In the United Kingdom, United States, and Western Europe, approximately 0.1% of all blood donors are chronic carriers of HBV.
While the death rate due to viral hepatitis is not high (in 1975 in the United Kingdom it was 3 per million), there are indications that as many as 5% of the population in the United Kingdom and 15% of the population in the United States have been infected. In many African and Asian countries, up to twenty percent of the population are chronic carriers of HBV, and over fifty percent of all adults in those countries have been or are infected with HBV.
;
,,...
V) .
AND THEIR METHOD OF PRODUCTION
TECHNICAL FIELD OF INVENTION
This invention relates to recombinant DNA
molecules and their method of production. This invention relates more particularly to recombinant DNA molecules expressed in appropriate host organisms. The recombinant DNA molecules disclosed herein are characterized by the DNA
that codes for polypeptides with the specificity of hepatitis B viral antigens. As will be appreciated from the disclosure to follow, the recombinant DNA molecules may ( be used in the detection of hepatitis B virus in man and the stimulation of antibodies in humans against this virus:
BACKGROUND ART
, The virus that causes hepatitis B or serum - - hepatitis appears to infect only man. Hepatitis B virus ("HBV") infection in humans is widespread. In the United Kingdom, United States, and Western Europe, approximately 0.1% of all blood donors are chronic carriers of HBV.
While the death rate due to viral hepatitis is not high (in 1975 in the United Kingdom it was 3 per million), there are indications that as many as 5% of the population in the United Kingdom and 15% of the population in the United States have been infected. In many African and Asian countries, up to twenty percent of the population are chronic carriers of HBV, and over fifty percent of all adults in those countries have been or are infected with HBV.
;
,,...
V) .
-2-The hepatitis infection is transmitted by three general mechanisms: (1) by parenteral inoculation of infected blood or body fluids, either in large amounts as in blood transfusions or in minute amounts as through an accidental skinprick; (2) by close family or ---sexual contact; and (3) by some mothers, who infected during pregnancy, transmit the virus to their new-born children.
Under natural conditions, HBV is not highly contagious.
Transmission by inhalation occurs rarely, if ever.
Most HBV infections are subclinical. The clinical illness usually lasts three to six weeks and ranges in severity from mild to acute fulminating hepatitis followed by cirrhosis or death. Recovery from clinical and subclinical HBV infections is usually complete. However, serious long term consequences result in some cases:
(1) approximately five percent of acute infections lead to chronic carriage of hepatitis B virus antigen with its continuing potential for infectivity to others and ongoing liver damage; and (2) it is likely that past infection with HBV may be wholly or partly responsible for the initiation of a significant proportion of HBV-seronegative cases of chronic active hepatitis, cirrhosis and primary liver carcinoma.
Recent advances in molecular biology have made it possible to introduce the DNA coding for specific non-bac-terial eukaryotic proteins into bacterial cells. In general, with DNA other than that prepared via chemical synthesis, the construction of the recombinant DNA
molecules comprises the steps of producing a single-stranded DNA copy (cDNA) of a purified messenger (mRNA) template for the desired protein; converting the cDNA to double-stranded DNA; linking the DNA 'to an appropriate site in an appropriate cloning vehicle and transforming an appropriate host with that recombinant DNA molecule. Such transformation permits the host to produce the desired protein. in addition, at least in the case of ovalbumin DNA, it is known that appropriate fusion of the particular
Under natural conditions, HBV is not highly contagious.
Transmission by inhalation occurs rarely, if ever.
Most HBV infections are subclinical. The clinical illness usually lasts three to six weeks and ranges in severity from mild to acute fulminating hepatitis followed by cirrhosis or death. Recovery from clinical and subclinical HBV infections is usually complete. However, serious long term consequences result in some cases:
(1) approximately five percent of acute infections lead to chronic carriage of hepatitis B virus antigen with its continuing potential for infectivity to others and ongoing liver damage; and (2) it is likely that past infection with HBV may be wholly or partly responsible for the initiation of a significant proportion of HBV-seronegative cases of chronic active hepatitis, cirrhosis and primary liver carcinoma.
Recent advances in molecular biology have made it possible to introduce the DNA coding for specific non-bac-terial eukaryotic proteins into bacterial cells. In general, with DNA other than that prepared via chemical synthesis, the construction of the recombinant DNA
molecules comprises the steps of producing a single-stranded DNA copy (cDNA) of a purified messenger (mRNA) template for the desired protein; converting the cDNA to double-stranded DNA; linking the DNA 'to an appropriate site in an appropriate cloning vehicle and transforming an appropriate host with that recombinant DNA molecule. Such transformation permits the host to produce the desired protein. in addition, at least in the case of ovalbumin DNA, it is known that appropriate fusion of the particular
-3-ti DNA to a strong bacterial promoter or expression control sequence produces larger amounts of the desired ovalbumin protein, i.e., about 0.5 to 1% of the total protein mass of an E. coil cell (0. Mercereau-Puijalon et al., "Synthesis Of An Ovalbumin-Like Protein By Escherichia COli K12 Har-boring A Recombinant Plasmid", Nature, 275, pp. 505-510 (1978) and T.H. Fraser and B.J. Bruce, "Chicken Ovalbumin Is Synthesized And Secreted By Escherichia coil", Proc.
Natl. Acad. Sci. USA, 75, pp. 5936-5940 (1978)).
Several non-bacterial proteins and genes have been synthesized using recombinant DNA technology. These include a protein displaying rat proinsulin antigenic determinants (L. Villa-Komaroff et al., "A Bacterial Clone = 'Synthesizing Proinsulin", Proc. Natl. Acad. Sci. USA, 75, pp. 3727-3731 (1978)), rat growth hormone (P.H. Seeburg et al., "Synthesis Of Growth Hormone By Bacteria", Nature, 276, pp. 795-798 (1978)), mouse dihydrofolate reductase (A.C.Y. Chang et al., "Phenotypic Expression in E. coil Of A DNA Sequence Coding For Mouse Dihydrofolate Reductase", = 20 Nature, 275, pp. 617-624 (1978)), somatostatin (K.
Itakura et al., "Expression In Escherichia coli Of A Chemically Synthesized Gene For The Hormone Somatostatin", Science, 198, 1056-1063 (1977), United Kingdom patent specifications 2,007,675A, 2,007,676A and 2,008,123A and cognate applica-tions in other countries), and the A and B polypeptide chains of human insulin (D.V. Goeddel et al., "Expression In Escherichia coil Of Chemically Synthesized Genes For Human Insulin", Proc. Natl. Acad. Sci. USA, 76, pp. 106-110 (1979) and the United Kingdom and related patent specifi-cations, supra).
None of the foregoing, however, is directed, as is this invention, toward the recombinant DNA synthesis of viral proteins such as hepatitis B virus antigens.
It is known that HBV infections cause development of antibodies to the antigens of the virus. These anti-bodies are the body's defense mechanism to the HBV infec-tion. The development of such antibodies prior to exposure or rapidly in the case of potential exposure should substan-tially reduce and combat virus growth and spread in the patient.
A problem however in the artificially stimulated development of antibodies to the antigens of hepatitis B
virus is the limited host range of the virus. For example, although highly infectious to man, experimental infection with hepatitis B virus has been achieved in only a few additional primates. And, the virus has not been propa-gated in tissue culture. This limited host range and inability to infect tissue culture cells has severely hampered both the characterization of the virus and the pathology of its infection and the development of rapid =detection means and effective means of infection control and prevention.
While there have been some attempts to employ authentic HBV viral antigens, isolated from victims of the HBV infection, for the development of antibodies and detec-tion of infection, these treatments are not generally available because of the limited supply of the active species. Furthermore, the use of human sources for these antigens is disfavored because of the well recognized contamination problems in using human isolates.
DISCLOSURE OF THE INVENTION
The present invention solves the problems referred to by providing in accordance with the invention a recombin-ant DNA molecule characterized by a structural gene coding for a polypeptide displaying HBV antigenicity.
By virtue of our invention, it is possible to obtain HBV antigens and genes in substantial and uncon-taminated quantities for vaccine preparations and for use in detection of the viral infection and determination of its pathology and molecular biology. Such supplies have not hitherto been available, because of the narrow host range of the virus and its inability to be grown in tissue culture. .
I ;
' 1111 ...5_ As will be appreciated from the disclosure to follow, the recombinant DNA molecule of the invention is capable of producing, in an appropriate host, at least one viral polypeptide displaying HBV antigenicity and the structural genes that code therefor. These recombinant DNA
molecules and hosts may be utilized to prepare polypeptides displaying HBV antigenicity and structural genes coding for these polypeptides. These products may also be identified and characterized and are useful either as produced in the host or after appropriate derivatization or modification in compositions and methods for improving production of these products themselves and for detecting HBV infection, tracing its pathology and stimulating the production of HBV anti-bodies in humans.
In accordance with the invention we also provide a process for producing a recombinant DNA molecule, charac-terized by linking a DNA sequence prepared from the endog-enous DNA of a_Dane particle to another DNA sequence pre-pared from a source other than the Dane particle.
= 20 Our process may be distinguished from the prior processes above mentioned in that none of the prior pro-cesses employs a natural gene or DNA for a particular protein for construction of the recombinant DNA molecule and production of that protein or gene. Instead, they employ either synthetic genes made by chemical synthesis or = artificial genes made by enzymatically copying the mRNA
isolated from the donor cell to produce cDNA sequences.
One reason that natural DNA has not been pre-viously employed directly in recombinant DNA synthesis of proteins is that natural DNA's from most higher organisms and at least some animal viruses contain Hintrons" or additional nucleotide sequences as part of the gene. These = introns do not form part of the final message of the gene.
Instead, they are removed in vivo in higher organisms by special processing enzymes acting upon the primary trans-cription product to afford the ultimate message (mRNA) of the gene. Bacteria are presumed to be unable to process such introns so that natural DNA would not be expected to -be expressed in bacterial hosts and the desired proteins would not be expected to be produced by these hosts.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a simplified diagram re-Presenting the structure of the endogenous DNA of a Dane particle. It displays the two complementary DNA strands, strand a and b, .the nick in strand a and the gap in strand b and the closing of that gap by DNA polymerase reaction.
Figure 2 is a schematic outline of the preferred embodiment of the process of this invention: fragments of the endogenous DNA isolated from the Dane particle are fused to the E. coli penicillinase gene on the plasmid =
'pBR322 at the Pst I site. After transformation into E.
coil HB101, the recombinant DNA molecule pBR322-Pst I dG:
HBV-KpnI dC directs the synthesis of polypeptides display-ing HBV antigenicity.
Figures 3-9 show the nucleotide sequence deter-mined for a part of a hepatitis B virus genome in accord-ance with this invention. The sequence is displayed with the stop codons in the three reading frames noted above it.
It is arbitrarily numbered from the initiation codon for the gene coding for HBcAg as determined in this invention.
Nucleotides -80 to -1 represent the leader sequence which precedes this gene. Nucleotides 1-549 represent the nucleo-tide sequence for the gene coding for hepatitis B virus core antigen, the amino acid sequence (reading frame 1) of this antigen being depicted above that sequence. Nucleo-tides 1437-2114 represent the nucleotide sequence deter-mined for the gene of hepatitis B virus surface antigen, the amino acid sequence (reading frame 3) of this antigen being depicted above that sequence. Various restriction endonuclease recognition sites in these genes are also depicted in Figures 3-9.
Figure 10 is a schematic outline of a process of this invention wherein a recombinant DNA molecule which is capable of producing a polypeptide displaying hepatitis B
=
I.. ' 11) -7-virus core antigenicity is fragmented and a fragment therefrom attached to an improved expression control sequence, the lac system.
5 Figure 11 is a schematic outline of a pro-cess of this invention wherein a recombinant DNA
molecule of this invention is fragmented and con-nected to a fragment of phage DNA for use in lyso-genizing host cells and thereby increasing the number of gene copies therein.
Figure 12 is a schematic outline of another embodiment of the process of this invention: a re-combinant DNA molecule of this invention which does not produce a polypeptide displaying hepatitis B
virus core antigenicity is fragmented to remove the structural gene for HBsAg and this structural gene employed to produce a recombinant DNA molecule which produces HBsAg in an appropriate host.
BEST MODE OF CARRYING OUT THE INVENTION
20 In order that the invention herein described may be more fully understood, the following detailed description is set forth.
In the description the following terms are employed:
25 Nucleotide--A monomeric unit of DNA or RNA
consisting of a sugar moiety (pentose), a phosphate, and a nitrogenous heterocyclic base. The base is = linked to the sugar moiety via the glycosidic carbon (1' carbon of the pentose) and that combination of 30 base and sugar is a nucleoside. The base character-izes the nucleotide. The four DNA bases are adenine ("A"), guanine ("G"), cytosine ("C") and thymine ("T"). The four RNA bases are A, G, C and uracil ("U").
35 DNA Sequence--A linear series of nucleo-tides connected one to the other by phosphodiester bonds between the 3' and 5' carbons of adjacent pentoses.
Codon--A DNA sequence of three nucleotides -7a-(a triplet) which encodes through mRNA an amino acid, a translational start signal or a translational ter-mination signal.
=
=
For example, example, the nucleotide triplets TTA, TTG, CTT, CTC, CTA and CTG encode for the amino acid leucine ("Leu"), TAG, TAA and TGA are translational stop signals and ATG is a translational start signal.
Reading Frame--The grouping of codon's during translation of mRNA into amino acid sequences.
During translation the proper reading frame must be maintained. For example, the sequence GCTGGTTGTAAG
may be translated in three reading frames or phases, each of which affords a different amino acid sequence:
GCT GGT TGT AAG--Ala-Gly-Cys-Lys G CTG GTT GTA AG--Leu-Val-Val GC TGG TTG TAA A--Trp-Leu-(STOP) Polypeptide--A linear series of amino acids connected one to the other by peptide bonds between the q-amino and carboxy groups of adjacent amino acids.
Genome--The entire DNA of a substance. It includes inter alia the structural genes encoding for the polypeptides of the substance, as well as operator, promoter and ribosome binding and interac-tion sequences including sequences such as the Shine-Dalgarno sequences.
Structural Gene--A DNA sequence which encodes through its template or messenger RNA ("mRNA") a sequence of amino acids characteristic of a specific polypeptide.
Transcription--The process of producing mRNA from a structural gene.
Translation--The process of producing a polypeptide from mRNA.
Expression--The process undergone by a structural gene to produce a polypeptide. It is a combination of transcription and translation.
S Plasmid--A non-chromosomal double-stranded DNA sequence comprising an intact "replicon" such that the plasmid is replicated in a host cell. When the plasmid is placed within a unicellular organism, the characteristics of that organism are changed or =
-8a-transformed as a result of the DNA of the plasmid.
For example, a plasmid carrying =
-the gene for tetracycline resistance (TetR) trans-forms a cell previously sensitive to tetracycline into one which is resistant to it. A cell trans-formed by a plasmid is called a "transformant".
Phage or Bacteriophage--Bacterial virus many of which include DNA sequences encapsidated in a protein envelope or coat ("capsid"). In a uni-cellular organism a phage replicates by a process called transfection.
Cloning Vehicle--A plasmid, phage DNA or other DNA sequences which are able to replicate in a host cell, characterized by one or a small number of endonuclease recognition sites at which such DNA
sequences may be cut in a determinable fashion with-out attendant loss of an essential biological func-tion of the DNA, e.g., replication, production of coat proteins or loss of promoter or binding sites, and which contain a marker suitable for use in the identification of transformed cells, e.g., tetra-cycline resistance or ampicillin resistance. A
cloning vehicle is often called a vector.
. Cloning--The process of asexual reproduc-= tion.
Recombinant DNA Molecule--A hybrid DNA
sequence comprising at least two nucleotide sequences, the first sequence not normally being found together in nature with the second.
Expression Control Sequence--A DNA sequence of nucleotides that controls and regulates expression of structural genes when operatively linked to those genes.
Referring now to Figure 1, we have shown therein a simplified diagram of the endogenous DNA
= of a Dane particle. Dane particles are detectable in the blood plasma of some patients with hepatitis B
viral infections (D. S. Dane and C. H. Cameron, "Virus-Like Particles In Serum Of Patients With Australia-Antiaen Assoniatpd Pann+4*4011 mu.
TWA--9a-1, pp. 695-698 (1970)). This particle is thought to be identical or closely related to the infective virion of hepatitis B. While its size is known (42 nanometers in diameter), its structure is not fully understood. Generally, the Dane particle is believed to consist of an outer layer and an inner core. The outer layer of the particle generally contains a polypeptide known as hepatitis B surface antigen ("HBsAg"). The inner core (27 nanometers in diameter) contains a second polypeptide, hepatitis B
core antigen ("HBcAg"), as well as an endogenous double-stranded, circular, DNA molecule of 2.1x106 daltons, which contains a single-stranded gap of varying length. A third polypeptide, 'e' antigen ("HBeAg"), may also be associated with the Dane par-ticles. In addition, the Dane particle is believed .
to include an endogenous DNA dependent DNA polymerase that partially closes the single-stranded gap in the endogenous DNA by a polymerase reaction employing that DNA as a primer/template. The structure and properties of the Dane particle and more especially its DNA have been the subject of several analyses.
E.g., W. S. Robinson, "The Genome of Hepatitis B
Virus", Ann. Rev. Microbiol., 31, pp. 357-377 (1977).
However, the actual structure of the various antigens or their genes had not been determined.
The Dane particle's endogenous DNA has been isolated by extraction from the Dane particle.
It consists of one nucleotide strand of constant length having a small nick or opening in it (Fig-ure 1, strand a, - 3000 nucleotides) and a second nucleotide strand of somewhat variable length (Fig-ure 1, 'strand b, - 2000 nucleotides). The second strand is complementary with the first and overlaps its nick to complete a circular structure by hydrogen bonding between complementary bases in the standard Watson-Crick manner. This overlap is apparent from Figure 1. The DNA polymerase of the Dane particle appears to use the second strand of the endogenous DNA as a primer and the first strand as a template to fill in the variable gap with nucleotides which ,i rc =
-10a-are complementary to the nucleotides of the first strand and afford a double-stranded DNA of about 3000 nucleotide pairs.
=
4111. A METHOD OF PREPARATION OF HEPATITIS B VIRUS DNA
Human serum from a,single H,BsAg positive, HBeAg positive donor (serotype adyw) was diluted with an equal volume of buffer (0.1 M tris-HC1, 0.1 M saline (NaC1), 0.1%
(weight/volume as used throughout 2-mercaptoethanol, 0.1%
bovine serum albumin, 0.001 M EDTA) at pH 7.4. This was centrifuged at 35,000 r.p.m. for 2 hours at 4 C. The pellet thus obtained was resuspended in 200 ul of the same buffer and layered at the top of a centrifuge tube contain-ing 20% sucrose. The suspension was centrifuged at 40,000 r.p.m. for 2 hours at 4 C. The pellet thus obtained was again resuspended in 100 ul of buffer (0.1 M tris-HC1, 0.1 M saline) at pH 7.5.
= The resultant DNA containing-pellet was then labelled with 3H or 32P as described by P. M. Kaplan et al., "DNA Polymerase Associated With Human Hepatitis B
Antigen", J. Virol., 12, pp. 995-1005 (1973) to facilitate = tracing it through subsequent steps of the process. This labelling results from the reaction of the concentrated Dane particles and 3H-or 32P-labelled deoxynucleoside triphosphates (dNTIlis) for 4 hours at 37 C. After this DNA
polymerase reaction, which results in partially closing the single-stranded gap in the DNA (See Figure 1), the labelled DNA material was layered at the top of a centrifuge tube containing 30% sucrose and centrifuged at 42,000 r.p.m. for 3 1/2 hours at 4 C.
The DNA was then extracted with phenol from the resulting pellet using the procedure of L. I. Lutwick and W. S. Robinson, ItroNA Synthesized In The Hepatitis B Dane Particle DNA Polymerase Reaction", J. Virol., 21, pp. 96-104 (1977). The extracted DNA was then dialyzed against a solution of 0.01 M tris-HC1, 0.001 M EDTA (pH 8.0) to eliminate the phenol solvent. The isolated DNA was then ' ready for restriction enzyme digestions. It displayed a , specific radio activity of Ad 108cpm4pg. The above discussed process is schematically depicted in Figure 2.
sf = -12-CLONING OF HEPATITIS B VIRUS DNA
A wide variety of host-cloning vehicle combina-tions may be usefully employed in cloning the double-stranded DNA isolated as above. For example, useful clon-ing vehicles may include chromosomal, non-chromosomal and synthetic DNA sequences such as various known bacterial plasmids such as pBR322, other E. coli plasmids and their derivatives and wider host range plasmids such as RP4, phage DNA such as the numerous derivatives of phage A, e.g., NM899 and vectors derived from combinations of plas-mids and phage DNA's such as plasmids which have been modified to employ phage DNA expression control sequences.
Useful hosts may include bacterial hosts such as E. coli .X1776, E. coli, X2282, E. coli HB101 and E. coli MRC1 and strains of Pseudomonas, Bacillus subtilis and other bacil-li, yeasts and other fungi, animal or plant hosts such as animal or plant cells in culture and other hosts. Of _ course, not-all hosts may be equally efficient. The par-ticular selection of host-cloning vehicle combination may be made by those of skill in the art after due considera-tion of the principles set forth above without departing from the scope of this invention.
Furthermore, within each specific vector, various sites may be selected for insertion of the isolated double-stranded DNA. These sites are usually designated by the restriction enzyme or endonuclease that cuts them. For example, in pBR322 the Pst I site is located in the gene for penicillinase between the nucleotide triplets that code for amino acids 181 and 182 of the penicillinase protein.
This site was employed by Villa-Komaroff et al., supra, in their synthesis of protein displaying rat proinsulin anti-genic determinants. A Hind II endonuclease recognition site is between the triplets coding for amino acids 101 and 102 and a Tact site at the triplet coding for amino acid 45 of that protein in pBR322. In similar fashion, the Eco RI
endonuclease recognition site for this plasmid is located between the genes coding for resistance to tetracycline and 111, ampicillin, respectively. This site was employed by Itakura et al. and Goeddel et al in their recombinant DNA
synthetic schemes, supra. Figures 2 and 11 display for illustrative purposes some of the restriction sites in plasmid pBR322 and phage )1/4 NM989.
The particular site chosen for insertion of the selected DNA fragment into the cloning vehicle to form a recombinant DNA molecule is determined by a variety of , factors. These include size and structure of the polypep-tide to be expressed, susceptibility of the desired poly-peptide to endoenzymatic degradation by the host cell components and contamination by its proteins, expression characteristics such as the location of start and stop .codons, and other factors recognized by those of skill in the art. Since the expression process is not fully under-stood, none of these factors alone absolutely controls the choice of insertion site for a particular polypeptide.
Rather the site chosen effects a balance of these factors and not all sites may be equally effective for a given protein.
Although several methods are known in the art for inserting foreign DNA into a cloning vehicle to form a recombinant DNA molecule, the method preferred in accord-ance with this invention is displayed in Figure 2. It is characterized by cleaving the Dane particle DNA with a restriction endonuclease, attaching poly-deoxyC sequences to the 3' termini (named by convention from the deoxyribose carbon of the DNA sugar backbone) of the cleaved DNA and linking the elongated DNA to a cloning vehicle which has been cut at a particular site by a restriction endonuclease and elongated at the 3' termini of that cut with poly deoxyG sequences, that are complementary to the poly-dC
sequences of the cleaved DNA. The complementary character of the 3' tails of the DNA and cloning vehicle permit cohesion of those termini.
.To be useful in the process of this invention a restriction enzyme chosen to cleave the HBV DNA should not 411, cleave the HBV DNA within an essential part of the gene that codes for polypeptides displaying HBV anti-genicity, and most suitably it should cleave the DNA
at a limited number of sites. Restriction enzymes that have been employed in this invention include <ipri I, Bgl II, Barn HI, Ava I and Eco RI. Other re-striction endonucleases may be similarly useful in accordance with this invention. Their selection may be made by those of skill in the art on due consider-ation of the factors set out above without departing from the scope of this invention. Figures 3-9 dis-play some of the restriction endonuclease recognition sites in part of the HBV genome.
Of course, other known methods of inserting DNA sequences into cloning vehicles to form recombi-nant DNA molecules are equally useful in this inven-tion. These include, for example, direct ligation wherein the same restriction endonuclease is employed to cleave the HBV DNA and the cloning vehicle. This procedure inherently provides complementary ends for cohesion.
It should, of course, be understood that the nucleotide sequence or gene fragment inserted at the selected restriction site of the cloning vehicle may include nucleotides which are not part of the actual structural gene for the desired protein or may include only a fragment of that structural gene.
= It is only required that whatever DNA sequence is inserted, the transformed host will produce a poly-peptide displaying HBV antigenicity.
The recombinant DNA molecule containing the hybrid gene may be employed to transform a host so as to permit that host (transformant) to express = the structural gene or fragment thereof and to pro-duce the polypeptide or portion thereof for which the hybrid DNA codes. The recombinant DNA molecule may also be employed to transform a host so as to L
....,,, III, -14a-permit that host on replication to produce additional recombinant DNA molecules as a source of HBV struc-tural genes and fragments thereof. The selection of _ -=
an appropriate host for either of these uses is controlled by a number of factors recognized by the art. These in-clude, for example, compatibility with the chosen vector, = toxicity of the co-products, ease of recovery of the desired polypeptide, expression characteristibs, biosafety and costs. Again, since the mechanisms of expression are not fully understood, no absolute choice of host may be made for a particular recombinant DNA molecule or polypep-tide from any of these factors alone. Instead, a balance of these factors must be struck with the realization that not all hosts may be equally effective for expression of a particular recombinant DNA molecule.
In the present synthesis, the preferred cloning .vehicle is the bacterial plasmid pBR322 and the preferred restriction endonuclease site therein is the Pst I site (Figure 2). This plasmid is a small (molecular weight approx. 2.6 megadaltons) plasmid carrying resistance genes ' to the antibiotics ampicillin (Amp) and tetracycline (Tet).
The plasmid has been fully characterized (F. Bolivar et al., "Construction and Characterization of New Cloning Vehicles II. A Multipurpose Cloning System", Gene, pp. 95-113 (1977)). 'The preferred host in accordance with this invention is E. coli HB101.
1. Preparation of dc-elongated Dane particle DNA
In actual practice of the preferred embodiment of this invention, the DNA isolated as above from the Dane particles was digested with restriction endonuclease Kpn I
(approximately 20 ng DNA in 10)11 10 mM tris-HC1, pH 7.5, 10 mM MgCl2, 10 mM 2-mercaptoethanol, 40 mM NaCl, 0.5)11 enzyme preparation) at 37 C for 90 minutes and the re-striction enzyme inactivated by heating at 70 C for 5 minutes. Poly-deoxyC sequences (for illustration purposes only depicted as CCC in Figure 2) were attached to the 3' termini of the digestion products by standard reaction with = 35 polynucleotide terminal transferase after removing the residual protein by extraction with phenol and chloroform (204p1 each). Phenol was removed from the aqueous phase by extraction with ether, and the DNA was precipitated by addition of 31 sodium acetate, pH 6.5 (5).11) and cold ethanol 0.1 ml. After storage at -70 C for one hour, the precipitated DNA was recovered by centrifugation at 10,000 r.p.m. for 20 minutes and the pellet dissolved in 10 mM
tris-HC1 pH 7.5 (10)p1). To this was added 3)11 of 400 mM
potassium cacodylate, pH 7.0, 4 mM 'cobalt chloride, 4 mM
deoxycnosine triphosphate (dCTP), 200 ,pg/m1 bovine serum albumin and the mixture incubated at 27 C for 10 minutes with 0.5,p1 of polynucleotide terminal transferase (6000 u/ml) and the reaction stopped by addition of 50 mM
EDTA (1 pl). ' 2. Preparation of Pst I Cleaved, dG-elongated pBR322 The plasmid pBR322 was digested with restriction endonuclease Pst I (for which the plasmid contains one target) and the products purified by phenol extraction and ethanol precipitation in the manner described above for HBV
DNA. Poly-dG sequences (for illustration purposes only , depicted as GGG in Figure 2) were added to the 3' termini of linear pBR322 molecules by terminal transferase as described for the addition of poly-dC sequences to HBV
except that the reaction was carried out at 37 C.
3. Cohesion of G-elongated pBR322 and C-elongated HBV DNA
Equimolar amounts of the pBR322-poly-dG and HBV
DNA-poly-dC were then mixed together and the complementary sequences allowed to link by incubation in 100 mM NaC1, 50 mM tris-HC1, pH 7.5, 5 mM EDTA (TNE), (50,p1) at 65 C for one hour, followed by 47 C for one hour, 37 C for one hour and 20 C for one hour and an equal volume of TNE and 20 ,11 of 100 mM MgC12, 100 mM CaCl2, 100 mM tris-HC1, pH 7.5 was added.
Natl. Acad. Sci. USA, 75, pp. 5936-5940 (1978)).
Several non-bacterial proteins and genes have been synthesized using recombinant DNA technology. These include a protein displaying rat proinsulin antigenic determinants (L. Villa-Komaroff et al., "A Bacterial Clone = 'Synthesizing Proinsulin", Proc. Natl. Acad. Sci. USA, 75, pp. 3727-3731 (1978)), rat growth hormone (P.H. Seeburg et al., "Synthesis Of Growth Hormone By Bacteria", Nature, 276, pp. 795-798 (1978)), mouse dihydrofolate reductase (A.C.Y. Chang et al., "Phenotypic Expression in E. coil Of A DNA Sequence Coding For Mouse Dihydrofolate Reductase", = 20 Nature, 275, pp. 617-624 (1978)), somatostatin (K.
Itakura et al., "Expression In Escherichia coli Of A Chemically Synthesized Gene For The Hormone Somatostatin", Science, 198, 1056-1063 (1977), United Kingdom patent specifications 2,007,675A, 2,007,676A and 2,008,123A and cognate applica-tions in other countries), and the A and B polypeptide chains of human insulin (D.V. Goeddel et al., "Expression In Escherichia coil Of Chemically Synthesized Genes For Human Insulin", Proc. Natl. Acad. Sci. USA, 76, pp. 106-110 (1979) and the United Kingdom and related patent specifi-cations, supra).
None of the foregoing, however, is directed, as is this invention, toward the recombinant DNA synthesis of viral proteins such as hepatitis B virus antigens.
It is known that HBV infections cause development of antibodies to the antigens of the virus. These anti-bodies are the body's defense mechanism to the HBV infec-tion. The development of such antibodies prior to exposure or rapidly in the case of potential exposure should substan-tially reduce and combat virus growth and spread in the patient.
A problem however in the artificially stimulated development of antibodies to the antigens of hepatitis B
virus is the limited host range of the virus. For example, although highly infectious to man, experimental infection with hepatitis B virus has been achieved in only a few additional primates. And, the virus has not been propa-gated in tissue culture. This limited host range and inability to infect tissue culture cells has severely hampered both the characterization of the virus and the pathology of its infection and the development of rapid =detection means and effective means of infection control and prevention.
While there have been some attempts to employ authentic HBV viral antigens, isolated from victims of the HBV infection, for the development of antibodies and detec-tion of infection, these treatments are not generally available because of the limited supply of the active species. Furthermore, the use of human sources for these antigens is disfavored because of the well recognized contamination problems in using human isolates.
DISCLOSURE OF THE INVENTION
The present invention solves the problems referred to by providing in accordance with the invention a recombin-ant DNA molecule characterized by a structural gene coding for a polypeptide displaying HBV antigenicity.
By virtue of our invention, it is possible to obtain HBV antigens and genes in substantial and uncon-taminated quantities for vaccine preparations and for use in detection of the viral infection and determination of its pathology and molecular biology. Such supplies have not hitherto been available, because of the narrow host range of the virus and its inability to be grown in tissue culture. .
I ;
' 1111 ...5_ As will be appreciated from the disclosure to follow, the recombinant DNA molecule of the invention is capable of producing, in an appropriate host, at least one viral polypeptide displaying HBV antigenicity and the structural genes that code therefor. These recombinant DNA
molecules and hosts may be utilized to prepare polypeptides displaying HBV antigenicity and structural genes coding for these polypeptides. These products may also be identified and characterized and are useful either as produced in the host or after appropriate derivatization or modification in compositions and methods for improving production of these products themselves and for detecting HBV infection, tracing its pathology and stimulating the production of HBV anti-bodies in humans.
In accordance with the invention we also provide a process for producing a recombinant DNA molecule, charac-terized by linking a DNA sequence prepared from the endog-enous DNA of a_Dane particle to another DNA sequence pre-pared from a source other than the Dane particle.
= 20 Our process may be distinguished from the prior processes above mentioned in that none of the prior pro-cesses employs a natural gene or DNA for a particular protein for construction of the recombinant DNA molecule and production of that protein or gene. Instead, they employ either synthetic genes made by chemical synthesis or = artificial genes made by enzymatically copying the mRNA
isolated from the donor cell to produce cDNA sequences.
One reason that natural DNA has not been pre-viously employed directly in recombinant DNA synthesis of proteins is that natural DNA's from most higher organisms and at least some animal viruses contain Hintrons" or additional nucleotide sequences as part of the gene. These = introns do not form part of the final message of the gene.
Instead, they are removed in vivo in higher organisms by special processing enzymes acting upon the primary trans-cription product to afford the ultimate message (mRNA) of the gene. Bacteria are presumed to be unable to process such introns so that natural DNA would not be expected to -be expressed in bacterial hosts and the desired proteins would not be expected to be produced by these hosts.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a simplified diagram re-Presenting the structure of the endogenous DNA of a Dane particle. It displays the two complementary DNA strands, strand a and b, .the nick in strand a and the gap in strand b and the closing of that gap by DNA polymerase reaction.
Figure 2 is a schematic outline of the preferred embodiment of the process of this invention: fragments of the endogenous DNA isolated from the Dane particle are fused to the E. coli penicillinase gene on the plasmid =
'pBR322 at the Pst I site. After transformation into E.
coil HB101, the recombinant DNA molecule pBR322-Pst I dG:
HBV-KpnI dC directs the synthesis of polypeptides display-ing HBV antigenicity.
Figures 3-9 show the nucleotide sequence deter-mined for a part of a hepatitis B virus genome in accord-ance with this invention. The sequence is displayed with the stop codons in the three reading frames noted above it.
It is arbitrarily numbered from the initiation codon for the gene coding for HBcAg as determined in this invention.
Nucleotides -80 to -1 represent the leader sequence which precedes this gene. Nucleotides 1-549 represent the nucleo-tide sequence for the gene coding for hepatitis B virus core antigen, the amino acid sequence (reading frame 1) of this antigen being depicted above that sequence. Nucleo-tides 1437-2114 represent the nucleotide sequence deter-mined for the gene of hepatitis B virus surface antigen, the amino acid sequence (reading frame 3) of this antigen being depicted above that sequence. Various restriction endonuclease recognition sites in these genes are also depicted in Figures 3-9.
Figure 10 is a schematic outline of a process of this invention wherein a recombinant DNA molecule which is capable of producing a polypeptide displaying hepatitis B
=
I.. ' 11) -7-virus core antigenicity is fragmented and a fragment therefrom attached to an improved expression control sequence, the lac system.
5 Figure 11 is a schematic outline of a pro-cess of this invention wherein a recombinant DNA
molecule of this invention is fragmented and con-nected to a fragment of phage DNA for use in lyso-genizing host cells and thereby increasing the number of gene copies therein.
Figure 12 is a schematic outline of another embodiment of the process of this invention: a re-combinant DNA molecule of this invention which does not produce a polypeptide displaying hepatitis B
virus core antigenicity is fragmented to remove the structural gene for HBsAg and this structural gene employed to produce a recombinant DNA molecule which produces HBsAg in an appropriate host.
BEST MODE OF CARRYING OUT THE INVENTION
20 In order that the invention herein described may be more fully understood, the following detailed description is set forth.
In the description the following terms are employed:
25 Nucleotide--A monomeric unit of DNA or RNA
consisting of a sugar moiety (pentose), a phosphate, and a nitrogenous heterocyclic base. The base is = linked to the sugar moiety via the glycosidic carbon (1' carbon of the pentose) and that combination of 30 base and sugar is a nucleoside. The base character-izes the nucleotide. The four DNA bases are adenine ("A"), guanine ("G"), cytosine ("C") and thymine ("T"). The four RNA bases are A, G, C and uracil ("U").
35 DNA Sequence--A linear series of nucleo-tides connected one to the other by phosphodiester bonds between the 3' and 5' carbons of adjacent pentoses.
Codon--A DNA sequence of three nucleotides -7a-(a triplet) which encodes through mRNA an amino acid, a translational start signal or a translational ter-mination signal.
=
=
For example, example, the nucleotide triplets TTA, TTG, CTT, CTC, CTA and CTG encode for the amino acid leucine ("Leu"), TAG, TAA and TGA are translational stop signals and ATG is a translational start signal.
Reading Frame--The grouping of codon's during translation of mRNA into amino acid sequences.
During translation the proper reading frame must be maintained. For example, the sequence GCTGGTTGTAAG
may be translated in three reading frames or phases, each of which affords a different amino acid sequence:
GCT GGT TGT AAG--Ala-Gly-Cys-Lys G CTG GTT GTA AG--Leu-Val-Val GC TGG TTG TAA A--Trp-Leu-(STOP) Polypeptide--A linear series of amino acids connected one to the other by peptide bonds between the q-amino and carboxy groups of adjacent amino acids.
Genome--The entire DNA of a substance. It includes inter alia the structural genes encoding for the polypeptides of the substance, as well as operator, promoter and ribosome binding and interac-tion sequences including sequences such as the Shine-Dalgarno sequences.
Structural Gene--A DNA sequence which encodes through its template or messenger RNA ("mRNA") a sequence of amino acids characteristic of a specific polypeptide.
Transcription--The process of producing mRNA from a structural gene.
Translation--The process of producing a polypeptide from mRNA.
Expression--The process undergone by a structural gene to produce a polypeptide. It is a combination of transcription and translation.
S Plasmid--A non-chromosomal double-stranded DNA sequence comprising an intact "replicon" such that the plasmid is replicated in a host cell. When the plasmid is placed within a unicellular organism, the characteristics of that organism are changed or =
-8a-transformed as a result of the DNA of the plasmid.
For example, a plasmid carrying =
-the gene for tetracycline resistance (TetR) trans-forms a cell previously sensitive to tetracycline into one which is resistant to it. A cell trans-formed by a plasmid is called a "transformant".
Phage or Bacteriophage--Bacterial virus many of which include DNA sequences encapsidated in a protein envelope or coat ("capsid"). In a uni-cellular organism a phage replicates by a process called transfection.
Cloning Vehicle--A plasmid, phage DNA or other DNA sequences which are able to replicate in a host cell, characterized by one or a small number of endonuclease recognition sites at which such DNA
sequences may be cut in a determinable fashion with-out attendant loss of an essential biological func-tion of the DNA, e.g., replication, production of coat proteins or loss of promoter or binding sites, and which contain a marker suitable for use in the identification of transformed cells, e.g., tetra-cycline resistance or ampicillin resistance. A
cloning vehicle is often called a vector.
. Cloning--The process of asexual reproduc-= tion.
Recombinant DNA Molecule--A hybrid DNA
sequence comprising at least two nucleotide sequences, the first sequence not normally being found together in nature with the second.
Expression Control Sequence--A DNA sequence of nucleotides that controls and regulates expression of structural genes when operatively linked to those genes.
Referring now to Figure 1, we have shown therein a simplified diagram of the endogenous DNA
= of a Dane particle. Dane particles are detectable in the blood plasma of some patients with hepatitis B
viral infections (D. S. Dane and C. H. Cameron, "Virus-Like Particles In Serum Of Patients With Australia-Antiaen Assoniatpd Pann+4*4011 mu.
TWA--9a-1, pp. 695-698 (1970)). This particle is thought to be identical or closely related to the infective virion of hepatitis B. While its size is known (42 nanometers in diameter), its structure is not fully understood. Generally, the Dane particle is believed to consist of an outer layer and an inner core. The outer layer of the particle generally contains a polypeptide known as hepatitis B surface antigen ("HBsAg"). The inner core (27 nanometers in diameter) contains a second polypeptide, hepatitis B
core antigen ("HBcAg"), as well as an endogenous double-stranded, circular, DNA molecule of 2.1x106 daltons, which contains a single-stranded gap of varying length. A third polypeptide, 'e' antigen ("HBeAg"), may also be associated with the Dane par-ticles. In addition, the Dane particle is believed .
to include an endogenous DNA dependent DNA polymerase that partially closes the single-stranded gap in the endogenous DNA by a polymerase reaction employing that DNA as a primer/template. The structure and properties of the Dane particle and more especially its DNA have been the subject of several analyses.
E.g., W. S. Robinson, "The Genome of Hepatitis B
Virus", Ann. Rev. Microbiol., 31, pp. 357-377 (1977).
However, the actual structure of the various antigens or their genes had not been determined.
The Dane particle's endogenous DNA has been isolated by extraction from the Dane particle.
It consists of one nucleotide strand of constant length having a small nick or opening in it (Fig-ure 1, strand a, - 3000 nucleotides) and a second nucleotide strand of somewhat variable length (Fig-ure 1, 'strand b, - 2000 nucleotides). The second strand is complementary with the first and overlaps its nick to complete a circular structure by hydrogen bonding between complementary bases in the standard Watson-Crick manner. This overlap is apparent from Figure 1. The DNA polymerase of the Dane particle appears to use the second strand of the endogenous DNA as a primer and the first strand as a template to fill in the variable gap with nucleotides which ,i rc =
-10a-are complementary to the nucleotides of the first strand and afford a double-stranded DNA of about 3000 nucleotide pairs.
=
4111. A METHOD OF PREPARATION OF HEPATITIS B VIRUS DNA
Human serum from a,single H,BsAg positive, HBeAg positive donor (serotype adyw) was diluted with an equal volume of buffer (0.1 M tris-HC1, 0.1 M saline (NaC1), 0.1%
(weight/volume as used throughout 2-mercaptoethanol, 0.1%
bovine serum albumin, 0.001 M EDTA) at pH 7.4. This was centrifuged at 35,000 r.p.m. for 2 hours at 4 C. The pellet thus obtained was resuspended in 200 ul of the same buffer and layered at the top of a centrifuge tube contain-ing 20% sucrose. The suspension was centrifuged at 40,000 r.p.m. for 2 hours at 4 C. The pellet thus obtained was again resuspended in 100 ul of buffer (0.1 M tris-HC1, 0.1 M saline) at pH 7.5.
= The resultant DNA containing-pellet was then labelled with 3H or 32P as described by P. M. Kaplan et al., "DNA Polymerase Associated With Human Hepatitis B
Antigen", J. Virol., 12, pp. 995-1005 (1973) to facilitate = tracing it through subsequent steps of the process. This labelling results from the reaction of the concentrated Dane particles and 3H-or 32P-labelled deoxynucleoside triphosphates (dNTIlis) for 4 hours at 37 C. After this DNA
polymerase reaction, which results in partially closing the single-stranded gap in the DNA (See Figure 1), the labelled DNA material was layered at the top of a centrifuge tube containing 30% sucrose and centrifuged at 42,000 r.p.m. for 3 1/2 hours at 4 C.
The DNA was then extracted with phenol from the resulting pellet using the procedure of L. I. Lutwick and W. S. Robinson, ItroNA Synthesized In The Hepatitis B Dane Particle DNA Polymerase Reaction", J. Virol., 21, pp. 96-104 (1977). The extracted DNA was then dialyzed against a solution of 0.01 M tris-HC1, 0.001 M EDTA (pH 8.0) to eliminate the phenol solvent. The isolated DNA was then ' ready for restriction enzyme digestions. It displayed a , specific radio activity of Ad 108cpm4pg. The above discussed process is schematically depicted in Figure 2.
sf = -12-CLONING OF HEPATITIS B VIRUS DNA
A wide variety of host-cloning vehicle combina-tions may be usefully employed in cloning the double-stranded DNA isolated as above. For example, useful clon-ing vehicles may include chromosomal, non-chromosomal and synthetic DNA sequences such as various known bacterial plasmids such as pBR322, other E. coli plasmids and their derivatives and wider host range plasmids such as RP4, phage DNA such as the numerous derivatives of phage A, e.g., NM899 and vectors derived from combinations of plas-mids and phage DNA's such as plasmids which have been modified to employ phage DNA expression control sequences.
Useful hosts may include bacterial hosts such as E. coli .X1776, E. coli, X2282, E. coli HB101 and E. coli MRC1 and strains of Pseudomonas, Bacillus subtilis and other bacil-li, yeasts and other fungi, animal or plant hosts such as animal or plant cells in culture and other hosts. Of _ course, not-all hosts may be equally efficient. The par-ticular selection of host-cloning vehicle combination may be made by those of skill in the art after due considera-tion of the principles set forth above without departing from the scope of this invention.
Furthermore, within each specific vector, various sites may be selected for insertion of the isolated double-stranded DNA. These sites are usually designated by the restriction enzyme or endonuclease that cuts them. For example, in pBR322 the Pst I site is located in the gene for penicillinase between the nucleotide triplets that code for amino acids 181 and 182 of the penicillinase protein.
This site was employed by Villa-Komaroff et al., supra, in their synthesis of protein displaying rat proinsulin anti-genic determinants. A Hind II endonuclease recognition site is between the triplets coding for amino acids 101 and 102 and a Tact site at the triplet coding for amino acid 45 of that protein in pBR322. In similar fashion, the Eco RI
endonuclease recognition site for this plasmid is located between the genes coding for resistance to tetracycline and 111, ampicillin, respectively. This site was employed by Itakura et al. and Goeddel et al in their recombinant DNA
synthetic schemes, supra. Figures 2 and 11 display for illustrative purposes some of the restriction sites in plasmid pBR322 and phage )1/4 NM989.
The particular site chosen for insertion of the selected DNA fragment into the cloning vehicle to form a recombinant DNA molecule is determined by a variety of , factors. These include size and structure of the polypep-tide to be expressed, susceptibility of the desired poly-peptide to endoenzymatic degradation by the host cell components and contamination by its proteins, expression characteristics such as the location of start and stop .codons, and other factors recognized by those of skill in the art. Since the expression process is not fully under-stood, none of these factors alone absolutely controls the choice of insertion site for a particular polypeptide.
Rather the site chosen effects a balance of these factors and not all sites may be equally effective for a given protein.
Although several methods are known in the art for inserting foreign DNA into a cloning vehicle to form a recombinant DNA molecule, the method preferred in accord-ance with this invention is displayed in Figure 2. It is characterized by cleaving the Dane particle DNA with a restriction endonuclease, attaching poly-deoxyC sequences to the 3' termini (named by convention from the deoxyribose carbon of the DNA sugar backbone) of the cleaved DNA and linking the elongated DNA to a cloning vehicle which has been cut at a particular site by a restriction endonuclease and elongated at the 3' termini of that cut with poly deoxyG sequences, that are complementary to the poly-dC
sequences of the cleaved DNA. The complementary character of the 3' tails of the DNA and cloning vehicle permit cohesion of those termini.
.To be useful in the process of this invention a restriction enzyme chosen to cleave the HBV DNA should not 411, cleave the HBV DNA within an essential part of the gene that codes for polypeptides displaying HBV anti-genicity, and most suitably it should cleave the DNA
at a limited number of sites. Restriction enzymes that have been employed in this invention include <ipri I, Bgl II, Barn HI, Ava I and Eco RI. Other re-striction endonucleases may be similarly useful in accordance with this invention. Their selection may be made by those of skill in the art on due consider-ation of the factors set out above without departing from the scope of this invention. Figures 3-9 dis-play some of the restriction endonuclease recognition sites in part of the HBV genome.
Of course, other known methods of inserting DNA sequences into cloning vehicles to form recombi-nant DNA molecules are equally useful in this inven-tion. These include, for example, direct ligation wherein the same restriction endonuclease is employed to cleave the HBV DNA and the cloning vehicle. This procedure inherently provides complementary ends for cohesion.
It should, of course, be understood that the nucleotide sequence or gene fragment inserted at the selected restriction site of the cloning vehicle may include nucleotides which are not part of the actual structural gene for the desired protein or may include only a fragment of that structural gene.
= It is only required that whatever DNA sequence is inserted, the transformed host will produce a poly-peptide displaying HBV antigenicity.
The recombinant DNA molecule containing the hybrid gene may be employed to transform a host so as to permit that host (transformant) to express = the structural gene or fragment thereof and to pro-duce the polypeptide or portion thereof for which the hybrid DNA codes. The recombinant DNA molecule may also be employed to transform a host so as to L
....,,, III, -14a-permit that host on replication to produce additional recombinant DNA molecules as a source of HBV struc-tural genes and fragments thereof. The selection of _ -=
an appropriate host for either of these uses is controlled by a number of factors recognized by the art. These in-clude, for example, compatibility with the chosen vector, = toxicity of the co-products, ease of recovery of the desired polypeptide, expression characteristibs, biosafety and costs. Again, since the mechanisms of expression are not fully understood, no absolute choice of host may be made for a particular recombinant DNA molecule or polypep-tide from any of these factors alone. Instead, a balance of these factors must be struck with the realization that not all hosts may be equally effective for expression of a particular recombinant DNA molecule.
In the present synthesis, the preferred cloning .vehicle is the bacterial plasmid pBR322 and the preferred restriction endonuclease site therein is the Pst I site (Figure 2). This plasmid is a small (molecular weight approx. 2.6 megadaltons) plasmid carrying resistance genes ' to the antibiotics ampicillin (Amp) and tetracycline (Tet).
The plasmid has been fully characterized (F. Bolivar et al., "Construction and Characterization of New Cloning Vehicles II. A Multipurpose Cloning System", Gene, pp. 95-113 (1977)). 'The preferred host in accordance with this invention is E. coli HB101.
1. Preparation of dc-elongated Dane particle DNA
In actual practice of the preferred embodiment of this invention, the DNA isolated as above from the Dane particles was digested with restriction endonuclease Kpn I
(approximately 20 ng DNA in 10)11 10 mM tris-HC1, pH 7.5, 10 mM MgCl2, 10 mM 2-mercaptoethanol, 40 mM NaCl, 0.5)11 enzyme preparation) at 37 C for 90 minutes and the re-striction enzyme inactivated by heating at 70 C for 5 minutes. Poly-deoxyC sequences (for illustration purposes only depicted as CCC in Figure 2) were attached to the 3' termini of the digestion products by standard reaction with = 35 polynucleotide terminal transferase after removing the residual protein by extraction with phenol and chloroform (204p1 each). Phenol was removed from the aqueous phase by extraction with ether, and the DNA was precipitated by addition of 31 sodium acetate, pH 6.5 (5).11) and cold ethanol 0.1 ml. After storage at -70 C for one hour, the precipitated DNA was recovered by centrifugation at 10,000 r.p.m. for 20 minutes and the pellet dissolved in 10 mM
tris-HC1 pH 7.5 (10)p1). To this was added 3)11 of 400 mM
potassium cacodylate, pH 7.0, 4 mM 'cobalt chloride, 4 mM
deoxycnosine triphosphate (dCTP), 200 ,pg/m1 bovine serum albumin and the mixture incubated at 27 C for 10 minutes with 0.5,p1 of polynucleotide terminal transferase (6000 u/ml) and the reaction stopped by addition of 50 mM
EDTA (1 pl). ' 2. Preparation of Pst I Cleaved, dG-elongated pBR322 The plasmid pBR322 was digested with restriction endonuclease Pst I (for which the plasmid contains one target) and the products purified by phenol extraction and ethanol precipitation in the manner described above for HBV
DNA. Poly-dG sequences (for illustration purposes only , depicted as GGG in Figure 2) were added to the 3' termini of linear pBR322 molecules by terminal transferase as described for the addition of poly-dC sequences to HBV
except that the reaction was carried out at 37 C.
3. Cohesion of G-elongated pBR322 and C-elongated HBV DNA
Equimolar amounts of the pBR322-poly-dG and HBV
DNA-poly-dC were then mixed together and the complementary sequences allowed to link by incubation in 100 mM NaC1, 50 mM tris-HC1, pH 7.5, 5 mM EDTA (TNE), (50,p1) at 65 C for one hour, followed by 47 C for one hour, 37 C for one hour and 20 C for one hour and an equal volume of TNE and 20 ,11 of 100 mM MgC12, 100 mM CaCl2, 100 mM tris-HC1, pH 7.5 was added.
4. Transformation of E. coli HB 101 Cultures of E. coli HB 101 competent, for trans-formation were prepared as described by E. M. Lederberg and ;
S. H. Cohen, "Transformation Of Salmonella typhimurium By'Plasmid Deoxyribonucleic Acid", J. Bacteriol., 119, pp. 1072-1074 (1974). 0.1 ml portions of the cells were mixed with 25 pl of the annealed DNA preparation
S. H. Cohen, "Transformation Of Salmonella typhimurium By'Plasmid Deoxyribonucleic Acid", J. Bacteriol., 119, pp. 1072-1074 (1974). 0.1 ml portions of the cells were mixed with 25 pl of the annealed DNA preparation
5 and incubated at 0 C for 20 minutes and then at 20 C
for 10 minutes before plating on L-agar plates con-taining tetracycline (50 pg/m1) for overnight incu-bation at 37 C. Since plasmid pBR322 includes the gene for tetracycline resistance, E.coli colonies 10 which have been transformed with that plasmid will grow in cultures containing that antibiotic to the exclusion of those E.coli colonies not so transformed.
Therefore, growth in tetracycline containing culture permits selection of properly transformed hosts.
15 5. Screening E.coli Colonies by Hybridization Bacterial colonies grown on the incubated tetracycline containing L-agar plates were tested for sensitivity to ampicillin. The plasmid pBR322 20 includes the gene for ampicillin resistance. This gene is the proposed site of hybrid gene insertion.
Therefore, colonies which have been transformed with a plasmid having DNA inserted at the chosen recogni-tion site will be sensitive to ampicillin, but will 25 retain their resistance to tetracycline. E.coli colonies that were sensitive to ampicillin but resist-ant to tetracycline were picked onto a disc of Milli-pore (Reg. Trade Mark) cellulose nitrate filter sup-ported on L-agar plates containing tetracycline.
30 After overnight growth at 37 C, the Millipore (Reg.
Trade Mark) filter was transferred to ,fresh L-agar plates containing tetracycline and chloramphenicol (150 pg/m1) and incubated for a further several hours at 37 C in order to amplify the number of copies of 35 the plasmid within the cells (Figure 2). The Milli-pore (Reg. Trade Mark) filters were then used for colony hybridization as described by M. Grunstein and D. S. Hogness, "Colony Hybridization: A Method For The Isolation Of Cloned DNAs That Contain A Speci-40 fic Gene", Proc. Natl. Acad. Sci. U.S.A., 72, pp. 3961-3965 (1975) with 32P-labelled HBV DNA pre-pared previously as the probe.
r-,1 Radioautography of the filters revealed the presence of colonies that contain DNA sequences complementary to authentic HBV DNA.
for 10 minutes before plating on L-agar plates con-taining tetracycline (50 pg/m1) for overnight incu-bation at 37 C. Since plasmid pBR322 includes the gene for tetracycline resistance, E.coli colonies 10 which have been transformed with that plasmid will grow in cultures containing that antibiotic to the exclusion of those E.coli colonies not so transformed.
Therefore, growth in tetracycline containing culture permits selection of properly transformed hosts.
15 5. Screening E.coli Colonies by Hybridization Bacterial colonies grown on the incubated tetracycline containing L-agar plates were tested for sensitivity to ampicillin. The plasmid pBR322 20 includes the gene for ampicillin resistance. This gene is the proposed site of hybrid gene insertion.
Therefore, colonies which have been transformed with a plasmid having DNA inserted at the chosen recogni-tion site will be sensitive to ampicillin, but will 25 retain their resistance to tetracycline. E.coli colonies that were sensitive to ampicillin but resist-ant to tetracycline were picked onto a disc of Milli-pore (Reg. Trade Mark) cellulose nitrate filter sup-ported on L-agar plates containing tetracycline.
30 After overnight growth at 37 C, the Millipore (Reg.
Trade Mark) filter was transferred to ,fresh L-agar plates containing tetracycline and chloramphenicol (150 pg/m1) and incubated for a further several hours at 37 C in order to amplify the number of copies of 35 the plasmid within the cells (Figure 2). The Milli-pore (Reg. Trade Mark) filters were then used for colony hybridization as described by M. Grunstein and D. S. Hogness, "Colony Hybridization: A Method For The Isolation Of Cloned DNAs That Contain A Speci-40 fic Gene", Proc. Natl. Acad. Sci. U.S.A., 72, pp. 3961-3965 (1975) with 32P-labelled HBV DNA pre-pared previously as the probe.
r-,1 Radioautography of the filters revealed the presence of colonies that contain DNA sequences complementary to authentic HBV DNA.
6. Detection of Colonies Synthesizing 5 Polypeptides with HBV Antigenicity Colonies that were resistant to tetracycline, sensitive to ampicillin and hybridized with authentic HBV DNA were tested for their ability to produce at least one polypeptide displaying HBV antigen speci-10 ficity or antigenicity. Colonies were again picked onto Millipore (Reg. Trade Mark) filters supported on L-agar plates containing tetracycline and incubated at 37 C overnight. A second plate of L-agar was covered with a culture of E.coli C600 (0.1 ml in = 15 2 ml soft agar) infected with a virulent strain of bacteriophage X vir (multiplicity of infection of about 1) and also incubated at 37 C overnight by which time the bacterial lawn was confluently lysed (i.e. substantially all of the hosts' cell walls had 20 burst) by the phage (X vir). =The Millipore (Reg.
Trade Mark) filter containing cells transformed in accordance with this invention was lifted from its plate and placed, colonies downwards, in contact with the surface of the L-agar plate of E.coli C600 25 that had been lysed with X vir. This contact was continued for about 10 minutes to permit infection of the cells residing on the Millipore (Reg. Trade Mark) filter by X vir. The Millipore (Reg. Trade Mark) filter was then transferred to a fresh plate of L-agar 30 and incubated at 37 C for a further 5 hours. Meanwhile polyvinyl discs were coated with HBV antibodies as described by S. Broome and W. Gilbert, "Immunological Screening Method To Detect Specific Translation Pro-ducts", Proc. Natl. Acad. Sci. U.S.A., 75, pp. 2746-35 2749 (1978). The Millipore (Reg. Trade Mark) filters on which the transformed bacterial colonies were now obviously lysed were placed with the colonies in contact with the coated polyvinyl discs and kept at 4 C for 3 hours. This contact results in any HBV
40 antigens lysed from the cells on the Millipore (Reg.
Trade Mark) filter binding with the HBV antibodies of the disc. The coated discs were separated from the Millipore (Reg. Trade Mark) filter and then washed and incubated with 125I-labelled HBV antibodies. This incubation results in radio-active labelling of those sites on the disc where HBV antigens from the Millipore (Reg. Trade Mark) 5 filter had previously become bound by the HBV anti-bodies of the disc. After washing and radioauto-graphy, as described by Broome and Gilbert, supra, colonies that had produced polypeptides with HBV
antigen specificity were located by reference to the 10 radioautograph.
USE OF POLYPEPTIDES AND GENES PRODUCED
FROM RECOMBINANT DNA MOLECULES IN DETECTING
THE PRESENCE OF HBV ANTIBODIES IN HUMANS
Polypeptides displaying HBV antigenicity 15 and the structural genes and fragments which code therefor may be used in methods and kits designed to detect the presence of HBV antibodies in humans and therefore recognize humans and blood samples which have been infected by this virus.
20 For example, the HBcAg produced by hosts transformed by recombinant DNA molecules of this invention can be used in the immunological diagnostic tests currently available for hepatitis B virus detection, that is radioimmunoassay or ELISA (enzyme 25 linked immunosorbent assay). In one type of radio-immunoassay anti-core antigen antibody, raised in a laboratory animal, is attached to a solid phase, for example, the inside of a test tube. HBcAg is then added to the tube so as to bind with the antibody.
30 To the tube coated with the antigen-antibody complex is added a sample of the patient's serum, together with a known amount of HBV anti-core antibody labelled with a radioactive isotope such as radioactive iodine.
Any HBV antibody in the patient's serum will compete 35 with the labelled antibody for the free binding sites on antigen-antibody complex. Once the serum has been allowed to interact, the excess liquid is removed, the test tube washed, and the amount of radioactivity measured. A positive result, i.e. that the patient's 40 serum contains HBV antibody, is indicated by a low radioactive count. In one type of Elisa test, a microtitre plate is coated with HBcAg and to this is added a sample of patient's serum. After a period of incubation permitting interaction of any antibody with the antigen, the plate is washed and a preparation of anti-human antibodies, raised in a laboratory animal, and which are linked to an enzyme label is added, incubated to allow reaction to take place, and the plate is then rewashed.
Thereafter, enzyme substrate is added to the microtitre plate and incubated for a period of time to allow the enzyme to work on the substrate, and the adsorbance of the final preparation is measured. A large change in adsorbance indicates a positive result.
IN VIVO ACTIVITY OF POLYPEPTIDES
PRODUCED FROM RECOMBINANT DNA MOLECULES
To test the biological activity of the antigenic polypeptides translated from the recombinant DNA molecule in the E. coli of this invention, sterile extracts of the bacterial cells which were shown to express HBcAg were injected into rabbits after mixing with an equal volume of Freund's adjuvant. Two animals received the crude bacte-rial extract and two were given samples of the same extract after fractionation on a Sephadex 650 column. Additional injections of the same sample, which had been frozen and stored (retaining full antigenic activity) were given two and five weeks after the first injection. The animals were bled at intervals several weeks after initial injection.
Immunodiffusion experiments, using the procedure described by 0. Ouchterlony, "Immunodiffusion and Immuno-electrophoresis", in Handbook of Experimental Immunology (D. W. Weir ed.) (Blackwell Scientific Publications, Oxford and Edinburgh) chap. 19 (1967), were performed to compare the rabbit sera (antibodies) to human hepatitis B virus core antibody ("HBcAb") using HBcAg derived from human liver (B. J. Cohen and Y. E. Cossart, "Application Of A
Screening Test For Antibody To Hepatitis B Core Antigen", J. Clin. Path., 30, pp. 709-713 (1977)). All four rabbit . .
Ill ' -21-.,_ sera gave precipitin lines with HBcAg identical to those formed between HBcAg and the HBcAb derived from human sources. Therefore, the core antigen synthesized in E.
coli with the recombinant DNA molecules of this invention is serologically active in vivo. This activity establishes the feasibility of compositions and methods using viral antigens synthesized in microbial cells for the stimulation of antibody formation in humans. Such compositions and methods are characterized by the polypeptides produced in hosts transformed by recombinant DNA molecules prepared in accordance with this invention. These polypeptides would be employed alone or with well known pharmaceutically acceptable carriers such as saline solutions or other .additives recognized by the art for use in compositions and methods for the treatment and prevention of viral infec-tions in humans.
As noted previously, restriction enzymes other than the Kpn I/Pst I combination described above may be usefully employed in preparing recombinant DNA molecules of this invention. Other restriction sites in plasmid pBR322 and part of the HBV genome are depicted in Figures 2 and 3, respectively. In illustration of these alternative, but less preferred embodiments, the following examples are set forth.
BAN HI, ECO RI, BGL II--PST I COMBINATIONS
The HBV DNA fragments produced by the use of Barn HI Eco RI and Bgl .II, unlike those produced by Kpn I, could not conveniently be directly tailed for cohesion owing to the presence of short 5' single-stranded pro-jections. They were therefore treated with )s exonuclease to remove these projections before the addition of poly-dC
sequences at 3' termini. This was done by incubation of the restricted DNA (10 )1l of a solution as described earlier) with 15 pl 100 mM sodium glycinate, pH 9.-5, 10 mM
MgCl2, 100 pg/m1 bovine serum albumin, 5 pl ). exonuclease at 0 C for 1.5 hours. The mixture was then extracted with = 1111 -22-phenol and chloroform and the process continued. In the preparation in which Barn HI was used, subsequent radioim-munoassay confirmed the production of polypeptides with HBV
antigen specificity.
DIRECT LIGATION: ECO RI--ECO RI AND BAN HI
Instead of tailing the DNA fragments for cohe-sion, as described above, direct ligation of the gene fragments may be employed in the processes of this inven-tion. For example, in two further preparations HBV DNA (20 ng) was restricted with restriction endonuclease Eco RI or Barn HI in 10 mM tris-HC1, pH 7.5, 10 mM MgCl2, 10 mM 2-mer-___ captoethanol, 50 mM NaC1 (10)41) at 37 C for 1.5 hours and mixed in excess with pBR322 that had been incubated with the same enzymes under identical conditions. A concen-trated buffer solution (660 mM tris-HC1, pH 7.5, 100 mM
MgCl2, 10 mM EDTA, 100 mM 2-mercaptoethanol, 400 mM NaCl, 1 mM ATP) (2 )11) was added and the mixture incubated with T4 DNA ligase (0.5)11) at 10 C for 3 hours followed by 0 C for at least 24 hours. The solution was diluted to 0.1 ml with 10 mM tris-HC1, pH 7.5 and used to transform competent cultures of E. coli HB 101 as described earlier. In the case of Barn HI prepared recombinant DNA molecules, the colonies were tested, before screening for hybridization, for sensitivity to tetracycline rather than to ampicillin, because the target for Barn HI in pBR322 is within the gene coding for tetracycline resistance and therefore successful insertion at this recognition site results in loss of this resistance instead of the ampicillin resistance as in the case of insertion at the Pst I site.
In the case of recombinant DNA molecules prepared via the Eco RI site, the colonies were tested, before screening for hybridization, for resistance to both ampi-cillin and tetracycline because the target for EcoRI in pBR322 is between the genes coding tetracycline and ampi-cillin resistance such that no inactivation of the genes for ampicillin or tetracycline resistance occurs on hybrid DNA insertion at this site.
Micro-organisms prepared by the =rocesses described herein are exemplified by culturzs deposited in the Culture Collection of the Microbiol=gical Research Establishment at Porton Down on D-cember 15, 1978 and identified as DBR322-HBV-A to F.
Specifically, these cultures are characterized as follows:
A: E. coil HB 101/pBR322-Pst I dG:HBV-Kpn I dc:
TetR Amps HBV* VA- [NCIB 11548]
B: E. coil HB 101/pBR322-Pst I dG:HBV-Bam HI dC:
TetR Amp' HBV- VA* [NCIB 11549]
C: E. coil HB 101/pBR322-Pst I dG:HBV-8g1 I dC:
Tee Amp HBV* [NCIB 1l550]
D: E coli HB 101/pBR322-Pst. I dG:HBV- PhI dC:
TetR Amps HBV* [NCIB 11551]
E: E_ coil MB 101/pBR322-Bam HI:HBV-Bam HI:
Tets Ai5 HBV* [NCIB 11552]
F: E oH MB 101/pBR322-Tco, RI:HBV-Eco RI:
TetR Amp' HBV* [NCIB 11553].
Portions of these identical cultures have .1so been deposited in the Culture Collection of the Iational Collection of Industrial Bacteria, Aberdee , Scotland on December 20, 1979, and were assigned the accession numbers shown above in brackets.
The above nomenclature for the cultures is a description of that culture as follows: Ho t/cloning vehicle - restriction site in cloning vehic e with indication of elongation nucleotide (if an ) :
hepatitis B virus restriction site in hepatitis B virus with indication of elongation nucleotide (if any) :
resistance (R) or sensitivity (S) to tetracicline (Tet), and ampicillin (.Amp), positive hybri=ization to HBv DNA in the colony hybridization test, s, ora, (HBV') and production of polypeptide displaying H7 anti-genicity, (VA-') Using this nomenclture culture pBR322-HBV-A designates an E. coli HE, 101 culture - 23a -containing as a plasmid a recombinant DNA molecule comprising pER322 cleaved at the .Pst I site and elongated with a poly =
/
111' dG tail at the 3' termini attached to HBV DNA cleaved with Kpn I and elongated with a poly-dC tail at the 3' termini, the culture displaying resistance to tetracycline, sensi-tivity to ampicillin, a positive HBV DNA hybridization test and producing a polypeptide displaying HBV antigenicity.
Of course, it is to be understood that the hybrid micro-organisms, recombinant DNA molecules and polypeptides and methods applicable to them of this invention are not limited to those described in the preferred embodiment above. Instead, the hybrid organisms, recombinant DNA
molecules and polypeptides may be modified during produc-tion or subsequently by known methods to good advantage.
For example, more efficient expression control sequences may be utilized for transcription of the HBV genes or hybrid genes, mutations to reduce the synthesis of unde-sired gene products may be introduced, the protease levels in the host cells may be reduced, thermo-inducible lysogens containing the HBV genes may be integrated into the host chromosome or other modifications and procedures may be carried out to increase the number of gene copies in the cell or to increase the cell's productivity in producing the desired polypeptides.
Apart from their use to produce polypeptides displaying HBV antigenicity, the hybrid micro-organisms of this invention are also useful for the production of large quantities of DNA containing all or part of the genome for hepatitis B virus. For example, amplification, e.g., by addition of chloramphenicol to the growth medium when the cell density has reached a suitable level, or the use of mutations to prevent lyais in bacteriophage hybrids that contain HBV sequences permit the preparation of HBV DNA in amounts previously unavailable.
HBV DNA prepared in this way may be used to determine the nucleotide sequence of the genome and from that the gene and amino acid sequences of the HBV antigens and structural genes themselves. Knowledge of these sequences aids in understanding the biology of the hepati-tis B virus and permits the modifications described above to be employed to best advantage.
DNA FRAGMENT MAPPING AND
, 5 NUCLEOTIDE SEQUENCE DETERMINATION_ 1. Hepatitis B Core Antigen A series of recombinant DNA molecules, made in accordance with the processes described above, which imparted to their host cells the ability to synthesize HBcAg as detected by solid phase radioimmunoassay were chosen for sequence analysis. Fragments were prepared from these recombinant DNA molecules by digestion with appro-priate restriction enzymes and the fragments labelled at their 5' termini with [o(-32P] ATP and T4 polynucleotide kinase. The nucleotide sequences of each fragment were then determined by well-known chemical degradative methods (A. M. Maxam and W. Gilbert, "A New Method For Sequencing DNA", Proc. Nat. Acad. Sci. U.S.A., 74, pp. 560-564 (1977).
The resulting nucleotide sequence is depicted in Figures 3-9. For reference, the-sequence is numbered from the A of the ATG translational initiation codon of the core gene. The nucleotide sequence of the gene for HBcAg and the amino acid sequence for the polypeptide deduced from this gene (Reading Frame 1) is depicted in Figures 3-9 between nucleotides 1-549. The size of the polypeptide encoded by this gene is close to the 19000 molecular weight observed for core antigens from Dane particles. However, the present structural determination does not exclude the possibility that some amino acids may be trimmed from the amino terminus of this polypeptide in vivo during formation of the authentic antigen. This structure also does not take into account any other or further modifications to the polypeptide caused by its interaction with other human enzymes, e.g., those enzymes which glycosolate proteins.
Therefore, the polypeptide structure determined herein may not be identical to that HBcAg found in vivo, but it will = -26-still elicit a very similar, if not identical, immuno response.
All of the recombinant DNA molecules examined had the HBV DNA inserted so that the core antigen gene was maintained in the same translational phase as-the gene for penicillin resistance of pBR322. Moreover, in the various recombinants the HBV DNA insert began within one or two nucleotides of the same position in the HBV sequence.
Because these recombinant DNA molecules originated from HBV
DNA digested with various restriction enzymes, e.g., Barn HI
and Kpn I, this unique attachment is surprising and may have resulted from inadvertent breakage or polynucleotide tailing of the HBV DNA at the nick in the endogenous DNA of the Dane particle (Figure 1). Recombinant DNA molecules having the HBV DNA insert in a different translational phase did not express the HBcAg gene and no polypeptide displaying HBV antigenicity was detected in hosts trans-formed with such recombinant DNA molecules. Of course, it is to be understood that such non-gene expressing hosts and recombinant DNA molecules are still useful for producing HBV DNA in accordance with this invention.
While it was expected that HBcAg or fragments thereof produced through expression of the HBV DNA inserts would be fused to the product ( fi -lactamase) of the gene for penicillinase resistance via a small number of glycine residues, that was not the case. Instead, the /8- lacta-mase was in each case fused through 5-8 glycines to an identical peptide sequence, but this sequence terminated after 25 amino acids. In this reading frame (Frame 1, Figure 3), a stop codon.(TAG) is followed three nucleotides later by an initiation codon from which translation con-tinues unhindered to give a polypeptide 183 amino acids in length (Figures 3-8). Therefore, core antigenic activity resides in a polypeptide of about 21,000 daltons translated de novo from the HBV sequence within the mRNA transcribed from the recombinant DNA molecule. This polypeptide remains within the host cell because it is not attached to the excreted penicillinase carrier protein due to the arrangement of the stop and start codons of the HBV DNA
insert.
2. Hepatitis B Surface Antigen The nucleotide sequence of the gene for HBsAg and the amino acid sequence for the polypeptide deduced from this gene (Reading Frame 3) is also depicted in Figures 6-8, between nucleotides 1437-2114. The amino acid sequence of this polypeptide begins at the N terminus with the sequence met-glu-asn-ile-thr-ser. This initial amino acid sequence was determined by D. L. Peterson et al., "Partial Amino Acid Sequence Of Two Major Component Polypeptides Of Hepatitis B Surface Antigen", Proc. Natl. Acad. Sci.
U.S.A., 74, pp. 1530-1534 (1977) from authentic human HBsAg. The amino acid sequence of this protein continues in Figures 6-8 to a stop codon 226 amino acids away. This 226 polypeptide sequence corresponds to a protein of 25,400 daltons.
The HBsAg amino acid sequence and length has been independently confirmed by sequencing appropriate recombi-nant DNA molecules by P. Valenzuela et al., "Nucleotide Sequence Of The Gene Coding For The Major Protein of Hepa-titis B Virus Surface Antigen", Nature, 280, pp. 815-819 (1979).
The nucleotide sequence determined for the recom-binant DNA molecules of this invention shows that none of those examined which expressed the gene for HBcAg can have the gene for HBsAg in a position from which it could also be expected to be expressed in an appropriate host cell.
In fact, no HBsAg was detected in the extracts of cells transformed with those plasmids found to express the gene for HBcAg. However, as noted, supra, knowledge of the nucleotide sequences of these genes permits modification of the expression process to improve its yield and effect and to favor the expression of genes and the production of polypeptides not previously expressed or detected.
lik = ' , PREPARATION OF IMPROVED RECOMBINANT DNA
MOLECULES FOR PRODUCTION OF HBV ANTIGENS
The gene and amino acid sequences of these anti-gens are useful in designing methods to increase the level of production of antigen or gene per bacterial cell.
The level of production of a protein is governed by two major factors: the number of copies of its gene within the cell and the efficiency with which these. gene copies are transcribed and translated. Efficiency of transcription and translation (which together comprise expression) is in turn dependent upon nucleotide sequences, normally situated ahead of the desired coding sequence.
These nucleotide sequences or expression control sequences , define, inter alia, the location at which RNA polymerase interacts to initiate transcription (the promoter sequence) and at which ribosomes bind and interact with the mRNA (the product of transcription) to initiate translation. Not all such expression control sequences function with equal efficiency. It is thus of advantage to separate the speci-fic coding sequences for the desired protein from their adjacent nucleotide sequences and fuse them instead to known expression control sequences so as to favor higher levels of expression. This having been achieved, the newly engineered DNA fragment may be inserted into a multicopy plasmid or a bacteriophage derivative in order to increase the number of gene copies within the cell and thereby further improve the yield of expressed protein.
For illUstrative purposes, the sequence deter- ' mined for the gene of HBcAg has been employed in this way to improve the production of HBcAg in E. coil. One such process is depicted in Figure 10.
Inspection of the DNA sequence for HBcAg in Figure 3 reveals that this gene is preceded by a target for Alu I at nucleotide-26 (AGCT) and contains targets for .
Eco RI* at nucleotides 22 and 1590, for Eco RII at 209 (CCTGG), for Hae III at 280 (GGCC), and for Ava II at 246 and 461 (GGACC, GGTCC). Given this complexity, it is Ilb convenient to transfer a core antigen coding sequence to more efficient expression control sequences in two stages. For example, digestion of a recombinant DNA molecule (pHBV 114), prepared in accordance with this invention, having an HBV DNA insert-of about 2350 base pairs (nucleotides), i.e., from nucleotides -80 to about 2270 of Figure 3, with both Alu I and Eco RI* gives, inter alia, the fragment between nu-cleotides -26 and 22 (Fragment A) while Eco RI* diges-tion alone gives the fragment from nucleotides 23 to 1589 (Fragment B) and low yields of a fragment from nucleotides 23 to 576 (Fragment B'). These fragments.
may be readily identified by reference to Figures 3-4 and are depicted schematically in Figure 10. Frag-ments A and B are fractionated by gel electrophoresis to purify them and joined via their Eco RI* cohesive ends to give a combined fragment (Fragment C). Frag-ment C is now attached to a new expression control sequence as appropriate via direct ligation to main-tain the correct translational phase, via the 3' tailing method described previously, or via synthetic oligo-nucleotide linkers. This attachment not only makes use of a better expression control sequence to improve protein production but it also permits the gene fragment coding for HBcAg to be attached closer to the expression control sequence itself so as to enhance control of that gene fragment's expression.
In like fashion, Fragments A and B' may be joined or numerous other fragments prepared and the resultant fragment employed as above. This process demonstrates that only a portion of the structural gene coding for a particular polypeptide needs to be employed in recombinant DNA molecules of this invention.
To shorten even further the distance be-tween the particular expression control sequence and the initiation codon of the chosen gene fragment, the particular fragment may be treated lightly with .
_ -.
-29a-a combination of nucleases acting specifically at or near its terminus or used in exonuclease and polymerase-linked repair reactions to - -=
-remove some or all of those nucleotides of the frag-ment preceding the fragment's start codon. Alterna-tively, a fragment, such as an Alu I fragment result-ing from cleavage at nucleotides -26 and 30 could be similarly shortened with exonuclease treatment or polymerase-linked repair reactions and then cleaved with Eco RI* to produce a fragment from between nucleotides -26 and 1 and nucleotide 22 to permit fusion to fragment B before attachment to the expres-sion control sequence. A further route might involve hybridization of fragment B, or an equivalent frag-ment, to an appropriate single stranded template from the parent recombinant DNA molecule for exten-sion in a series of reactions with DNA polymerase and a limited number of nucleoside triphosphates so that the fragment strand could be reconstructed to the beginning of the coding sequence. The template strand would then be cleaved at the position of its association to the extended fragment strand with endonuclease Si and the fragment so obtained attached to the expression control sequence.
Several expression control sequences may be employed as described above. These include the operator, promoter and ribosome binding and inter-action sequences (including sequences such as the Shine-Dalgarno sequences) of the lactose operon of E.
coli ("the lac system"), the corresponding sequences of the tryptophan synthetase system of E. coli ("the trp system"), the major operator and promoter regions of phage X (0LPL and 0R PI), and the control region R
of the phage fd coat protein. DNA fragments contain-ing these sequences are excised by cleavage with restriction enzymes from the DNA isolated from trans-ducing phages that carry the lac or trp operons, or from the DNA of phage A or fd. These fragments are then manipulated in the manner outlined for the HBV
antigen sequences in order to obtain a limited popu--30a-lation of molecules such that the essential con-trolling sequences can be joined very close to, or in juxtaposition with the initiation codon of the coding sequence for the desired antigen as de-scribed above for Fragment C.
The fusion product is then inserted as before into a cloning vehicle for transformation of the appropriate hosts and the level of antigen pro-duction measured by radioimmunoassay after lysis of the cells. Cells giving the most efficient expression may be thus selected. Alternatively, cloning vehicles carrying the lac, trp or X PI, control system attached to an initiation codon may be employed and fused to a fragment containing a sequence coding for a poly-peptide displaying HBV antigenicity such that the structural gene fragment is correctly translated from the initiation codon of the cloning vehicle.
While these experiments have related to the production of HBV core antigen specifically, they are applicable to the improvement of the ex-pression of other genes such as HBsAg and HBeAg genes and fragments thereof.
Further increases in the cellular yield of these particular antigens depend upon an increase in the number of genes that can be utilized in the cell.
This has been achieved for illustration purposes by insertion of recombinant DNA molecules engineered in the way described previously into the temperate bac-teriophage X (NM989), most simply by digestion of the plasmid with a restriction enzyme, e.g., Eco RI
or Hind III, to give a linear molecule which was then mixed with a restricted phage X cloning vehicle (e.g., of the type described by N. E. Murray et al., "Lambdoid Phages That Simplify The Recovery Of In Vitro Recombinants", Molec. gen. Genet., 150, pp. 53-61 (1977) and N. E. Murray et al., "Molecular Cloning Of The DNA Ligase Gene From Bacteriophage T4", J. Mol. Biol., 132, pp. 493-505 (1979)) and the recombinant DNA molecule produced by incubation with DNA ligase. Such a procedure is depicted in Figure 11.
. I
µ=
. .
-31a-The desired recombinant phage was then.selected by radioimmunoassay for the particular antigen or by hybridization with radioactively labelled HBV DNA
sequences and used to.lysogenise a host strain of E.coli.
,.
. =
= =
= =
. .
. . .
.. . =
. .
. . .
, .
II)-32-Particularly useful A cloning vehicles contain a temperature-sensitive mutation in the repression gene cI
and suppressible mutations in gene S. the product of which is necessary for lysis of the host cell, and gene E, the product which is the major capsid protein of-the virus.
With this system the lysogenic cells are grown at 32 C and then heated to 45 C to induce excision of the prophage.
Prolonged growth at 37 C leads to high levels of production of the antigen, which is retained within the cells, since these are not lysed by phage gene products in the normal way, and since the phage gene insert is not encapsidated it remains available for transcription. Artificial lysis of the cells then releases the antigen in high yield .(Figure 11).
In addition to the E. coli systems with which these examples have been principally concerned, the same type of manipulations can be performed to increase the level of antigen production in other microbial cells, such as B. subtilis, thermophilic bacteria or yeasts and fungi, or in animal or plant cells in culture. In the case of B. subtilis a plasmid that carries the determinants for penicillinase'isolated from B. licheniformis offers a useful expression control sequence for these purposes.
PREPARATION OF IMPROVED RECOMBINANT DNA MOLECULES
FOR EXPRESSION OF THE GENE CODING FOR HBsAg The gene and amino acid sequences determined for HBsAg are useful in designing methods to permit expression of the gene for HBsAg by the process of this invention.
Such expression was not previously observed in hosts trans-formed with recombinant DNA molecules that produced poly-peptides displaying HBV antigenicity.
The nucleotide sequence of Figures 6-8 displays the gene coding for HBsAg between nucleotides 1437 and 2114. This gene is in a different translational phase (Reading Frame 3) than the gene coding for HBcAg (Reading Frame 1) in the HBV genome of Figures 3-9. Therefore, a recombinant DNA molecule that did not produce HBcAg when used to transform an appropriate host, but contained an HBV
DNA insert of about 2350 nucleotides (corresponding to about nucleotides -80 to 2270 of the sequence of Figures 3-8) was chosen for use in HBsAg production.
The selected recombinant DNA molecule contained, inter alia, the entire gene coding for HBsAg. Examination of the 5ucleotide sequence of the HBV DNA ,insert of this recombinant DNA molecule reveals several restriction endo-nuclease targets that permit excision of fragments contain-ing the gene coding for HBsAg. E.g., nucleotide 1409 (Xho), nucleotide 1410 (Taq), nucleotide 1409 (Ava I) and nucleotide 1428 (Hha I) (Figure 6). Of these the latter (Hha I) is particularly useful because Cleavage of the HBV
DNA insert occurs between nucleotides 1430 and 1431, a cut only six nucleotides in advance of the translational ini-tiation codon (ATG) of the gene for HBsAg itself. In all four cases, the excised fragment will bextend beyond the chosen HBV DNA insert to a target for the particular restriction endonuclease located within the nucleotide sequence of the pBR322 portion of the recombinant DNA
molecule. Therefore, the use of these restriction endonu-, cleases and others similarly useful, alone or in combina-tion, permit the excision of the gene coding for HBsAg near but in advance of its initiation codon and after its trans-lational termination codon. Of course, it is to be under-stood that as illustrated in the case of the gene for HBcAg, only fragments of the entire gene need be actually employed in recombinant DNA molecules to produce polypep-tides displaying HBsAg antigenicity in appropriate hosts.
ln those described described for the gene and gene fragments coding for core antigen. For example, the gene fragment may be inserted into the gene coding for penicillin resistance of pBR322 (e.g., Pst I recognition site, Figure 2) producing the polypeptide displaying HBsAg antigenicity-in fusion with /7 -lactamase (the product of the penicillinase gene), the gene fragment may be inserted between the genes coding for penicillin resistance and tetracycline resist-ance in pBR322 (Eco RI or Hind III recognition sites, Figure 2) and joined there or before insertion to the lac system expression control sequence thereby producing the polypeptide displaying HBsAg antigenicity fused to the /? -galactosidase protein of the lac system, the gene fragment may be inserted into a cloning vehicle as close as possible to the chosen expression control sequence itself as was described previously for the gene coding for HBcAg, or the gene fragment may be otherwise cloned as described in accordance with this invention.
For illustration purposes, one such scheme is depicted in Figure 12. There, the selected recombinant DNA
molecule having an HBV DNA insert extending between about nucleotides -80 and 2270 (Figures 3-8) was fragmented by digestion with Hha I. The resulting fragment was inserted , via the 3' tailing method described previously at the Pet I
site of pBR322. Hosts transformed with this recombinant DNA molecule produced polypeptides displaying HBsAg anti-genicity.
Other fragments (e.g., Ava I, Tag, Xho, or Pet I
(partial digest)) have also been inserted at the Pet I
recognition site or other sites in pBR322 to produce recom-binant DNA molecules which are useful for the production of polypeptides displaying HBsAg antigenicity or gene frag-ments coding for HBsAg.
In addition, such gene fragments have been inserted in pBR322 which had been cleaved by Pet I and the resulting .fragment of the gene coding for penicillinase , then chewed away from the codon coding for amino acid 182 of penicillinase or /7 -lactamase in one case to the codon coding for amino acid 32 and in another case to the codon coding for amino acid 13 of that polypeptide. These deny-ative plasmids of pBR322-Pst I have also been- employed with HBsAg gene fragments that extended from nucleotides 1447 to 1796 (Figures 6-7) to produce polypeptides displaying HBsAg antigen;city.
The nucleotide sequence between nucleotides 948 and 1437, immediately preceding the structural gene' coding for HBsAg, may also be included in the fragments employed to produce useful recombinant DNA molecules for production of polypeptides displaying HBsAg antigenicity and gene .fragments of the structural gene for HBsAg. This nucleo-tide sequence is referred to as the precursor sequence of the gene. Gene fragments including both this precursor sequence and the structural gene for HBsAg may be excised with Alu I (nucleotide 939), Hha I (nucleotide 900) or Hae II (nucleotide 899). In the case of Alu I and Hha I
partial digestion and separation of the resulting frag-ments, e.g., by gel electrophoresis), is necessary because recognition sites for these enzymes also occur within the precursor sequence at nucleotide 1091 for Alu I and at nucleotide 1428 for Hha II.
While we have hereinbefore presented a number of embodiments of this invention, it is apparent that our basic construction can be altered to provide other embodi-ments which utilize the process of this invention. There-fore, it will be appreciated that the scope of this inven-tion is to be defined by the claims appended hereto rather than the specific embodiments which have been presented hereinbefore by way of example.
Micro-organisms prepared by the proce ses described herein are exemplified by cultur is deposited J
in the Culture Collection of the National Collection of Industrial Bacteria, Aberdeen, Scotland on December 19, 1979, and identified as pBR322-HBV-G-L and characterized as follows:
G: J ___________________ coli HB101/pBR322-Pst I dG:haff-Kon I dC
(hereinafter "pHBV114"):TetR Amps HBV" [NCIB 11554]
H: JE coli HB101/pBR322-Pst I' dG:pHBV114-Plst I : Teta Amps HBV" VA" [NCIB 11555 and 11556]
I: E. coli HB101/((pBR322-Eco RI, Bam HI: lac promoter sequence)-Hind III linkers:pHBV114-Hha, I Bam HI):
Tets Ampa HBV VA' [NCII3 11557]
coli HB101/pUR2*-Eco RI:pHBV114-Hha I! Eco RI
linkers:Tets Ampa HBV' VA' NCIB 11558]
K: E. coli HB101/pBR322-Pst I dG:pHBV114-Avbõ.I dC =
TetR Amps HBV-* VA*
.(NCIB 11559]
;
L: E. coli HB101/pBR322-Pst I dG:DHBV114-Tab dC
Teti' Amps HBV" VA' tNCIE 11560]
i * A derivative of pBR322 having a DNA sequence including the gene coding for the polypeptide for tetracycline resistance replaced with a smaller DNA
sequence including the Ei_r_ coli lac system. :
, .:=
=
' =
:
' , ;
= ;
:
=
, =
. .
X 2) .7. =
Trade Mark) filter containing cells transformed in accordance with this invention was lifted from its plate and placed, colonies downwards, in contact with the surface of the L-agar plate of E.coli C600 25 that had been lysed with X vir. This contact was continued for about 10 minutes to permit infection of the cells residing on the Millipore (Reg. Trade Mark) filter by X vir. The Millipore (Reg. Trade Mark) filter was then transferred to a fresh plate of L-agar 30 and incubated at 37 C for a further 5 hours. Meanwhile polyvinyl discs were coated with HBV antibodies as described by S. Broome and W. Gilbert, "Immunological Screening Method To Detect Specific Translation Pro-ducts", Proc. Natl. Acad. Sci. U.S.A., 75, pp. 2746-35 2749 (1978). The Millipore (Reg. Trade Mark) filters on which the transformed bacterial colonies were now obviously lysed were placed with the colonies in contact with the coated polyvinyl discs and kept at 4 C for 3 hours. This contact results in any HBV
40 antigens lysed from the cells on the Millipore (Reg.
Trade Mark) filter binding with the HBV antibodies of the disc. The coated discs were separated from the Millipore (Reg. Trade Mark) filter and then washed and incubated with 125I-labelled HBV antibodies. This incubation results in radio-active labelling of those sites on the disc where HBV antigens from the Millipore (Reg. Trade Mark) 5 filter had previously become bound by the HBV anti-bodies of the disc. After washing and radioauto-graphy, as described by Broome and Gilbert, supra, colonies that had produced polypeptides with HBV
antigen specificity were located by reference to the 10 radioautograph.
USE OF POLYPEPTIDES AND GENES PRODUCED
FROM RECOMBINANT DNA MOLECULES IN DETECTING
THE PRESENCE OF HBV ANTIBODIES IN HUMANS
Polypeptides displaying HBV antigenicity 15 and the structural genes and fragments which code therefor may be used in methods and kits designed to detect the presence of HBV antibodies in humans and therefore recognize humans and blood samples which have been infected by this virus.
20 For example, the HBcAg produced by hosts transformed by recombinant DNA molecules of this invention can be used in the immunological diagnostic tests currently available for hepatitis B virus detection, that is radioimmunoassay or ELISA (enzyme 25 linked immunosorbent assay). In one type of radio-immunoassay anti-core antigen antibody, raised in a laboratory animal, is attached to a solid phase, for example, the inside of a test tube. HBcAg is then added to the tube so as to bind with the antibody.
30 To the tube coated with the antigen-antibody complex is added a sample of the patient's serum, together with a known amount of HBV anti-core antibody labelled with a radioactive isotope such as radioactive iodine.
Any HBV antibody in the patient's serum will compete 35 with the labelled antibody for the free binding sites on antigen-antibody complex. Once the serum has been allowed to interact, the excess liquid is removed, the test tube washed, and the amount of radioactivity measured. A positive result, i.e. that the patient's 40 serum contains HBV antibody, is indicated by a low radioactive count. In one type of Elisa test, a microtitre plate is coated with HBcAg and to this is added a sample of patient's serum. After a period of incubation permitting interaction of any antibody with the antigen, the plate is washed and a preparation of anti-human antibodies, raised in a laboratory animal, and which are linked to an enzyme label is added, incubated to allow reaction to take place, and the plate is then rewashed.
Thereafter, enzyme substrate is added to the microtitre plate and incubated for a period of time to allow the enzyme to work on the substrate, and the adsorbance of the final preparation is measured. A large change in adsorbance indicates a positive result.
IN VIVO ACTIVITY OF POLYPEPTIDES
PRODUCED FROM RECOMBINANT DNA MOLECULES
To test the biological activity of the antigenic polypeptides translated from the recombinant DNA molecule in the E. coli of this invention, sterile extracts of the bacterial cells which were shown to express HBcAg were injected into rabbits after mixing with an equal volume of Freund's adjuvant. Two animals received the crude bacte-rial extract and two were given samples of the same extract after fractionation on a Sephadex 650 column. Additional injections of the same sample, which had been frozen and stored (retaining full antigenic activity) were given two and five weeks after the first injection. The animals were bled at intervals several weeks after initial injection.
Immunodiffusion experiments, using the procedure described by 0. Ouchterlony, "Immunodiffusion and Immuno-electrophoresis", in Handbook of Experimental Immunology (D. W. Weir ed.) (Blackwell Scientific Publications, Oxford and Edinburgh) chap. 19 (1967), were performed to compare the rabbit sera (antibodies) to human hepatitis B virus core antibody ("HBcAb") using HBcAg derived from human liver (B. J. Cohen and Y. E. Cossart, "Application Of A
Screening Test For Antibody To Hepatitis B Core Antigen", J. Clin. Path., 30, pp. 709-713 (1977)). All four rabbit . .
Ill ' -21-.,_ sera gave precipitin lines with HBcAg identical to those formed between HBcAg and the HBcAb derived from human sources. Therefore, the core antigen synthesized in E.
coli with the recombinant DNA molecules of this invention is serologically active in vivo. This activity establishes the feasibility of compositions and methods using viral antigens synthesized in microbial cells for the stimulation of antibody formation in humans. Such compositions and methods are characterized by the polypeptides produced in hosts transformed by recombinant DNA molecules prepared in accordance with this invention. These polypeptides would be employed alone or with well known pharmaceutically acceptable carriers such as saline solutions or other .additives recognized by the art for use in compositions and methods for the treatment and prevention of viral infec-tions in humans.
As noted previously, restriction enzymes other than the Kpn I/Pst I combination described above may be usefully employed in preparing recombinant DNA molecules of this invention. Other restriction sites in plasmid pBR322 and part of the HBV genome are depicted in Figures 2 and 3, respectively. In illustration of these alternative, but less preferred embodiments, the following examples are set forth.
BAN HI, ECO RI, BGL II--PST I COMBINATIONS
The HBV DNA fragments produced by the use of Barn HI Eco RI and Bgl .II, unlike those produced by Kpn I, could not conveniently be directly tailed for cohesion owing to the presence of short 5' single-stranded pro-jections. They were therefore treated with )s exonuclease to remove these projections before the addition of poly-dC
sequences at 3' termini. This was done by incubation of the restricted DNA (10 )1l of a solution as described earlier) with 15 pl 100 mM sodium glycinate, pH 9.-5, 10 mM
MgCl2, 100 pg/m1 bovine serum albumin, 5 pl ). exonuclease at 0 C for 1.5 hours. The mixture was then extracted with = 1111 -22-phenol and chloroform and the process continued. In the preparation in which Barn HI was used, subsequent radioim-munoassay confirmed the production of polypeptides with HBV
antigen specificity.
DIRECT LIGATION: ECO RI--ECO RI AND BAN HI
Instead of tailing the DNA fragments for cohe-sion, as described above, direct ligation of the gene fragments may be employed in the processes of this inven-tion. For example, in two further preparations HBV DNA (20 ng) was restricted with restriction endonuclease Eco RI or Barn HI in 10 mM tris-HC1, pH 7.5, 10 mM MgCl2, 10 mM 2-mer-___ captoethanol, 50 mM NaC1 (10)41) at 37 C for 1.5 hours and mixed in excess with pBR322 that had been incubated with the same enzymes under identical conditions. A concen-trated buffer solution (660 mM tris-HC1, pH 7.5, 100 mM
MgCl2, 10 mM EDTA, 100 mM 2-mercaptoethanol, 400 mM NaCl, 1 mM ATP) (2 )11) was added and the mixture incubated with T4 DNA ligase (0.5)11) at 10 C for 3 hours followed by 0 C for at least 24 hours. The solution was diluted to 0.1 ml with 10 mM tris-HC1, pH 7.5 and used to transform competent cultures of E. coli HB 101 as described earlier. In the case of Barn HI prepared recombinant DNA molecules, the colonies were tested, before screening for hybridization, for sensitivity to tetracycline rather than to ampicillin, because the target for Barn HI in pBR322 is within the gene coding for tetracycline resistance and therefore successful insertion at this recognition site results in loss of this resistance instead of the ampicillin resistance as in the case of insertion at the Pst I site.
In the case of recombinant DNA molecules prepared via the Eco RI site, the colonies were tested, before screening for hybridization, for resistance to both ampi-cillin and tetracycline because the target for EcoRI in pBR322 is between the genes coding tetracycline and ampi-cillin resistance such that no inactivation of the genes for ampicillin or tetracycline resistance occurs on hybrid DNA insertion at this site.
Micro-organisms prepared by the =rocesses described herein are exemplified by culturzs deposited in the Culture Collection of the Microbiol=gical Research Establishment at Porton Down on D-cember 15, 1978 and identified as DBR322-HBV-A to F.
Specifically, these cultures are characterized as follows:
A: E. coil HB 101/pBR322-Pst I dG:HBV-Kpn I dc:
TetR Amps HBV* VA- [NCIB 11548]
B: E. coil HB 101/pBR322-Pst I dG:HBV-Bam HI dC:
TetR Amp' HBV- VA* [NCIB 11549]
C: E. coil HB 101/pBR322-Pst I dG:HBV-8g1 I dC:
Tee Amp HBV* [NCIB 1l550]
D: E coli HB 101/pBR322-Pst. I dG:HBV- PhI dC:
TetR Amps HBV* [NCIB 11551]
E: E_ coil MB 101/pBR322-Bam HI:HBV-Bam HI:
Tets Ai5 HBV* [NCIB 11552]
F: E oH MB 101/pBR322-Tco, RI:HBV-Eco RI:
TetR Amp' HBV* [NCIB 11553].
Portions of these identical cultures have .1so been deposited in the Culture Collection of the Iational Collection of Industrial Bacteria, Aberdee , Scotland on December 20, 1979, and were assigned the accession numbers shown above in brackets.
The above nomenclature for the cultures is a description of that culture as follows: Ho t/cloning vehicle - restriction site in cloning vehic e with indication of elongation nucleotide (if an ) :
hepatitis B virus restriction site in hepatitis B virus with indication of elongation nucleotide (if any) :
resistance (R) or sensitivity (S) to tetracicline (Tet), and ampicillin (.Amp), positive hybri=ization to HBv DNA in the colony hybridization test, s, ora, (HBV') and production of polypeptide displaying H7 anti-genicity, (VA-') Using this nomenclture culture pBR322-HBV-A designates an E. coli HE, 101 culture - 23a -containing as a plasmid a recombinant DNA molecule comprising pER322 cleaved at the .Pst I site and elongated with a poly =
/
111' dG tail at the 3' termini attached to HBV DNA cleaved with Kpn I and elongated with a poly-dC tail at the 3' termini, the culture displaying resistance to tetracycline, sensi-tivity to ampicillin, a positive HBV DNA hybridization test and producing a polypeptide displaying HBV antigenicity.
Of course, it is to be understood that the hybrid micro-organisms, recombinant DNA molecules and polypeptides and methods applicable to them of this invention are not limited to those described in the preferred embodiment above. Instead, the hybrid organisms, recombinant DNA
molecules and polypeptides may be modified during produc-tion or subsequently by known methods to good advantage.
For example, more efficient expression control sequences may be utilized for transcription of the HBV genes or hybrid genes, mutations to reduce the synthesis of unde-sired gene products may be introduced, the protease levels in the host cells may be reduced, thermo-inducible lysogens containing the HBV genes may be integrated into the host chromosome or other modifications and procedures may be carried out to increase the number of gene copies in the cell or to increase the cell's productivity in producing the desired polypeptides.
Apart from their use to produce polypeptides displaying HBV antigenicity, the hybrid micro-organisms of this invention are also useful for the production of large quantities of DNA containing all or part of the genome for hepatitis B virus. For example, amplification, e.g., by addition of chloramphenicol to the growth medium when the cell density has reached a suitable level, or the use of mutations to prevent lyais in bacteriophage hybrids that contain HBV sequences permit the preparation of HBV DNA in amounts previously unavailable.
HBV DNA prepared in this way may be used to determine the nucleotide sequence of the genome and from that the gene and amino acid sequences of the HBV antigens and structural genes themselves. Knowledge of these sequences aids in understanding the biology of the hepati-tis B virus and permits the modifications described above to be employed to best advantage.
DNA FRAGMENT MAPPING AND
, 5 NUCLEOTIDE SEQUENCE DETERMINATION_ 1. Hepatitis B Core Antigen A series of recombinant DNA molecules, made in accordance with the processes described above, which imparted to their host cells the ability to synthesize HBcAg as detected by solid phase radioimmunoassay were chosen for sequence analysis. Fragments were prepared from these recombinant DNA molecules by digestion with appro-priate restriction enzymes and the fragments labelled at their 5' termini with [o(-32P] ATP and T4 polynucleotide kinase. The nucleotide sequences of each fragment were then determined by well-known chemical degradative methods (A. M. Maxam and W. Gilbert, "A New Method For Sequencing DNA", Proc. Nat. Acad. Sci. U.S.A., 74, pp. 560-564 (1977).
The resulting nucleotide sequence is depicted in Figures 3-9. For reference, the-sequence is numbered from the A of the ATG translational initiation codon of the core gene. The nucleotide sequence of the gene for HBcAg and the amino acid sequence for the polypeptide deduced from this gene (Reading Frame 1) is depicted in Figures 3-9 between nucleotides 1-549. The size of the polypeptide encoded by this gene is close to the 19000 molecular weight observed for core antigens from Dane particles. However, the present structural determination does not exclude the possibility that some amino acids may be trimmed from the amino terminus of this polypeptide in vivo during formation of the authentic antigen. This structure also does not take into account any other or further modifications to the polypeptide caused by its interaction with other human enzymes, e.g., those enzymes which glycosolate proteins.
Therefore, the polypeptide structure determined herein may not be identical to that HBcAg found in vivo, but it will = -26-still elicit a very similar, if not identical, immuno response.
All of the recombinant DNA molecules examined had the HBV DNA inserted so that the core antigen gene was maintained in the same translational phase as-the gene for penicillin resistance of pBR322. Moreover, in the various recombinants the HBV DNA insert began within one or two nucleotides of the same position in the HBV sequence.
Because these recombinant DNA molecules originated from HBV
DNA digested with various restriction enzymes, e.g., Barn HI
and Kpn I, this unique attachment is surprising and may have resulted from inadvertent breakage or polynucleotide tailing of the HBV DNA at the nick in the endogenous DNA of the Dane particle (Figure 1). Recombinant DNA molecules having the HBV DNA insert in a different translational phase did not express the HBcAg gene and no polypeptide displaying HBV antigenicity was detected in hosts trans-formed with such recombinant DNA molecules. Of course, it is to be understood that such non-gene expressing hosts and recombinant DNA molecules are still useful for producing HBV DNA in accordance with this invention.
While it was expected that HBcAg or fragments thereof produced through expression of the HBV DNA inserts would be fused to the product ( fi -lactamase) of the gene for penicillinase resistance via a small number of glycine residues, that was not the case. Instead, the /8- lacta-mase was in each case fused through 5-8 glycines to an identical peptide sequence, but this sequence terminated after 25 amino acids. In this reading frame (Frame 1, Figure 3), a stop codon.(TAG) is followed three nucleotides later by an initiation codon from which translation con-tinues unhindered to give a polypeptide 183 amino acids in length (Figures 3-8). Therefore, core antigenic activity resides in a polypeptide of about 21,000 daltons translated de novo from the HBV sequence within the mRNA transcribed from the recombinant DNA molecule. This polypeptide remains within the host cell because it is not attached to the excreted penicillinase carrier protein due to the arrangement of the stop and start codons of the HBV DNA
insert.
2. Hepatitis B Surface Antigen The nucleotide sequence of the gene for HBsAg and the amino acid sequence for the polypeptide deduced from this gene (Reading Frame 3) is also depicted in Figures 6-8, between nucleotides 1437-2114. The amino acid sequence of this polypeptide begins at the N terminus with the sequence met-glu-asn-ile-thr-ser. This initial amino acid sequence was determined by D. L. Peterson et al., "Partial Amino Acid Sequence Of Two Major Component Polypeptides Of Hepatitis B Surface Antigen", Proc. Natl. Acad. Sci.
U.S.A., 74, pp. 1530-1534 (1977) from authentic human HBsAg. The amino acid sequence of this protein continues in Figures 6-8 to a stop codon 226 amino acids away. This 226 polypeptide sequence corresponds to a protein of 25,400 daltons.
The HBsAg amino acid sequence and length has been independently confirmed by sequencing appropriate recombi-nant DNA molecules by P. Valenzuela et al., "Nucleotide Sequence Of The Gene Coding For The Major Protein of Hepa-titis B Virus Surface Antigen", Nature, 280, pp. 815-819 (1979).
The nucleotide sequence determined for the recom-binant DNA molecules of this invention shows that none of those examined which expressed the gene for HBcAg can have the gene for HBsAg in a position from which it could also be expected to be expressed in an appropriate host cell.
In fact, no HBsAg was detected in the extracts of cells transformed with those plasmids found to express the gene for HBcAg. However, as noted, supra, knowledge of the nucleotide sequences of these genes permits modification of the expression process to improve its yield and effect and to favor the expression of genes and the production of polypeptides not previously expressed or detected.
lik = ' , PREPARATION OF IMPROVED RECOMBINANT DNA
MOLECULES FOR PRODUCTION OF HBV ANTIGENS
The gene and amino acid sequences of these anti-gens are useful in designing methods to increase the level of production of antigen or gene per bacterial cell.
The level of production of a protein is governed by two major factors: the number of copies of its gene within the cell and the efficiency with which these. gene copies are transcribed and translated. Efficiency of transcription and translation (which together comprise expression) is in turn dependent upon nucleotide sequences, normally situated ahead of the desired coding sequence.
These nucleotide sequences or expression control sequences , define, inter alia, the location at which RNA polymerase interacts to initiate transcription (the promoter sequence) and at which ribosomes bind and interact with the mRNA (the product of transcription) to initiate translation. Not all such expression control sequences function with equal efficiency. It is thus of advantage to separate the speci-fic coding sequences for the desired protein from their adjacent nucleotide sequences and fuse them instead to known expression control sequences so as to favor higher levels of expression. This having been achieved, the newly engineered DNA fragment may be inserted into a multicopy plasmid or a bacteriophage derivative in order to increase the number of gene copies within the cell and thereby further improve the yield of expressed protein.
For illUstrative purposes, the sequence deter- ' mined for the gene of HBcAg has been employed in this way to improve the production of HBcAg in E. coil. One such process is depicted in Figure 10.
Inspection of the DNA sequence for HBcAg in Figure 3 reveals that this gene is preceded by a target for Alu I at nucleotide-26 (AGCT) and contains targets for .
Eco RI* at nucleotides 22 and 1590, for Eco RII at 209 (CCTGG), for Hae III at 280 (GGCC), and for Ava II at 246 and 461 (GGACC, GGTCC). Given this complexity, it is Ilb convenient to transfer a core antigen coding sequence to more efficient expression control sequences in two stages. For example, digestion of a recombinant DNA molecule (pHBV 114), prepared in accordance with this invention, having an HBV DNA insert-of about 2350 base pairs (nucleotides), i.e., from nucleotides -80 to about 2270 of Figure 3, with both Alu I and Eco RI* gives, inter alia, the fragment between nu-cleotides -26 and 22 (Fragment A) while Eco RI* diges-tion alone gives the fragment from nucleotides 23 to 1589 (Fragment B) and low yields of a fragment from nucleotides 23 to 576 (Fragment B'). These fragments.
may be readily identified by reference to Figures 3-4 and are depicted schematically in Figure 10. Frag-ments A and B are fractionated by gel electrophoresis to purify them and joined via their Eco RI* cohesive ends to give a combined fragment (Fragment C). Frag-ment C is now attached to a new expression control sequence as appropriate via direct ligation to main-tain the correct translational phase, via the 3' tailing method described previously, or via synthetic oligo-nucleotide linkers. This attachment not only makes use of a better expression control sequence to improve protein production but it also permits the gene fragment coding for HBcAg to be attached closer to the expression control sequence itself so as to enhance control of that gene fragment's expression.
In like fashion, Fragments A and B' may be joined or numerous other fragments prepared and the resultant fragment employed as above. This process demonstrates that only a portion of the structural gene coding for a particular polypeptide needs to be employed in recombinant DNA molecules of this invention.
To shorten even further the distance be-tween the particular expression control sequence and the initiation codon of the chosen gene fragment, the particular fragment may be treated lightly with .
_ -.
-29a-a combination of nucleases acting specifically at or near its terminus or used in exonuclease and polymerase-linked repair reactions to - -=
-remove some or all of those nucleotides of the frag-ment preceding the fragment's start codon. Alterna-tively, a fragment, such as an Alu I fragment result-ing from cleavage at nucleotides -26 and 30 could be similarly shortened with exonuclease treatment or polymerase-linked repair reactions and then cleaved with Eco RI* to produce a fragment from between nucleotides -26 and 1 and nucleotide 22 to permit fusion to fragment B before attachment to the expres-sion control sequence. A further route might involve hybridization of fragment B, or an equivalent frag-ment, to an appropriate single stranded template from the parent recombinant DNA molecule for exten-sion in a series of reactions with DNA polymerase and a limited number of nucleoside triphosphates so that the fragment strand could be reconstructed to the beginning of the coding sequence. The template strand would then be cleaved at the position of its association to the extended fragment strand with endonuclease Si and the fragment so obtained attached to the expression control sequence.
Several expression control sequences may be employed as described above. These include the operator, promoter and ribosome binding and inter-action sequences (including sequences such as the Shine-Dalgarno sequences) of the lactose operon of E.
coli ("the lac system"), the corresponding sequences of the tryptophan synthetase system of E. coli ("the trp system"), the major operator and promoter regions of phage X (0LPL and 0R PI), and the control region R
of the phage fd coat protein. DNA fragments contain-ing these sequences are excised by cleavage with restriction enzymes from the DNA isolated from trans-ducing phages that carry the lac or trp operons, or from the DNA of phage A or fd. These fragments are then manipulated in the manner outlined for the HBV
antigen sequences in order to obtain a limited popu--30a-lation of molecules such that the essential con-trolling sequences can be joined very close to, or in juxtaposition with the initiation codon of the coding sequence for the desired antigen as de-scribed above for Fragment C.
The fusion product is then inserted as before into a cloning vehicle for transformation of the appropriate hosts and the level of antigen pro-duction measured by radioimmunoassay after lysis of the cells. Cells giving the most efficient expression may be thus selected. Alternatively, cloning vehicles carrying the lac, trp or X PI, control system attached to an initiation codon may be employed and fused to a fragment containing a sequence coding for a poly-peptide displaying HBV antigenicity such that the structural gene fragment is correctly translated from the initiation codon of the cloning vehicle.
While these experiments have related to the production of HBV core antigen specifically, they are applicable to the improvement of the ex-pression of other genes such as HBsAg and HBeAg genes and fragments thereof.
Further increases in the cellular yield of these particular antigens depend upon an increase in the number of genes that can be utilized in the cell.
This has been achieved for illustration purposes by insertion of recombinant DNA molecules engineered in the way described previously into the temperate bac-teriophage X (NM989), most simply by digestion of the plasmid with a restriction enzyme, e.g., Eco RI
or Hind III, to give a linear molecule which was then mixed with a restricted phage X cloning vehicle (e.g., of the type described by N. E. Murray et al., "Lambdoid Phages That Simplify The Recovery Of In Vitro Recombinants", Molec. gen. Genet., 150, pp. 53-61 (1977) and N. E. Murray et al., "Molecular Cloning Of The DNA Ligase Gene From Bacteriophage T4", J. Mol. Biol., 132, pp. 493-505 (1979)) and the recombinant DNA molecule produced by incubation with DNA ligase. Such a procedure is depicted in Figure 11.
. I
µ=
. .
-31a-The desired recombinant phage was then.selected by radioimmunoassay for the particular antigen or by hybridization with radioactively labelled HBV DNA
sequences and used to.lysogenise a host strain of E.coli.
,.
. =
= =
= =
. .
. . .
.. . =
. .
. . .
, .
II)-32-Particularly useful A cloning vehicles contain a temperature-sensitive mutation in the repression gene cI
and suppressible mutations in gene S. the product of which is necessary for lysis of the host cell, and gene E, the product which is the major capsid protein of-the virus.
With this system the lysogenic cells are grown at 32 C and then heated to 45 C to induce excision of the prophage.
Prolonged growth at 37 C leads to high levels of production of the antigen, which is retained within the cells, since these are not lysed by phage gene products in the normal way, and since the phage gene insert is not encapsidated it remains available for transcription. Artificial lysis of the cells then releases the antigen in high yield .(Figure 11).
In addition to the E. coli systems with which these examples have been principally concerned, the same type of manipulations can be performed to increase the level of antigen production in other microbial cells, such as B. subtilis, thermophilic bacteria or yeasts and fungi, or in animal or plant cells in culture. In the case of B. subtilis a plasmid that carries the determinants for penicillinase'isolated from B. licheniformis offers a useful expression control sequence for these purposes.
PREPARATION OF IMPROVED RECOMBINANT DNA MOLECULES
FOR EXPRESSION OF THE GENE CODING FOR HBsAg The gene and amino acid sequences determined for HBsAg are useful in designing methods to permit expression of the gene for HBsAg by the process of this invention.
Such expression was not previously observed in hosts trans-formed with recombinant DNA molecules that produced poly-peptides displaying HBV antigenicity.
The nucleotide sequence of Figures 6-8 displays the gene coding for HBsAg between nucleotides 1437 and 2114. This gene is in a different translational phase (Reading Frame 3) than the gene coding for HBcAg (Reading Frame 1) in the HBV genome of Figures 3-9. Therefore, a recombinant DNA molecule that did not produce HBcAg when used to transform an appropriate host, but contained an HBV
DNA insert of about 2350 nucleotides (corresponding to about nucleotides -80 to 2270 of the sequence of Figures 3-8) was chosen for use in HBsAg production.
The selected recombinant DNA molecule contained, inter alia, the entire gene coding for HBsAg. Examination of the 5ucleotide sequence of the HBV DNA ,insert of this recombinant DNA molecule reveals several restriction endo-nuclease targets that permit excision of fragments contain-ing the gene coding for HBsAg. E.g., nucleotide 1409 (Xho), nucleotide 1410 (Taq), nucleotide 1409 (Ava I) and nucleotide 1428 (Hha I) (Figure 6). Of these the latter (Hha I) is particularly useful because Cleavage of the HBV
DNA insert occurs between nucleotides 1430 and 1431, a cut only six nucleotides in advance of the translational ini-tiation codon (ATG) of the gene for HBsAg itself. In all four cases, the excised fragment will bextend beyond the chosen HBV DNA insert to a target for the particular restriction endonuclease located within the nucleotide sequence of the pBR322 portion of the recombinant DNA
molecule. Therefore, the use of these restriction endonu-, cleases and others similarly useful, alone or in combina-tion, permit the excision of the gene coding for HBsAg near but in advance of its initiation codon and after its trans-lational termination codon. Of course, it is to be under-stood that as illustrated in the case of the gene for HBcAg, only fragments of the entire gene need be actually employed in recombinant DNA molecules to produce polypep-tides displaying HBsAg antigenicity in appropriate hosts.
ln those described described for the gene and gene fragments coding for core antigen. For example, the gene fragment may be inserted into the gene coding for penicillin resistance of pBR322 (e.g., Pst I recognition site, Figure 2) producing the polypeptide displaying HBsAg antigenicity-in fusion with /7 -lactamase (the product of the penicillinase gene), the gene fragment may be inserted between the genes coding for penicillin resistance and tetracycline resist-ance in pBR322 (Eco RI or Hind III recognition sites, Figure 2) and joined there or before insertion to the lac system expression control sequence thereby producing the polypeptide displaying HBsAg antigenicity fused to the /? -galactosidase protein of the lac system, the gene fragment may be inserted into a cloning vehicle as close as possible to the chosen expression control sequence itself as was described previously for the gene coding for HBcAg, or the gene fragment may be otherwise cloned as described in accordance with this invention.
For illustration purposes, one such scheme is depicted in Figure 12. There, the selected recombinant DNA
molecule having an HBV DNA insert extending between about nucleotides -80 and 2270 (Figures 3-8) was fragmented by digestion with Hha I. The resulting fragment was inserted , via the 3' tailing method described previously at the Pet I
site of pBR322. Hosts transformed with this recombinant DNA molecule produced polypeptides displaying HBsAg anti-genicity.
Other fragments (e.g., Ava I, Tag, Xho, or Pet I
(partial digest)) have also been inserted at the Pet I
recognition site or other sites in pBR322 to produce recom-binant DNA molecules which are useful for the production of polypeptides displaying HBsAg antigenicity or gene frag-ments coding for HBsAg.
In addition, such gene fragments have been inserted in pBR322 which had been cleaved by Pet I and the resulting .fragment of the gene coding for penicillinase , then chewed away from the codon coding for amino acid 182 of penicillinase or /7 -lactamase in one case to the codon coding for amino acid 32 and in another case to the codon coding for amino acid 13 of that polypeptide. These deny-ative plasmids of pBR322-Pst I have also been- employed with HBsAg gene fragments that extended from nucleotides 1447 to 1796 (Figures 6-7) to produce polypeptides displaying HBsAg antigen;city.
The nucleotide sequence between nucleotides 948 and 1437, immediately preceding the structural gene' coding for HBsAg, may also be included in the fragments employed to produce useful recombinant DNA molecules for production of polypeptides displaying HBsAg antigenicity and gene .fragments of the structural gene for HBsAg. This nucleo-tide sequence is referred to as the precursor sequence of the gene. Gene fragments including both this precursor sequence and the structural gene for HBsAg may be excised with Alu I (nucleotide 939), Hha I (nucleotide 900) or Hae II (nucleotide 899). In the case of Alu I and Hha I
partial digestion and separation of the resulting frag-ments, e.g., by gel electrophoresis), is necessary because recognition sites for these enzymes also occur within the precursor sequence at nucleotide 1091 for Alu I and at nucleotide 1428 for Hha II.
While we have hereinbefore presented a number of embodiments of this invention, it is apparent that our basic construction can be altered to provide other embodi-ments which utilize the process of this invention. There-fore, it will be appreciated that the scope of this inven-tion is to be defined by the claims appended hereto rather than the specific embodiments which have been presented hereinbefore by way of example.
Micro-organisms prepared by the proce ses described herein are exemplified by cultur is deposited J
in the Culture Collection of the National Collection of Industrial Bacteria, Aberdeen, Scotland on December 19, 1979, and identified as pBR322-HBV-G-L and characterized as follows:
G: J ___________________ coli HB101/pBR322-Pst I dG:haff-Kon I dC
(hereinafter "pHBV114"):TetR Amps HBV" [NCIB 11554]
H: JE coli HB101/pBR322-Pst I' dG:pHBV114-Plst I : Teta Amps HBV" VA" [NCIB 11555 and 11556]
I: E. coli HB101/((pBR322-Eco RI, Bam HI: lac promoter sequence)-Hind III linkers:pHBV114-Hha, I Bam HI):
Tets Ampa HBV VA' [NCII3 11557]
coli HB101/pUR2*-Eco RI:pHBV114-Hha I! Eco RI
linkers:Tets Ampa HBV' VA' NCIB 11558]
K: E. coli HB101/pBR322-Pst I dG:pHBV114-Avbõ.I dC =
TetR Amps HBV-* VA*
.(NCIB 11559]
;
L: E. coli HB101/pBR322-Pst I dG:DHBV114-Tab dC
Teti' Amps HBV" VA' tNCIE 11560]
i * A derivative of pBR322 having a DNA sequence including the gene coding for the polypeptide for tetracycline resistance replaced with a smaller DNA
sequence including the Ei_r_ coli lac system. :
, .:=
=
' =
:
' , ;
= ;
:
=
, =
. .
X 2) .7. =
Claims (120)
1. A method for producing a recombinant DNA molecule characterized by the step of introducing into a cloning vehicle a DNA sequence, said DNA
sequence being operatively linked to an expression control sequence in said recombinant DNA molecule and being expressed to produce a polypeptide or fragment thereof displaying HBV antigenicity or HBV antigen specificity when an appropriate host cell transformed with said recombinant DNA
molecule is cultured.
sequence being operatively linked to an expression control sequence in said recombinant DNA molecule and being expressed to produce a polypeptide or fragment thereof displaying HBV antigenicity or HBV antigen specificity when an appropriate host cell transformed with said recombinant DNA
molecule is cultured.
2. A method for producing a recombinant DNA molecule characterized by the step of introducing into a cloning vehicle a DNA sequence, said DNA
sequence being operatively linked to an expression control sequence in said recombinant DNA molecule and being expressed to produce a polypeptide or fragment thereof displaying the HBV antigenicity or HBV antigen specificity of a hepatitis B virus core antigen when an appropriate host cell transformed with said recombinant DNA molecule is cultured.
sequence being operatively linked to an expression control sequence in said recombinant DNA molecule and being expressed to produce a polypeptide or fragment thereof displaying the HBV antigenicity or HBV antigen specificity of a hepatitis B virus core antigen when an appropriate host cell transformed with said recombinant DNA molecule is cultured.
3. A method for producing a recombinant DNA molecule characterized by the step of introducing into a cloning vehicle a DNA sequence, said DNA
sequence being operatively linked to an expression control sequence in said recombinant DNA molecule and being expressed to produce a polypeptide or fragment thereof displaying the HBV antigen specificity of a hepatitis B virus surface antigen when an appropriate host cell transformed with said recombinant DNA molecule is cultured.
sequence being operatively linked to an expression control sequence in said recombinant DNA molecule and being expressed to produce a polypeptide or fragment thereof displaying the HBV antigen specificity of a hepatitis B virus surface antigen when an appropriate host cell transformed with said recombinant DNA molecule is cultured.
4. The method according to claim 1 or 2, wherein the DNA sequence is selected from the endogenous DNA of a Dane particle, fragments thereof which encode polypeptides displaying the HBV antigenicity or HBV antigen specificity of a hepatitis B virus core antigen and synthetically prepared equivalents thereto.
5. The method according to claim 1 or 3, wherein the DNA sequence is selected from the endogenous DNA of a Dane particle, fragments thereof which encode polypeptides displaying the HBV antigenicity or HBV antigen specificity of a hepatitis B virus surface antigen and synthetically prepared equivalents thereto.
- 38.-
- 38.-
6. The method according to claim 1 or 2, wherein the host cell is selected from strains of E.coli, Pseudomonas, Bacillus subtilis, other Bacilli, yeast, other fungi or animal or plant cells in culture.
7. The method according to claim 1 or 3, wherein the host cell is selected from strains of E.coli, Pseudomonas, Bacillus subtilis, other Bacilli, yeast, other fungi or animal or plant cells in culture.
8. The method according to claim 1 or 2, wherein the expression control sequence is selected from the lac system, the trp system, the major operator and promoter regions of phage A, and the= control region of phage fd coat protein.
9. The method according to claim 1 or 3, wherein the expression control sequence is selected from the lac system, the trp system, the major operator and promoter regions of phage and the control region of phage fd coat protein.
10. The method according to claim 1 or 2, wherein the DNA sequence is selected from DNA sequences of the formulae:
(a) ATGGACATTGACCCTTATAAAGAATTTGGAGCTACTGTGGAGTTACTCTCGT
TTTTGCCTTCTGACTTCTTTCCTTCCGTACGAGATCTTCTAGATACCGCCGC
AGCTCTGTATCGGGATGCCTTAGAGTCTCCTGAGCATTGTTCACCTCACCAT
ACTGCACTCAGGCAAGCAATTCTTTGCTGGGGAGACTTAATGACTCTAGCTA
CCTGGGTGGGTACTAATTTAGAAGATCCAGCATCTAGGGACCTAGTAGTCAG
TTATGTCAACACTAATGTGGGCCTAAAGTTCAGACAATTATTGTGGTTTCAC
ATTTCTTGTCTCACTTTTGGAAGAGAAACGGTTCTAGAGTATTTGGTGTCTT
TTGGAGTGTGGATTCGCACTCCTCCAGCTTATAGACCACCAAATGCCCCTAT
CCTATCAACGCTTCCGGAGACTACTGTTGTTAGACGACGAGGCAGGTCCCCT
AGAAGAAGAACTCCCTCGCCTCGCAGACGAAGATCTCAATCGCCGCGTCGCA
GAAGATCTCAATCTCGGGAATCTCAATGT and fragments thereof which encode polypeptides displaying the HBV antigenicity or HBV antigen specificity of a hepatitis B virus core antigen; and (b) DNA sequences which are degenerate as a result of the genetic code to any of the foregoing DNA sequences and which encode polypeptides displaying the HBV antigenicity or HBV antigen specificity of a hepatitis B virus core antigen.
(a) ATGGACATTGACCCTTATAAAGAATTTGGAGCTACTGTGGAGTTACTCTCGT
TTTTGCCTTCTGACTTCTTTCCTTCCGTACGAGATCTTCTAGATACCGCCGC
AGCTCTGTATCGGGATGCCTTAGAGTCTCCTGAGCATTGTTCACCTCACCAT
ACTGCACTCAGGCAAGCAATTCTTTGCTGGGGAGACTTAATGACTCTAGCTA
CCTGGGTGGGTACTAATTTAGAAGATCCAGCATCTAGGGACCTAGTAGTCAG
TTATGTCAACACTAATGTGGGCCTAAAGTTCAGACAATTATTGTGGTTTCAC
ATTTCTTGTCTCACTTTTGGAAGAGAAACGGTTCTAGAGTATTTGGTGTCTT
TTGGAGTGTGGATTCGCACTCCTCCAGCTTATAGACCACCAAATGCCCCTAT
CCTATCAACGCTTCCGGAGACTACTGTTGTTAGACGACGAGGCAGGTCCCCT
AGAAGAAGAACTCCCTCGCCTCGCAGACGAAGATCTCAATCGCCGCGTCGCA
GAAGATCTCAATCTCGGGAATCTCAATGT and fragments thereof which encode polypeptides displaying the HBV antigenicity or HBV antigen specificity of a hepatitis B virus core antigen; and (b) DNA sequences which are degenerate as a result of the genetic code to any of the foregoing DNA sequences and which encode polypeptides displaying the HBV antigenicity or HBV antigen specificity of a hepatitis B virus core antigen.
11. The method according to claim 1 or 3, wherein the DNA sequence is selected from DNA sequences of the formulae:
(a) ATGGAGAACATCACATCAGGATTCCTAGGACCCCTGCTCGTGTTACAGGCGG
GGTTTTTCTTGTTGACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTG
GTGGACTTCTCTCAATTTTCTAGGGGGAACTACCGTGTGTCTTGGCCAAAAT
TCGCAGTCCCCAATCTCCAATCACTCACCAACCTCCTGTCCTCCAACTTGTC
CTGGTTATCGCTGGATGTGTCTGCGGCGTTTTATCATCTTCCTCTTCATCCT
GCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTGGACTATCAAGGTATGTTG
CCCGTTTGTCCTCTAATTCCAGGATCATCAACCACCAGCACGGGATCCTGCA
GAACCTGCACGACTCCTGCTCAAGGAATCTCTATGTATCCCTCCTGTTGCTG
TACAAAACCTTCGGATGGAAACTGCACCTGTATTCCCATCCCATCATCCTGG
GCTTTCGGAAAATTCCTATGGGAGTGGGCCTCAGCCCGTTTCTCTTGGCTCA
GTTTACTAGTGCCATTTGTTCAGTGGTTCGTAGGGCTTTCCCCCATTGTTTG
GCTTTCAGTTATATGGATGATGTGGTATTGGGGGCCAAGTCTGTACAGCATC
TTGAGTCCCTTTTTACCGCTGTTACCAATTTTCTTTTGTCTTTGGGCATACA
TT and fragments thereof which encode polypeptides displaying the HBV
antigenicity or HBV antigen specificity of a hepatitis B virus surface antigen;
and (b) DNA sequences which are degenerate as a result of the genetic code to any of the foregoing DNA sequences and which encode polypeptides displaying the HBV antigenicity or HBV antigen specificity of a hepatitis B virus surface antigen.
(a) ATGGAGAACATCACATCAGGATTCCTAGGACCCCTGCTCGTGTTACAGGCGG
GGTTTTTCTTGTTGACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTG
GTGGACTTCTCTCAATTTTCTAGGGGGAACTACCGTGTGTCTTGGCCAAAAT
TCGCAGTCCCCAATCTCCAATCACTCACCAACCTCCTGTCCTCCAACTTGTC
CTGGTTATCGCTGGATGTGTCTGCGGCGTTTTATCATCTTCCTCTTCATCCT
GCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTGGACTATCAAGGTATGTTG
CCCGTTTGTCCTCTAATTCCAGGATCATCAACCACCAGCACGGGATCCTGCA
GAACCTGCACGACTCCTGCTCAAGGAATCTCTATGTATCCCTCCTGTTGCTG
TACAAAACCTTCGGATGGAAACTGCACCTGTATTCCCATCCCATCATCCTGG
GCTTTCGGAAAATTCCTATGGGAGTGGGCCTCAGCCCGTTTCTCTTGGCTCA
GTTTACTAGTGCCATTTGTTCAGTGGTTCGTAGGGCTTTCCCCCATTGTTTG
GCTTTCAGTTATATGGATGATGTGGTATTGGGGGCCAAGTCTGTACAGCATC
TTGAGTCCCTTTTTACCGCTGTTACCAATTTTCTTTTGTCTTTGGGCATACA
TT and fragments thereof which encode polypeptides displaying the HBV
antigenicity or HBV antigen specificity of a hepatitis B virus surface antigen;
and (b) DNA sequences which are degenerate as a result of the genetic code to any of the foregoing DNA sequences and which encode polypeptides displaying the HBV antigenicity or HBV antigen specificity of a hepatitis B virus surface antigen.
12. The method according to claim 1 or 2, wherein the recombinant DNA
molecule is selected from the recombinant DNA molecules contained in the transformed E.coli HB101 strains identified by accession numbers NCIB 11548 and 11549.
molecule is selected from the recombinant DNA molecules contained in the transformed E.coli HB101 strains identified by accession numbers NCIB 11548 and 11549.
13. The method according to claim 1 or 3, wherein the recombinant DNA
molecule is selected from the recombinant DNA molecules contained in the transformed E.coli HB101 strains identified by accession numbers NCIB 11558, 11559 and 11560.
molecule is selected from the recombinant DNA molecules contained in the transformed E.coli HB101 strains identified by accession numbers NCIB 11558, 11559 and 11560.
14. A method for transforming a host cell characterized by the step of introducing into said host cell the recombinant DNA molecule produced by the method according to claim 1.
15. A method for transforming a host cell characterized by the step of introducing into said host cell the recombinant DNA molecule produced by the method according to claim 2.
16. A method for transforming a host cell characterized by the step of introducing into said host cell the recombinant DNA molecule produced by the method according to claim 3.
17. A recombinant DNA molecule characterized by a DNA sequence coding for a polypeptide or fragment thereof displaying HBV antigenicity or HBV antigen specificity, said DNA sequence being operatively linked to an expression control sequence in the recombinant DNA molecule and being expressed to produce the polypeptide or fragment thereof displaying HBV antigenicity or HBV antigen specificity when an appropriate host cell transformed with said recombinant DNA molecule is cultured.
18. A recombinant DNA molecule characterized by a DNA sequence coding for a polypeptide or fragment thereof displaying the HBV antigenicity or HBV
antigen specificity of a hepatitis B virus core antigen, said DNA sequence being operatively linked to an expression control sequence in the recombinant DNA molecule and being expressed to produce the polypeptide or fragment thereof displaying the HBV antigenicity or HBV antigen specificity of a hepatitis B virus core antigen when an appropriate host cell transformed with said recombinant DNA molecule is cultured.
antigen specificity of a hepatitis B virus core antigen, said DNA sequence being operatively linked to an expression control sequence in the recombinant DNA molecule and being expressed to produce the polypeptide or fragment thereof displaying the HBV antigenicity or HBV antigen specificity of a hepatitis B virus core antigen when an appropriate host cell transformed with said recombinant DNA molecule is cultured.
19. A recombinant DNA molecule characterized by a DNA sequence coding for a polypeptide or fragment thereof displaying the HBV antigenicity or HBV
antigen specificity of a hepatitis B virus surface antigen, said DNA sequence being operatively linked to an expression control sequence in the recombinant DNA molecule and being expressed to produce the polypeptide or fragment thereof displaying the 1-1BV antigenicity or HBV antigen specificity of a hepatitis B virus surface antigen when an appropriate host cell transformed with said recombinant DNA molecule is cultured.
antigen specificity of a hepatitis B virus surface antigen, said DNA sequence being operatively linked to an expression control sequence in the recombinant DNA molecule and being expressed to produce the polypeptide or fragment thereof displaying the 1-1BV antigenicity or HBV antigen specificity of a hepatitis B virus surface antigen when an appropriate host cell transformed with said recombinant DNA molecule is cultured.
20. The recombinant DNA molecule according to claim 17 or 18, wherein the DNA sequence is selected from DNA sequences of the formulae:
( a ) ATGGACATTGACCCTTATAAAGAATTTGGAGCTACTGTGGAGTTACTCTCGT
TTTTGCCTTCTGACTTCTTTCCTTCCGTACGAGATCTTCTAGATACCGCCGC
AGCTCTGTATCGGGATGCCTTAGAGTCTCCTGAGCATTGTTCACCTCACCAT
ACTGCACTCAGGCAAGCAATTCTTTGCTGGGGAGACTTAATGACTCTAGCTA
CCTGGGTGGGTACTAATTTAGAAGATCCAGCATCTAGGGACCTAGTAGTCAG
TTATGTCAACACTAATGTGGGCCTAAAGTTCAGACAATTATTGTGGTTTCAC
ATTTCTTGTCTCACTTTTGGAAGAGAAACGGTTCTAGAGTATTTGGTGTCTT
TTGGAGTGTGGATTCGCACTCCTCCAGCTTATAGACCACCAAATGCCCCTAT
CCTATCAACGCTTCCGGAGACTACTGTTGTTAGACGACGAGGCAGGTCCCCT
AGAAGAAGAACTCCCTCGCCTCGCAGACGAAGATCTCAATCGCCGCGTCGCA
GAAGATCTCAATCTCGGGAATCTCAATGT and fragments thereof which encode polypeptides displaying the HBV antigenicity or HBV antigen specificity of a hepatitis B virus core antigen; and (b) DNA sequences which are degenerate as a result of the genetic code to any of the foregoing DNA sequences and which encode polypeptides displaying the HBV antigenicity or HBV antigen specificity of a hepatitis B virus core antigen.
( a ) ATGGACATTGACCCTTATAAAGAATTTGGAGCTACTGTGGAGTTACTCTCGT
TTTTGCCTTCTGACTTCTTTCCTTCCGTACGAGATCTTCTAGATACCGCCGC
AGCTCTGTATCGGGATGCCTTAGAGTCTCCTGAGCATTGTTCACCTCACCAT
ACTGCACTCAGGCAAGCAATTCTTTGCTGGGGAGACTTAATGACTCTAGCTA
CCTGGGTGGGTACTAATTTAGAAGATCCAGCATCTAGGGACCTAGTAGTCAG
TTATGTCAACACTAATGTGGGCCTAAAGTTCAGACAATTATTGTGGTTTCAC
ATTTCTTGTCTCACTTTTGGAAGAGAAACGGTTCTAGAGTATTTGGTGTCTT
TTGGAGTGTGGATTCGCACTCCTCCAGCTTATAGACCACCAAATGCCCCTAT
CCTATCAACGCTTCCGGAGACTACTGTTGTTAGACGACGAGGCAGGTCCCCT
AGAAGAAGAACTCCCTCGCCTCGCAGACGAAGATCTCAATCGCCGCGTCGCA
GAAGATCTCAATCTCGGGAATCTCAATGT and fragments thereof which encode polypeptides displaying the HBV antigenicity or HBV antigen specificity of a hepatitis B virus core antigen; and (b) DNA sequences which are degenerate as a result of the genetic code to any of the foregoing DNA sequences and which encode polypeptides displaying the HBV antigenicity or HBV antigen specificity of a hepatitis B virus core antigen.
21. The recombinant DNA molecule according to claim 17 or 19, wherein the DNA sequence is selected from DNA sequences of the formulae:
(a) ATGGAGAACATCACATCAGGATTCCTAGGACCCCTGCTCGTGTTACAGGCGG
GGTTTTTCTTGTTGACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTG
GTGGACTTCTCTCAATTTTCTAGGGGGAACTACCGTGTGTCTTGGCCAAAAT
TCGCAGTCCCCAATCTCCAATCACTCACCAACCTCCTGTCCTCCAACTTGTC
CTGGTTATCGCTGGATGTGTCTGCGGCGTTTTATCATCTTCCTCTTCATCCT
GCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTGGACTATCAAGGTATGTTG
CCCGTTTGTCCTCTAATTCCAGGATCATCAACCACCAGCACGGGATCCTGCA
GAACCTGCACGACTCCTGCTCAAGGAATCTCTATGTATCCCTCCTGTTGCTG
TACAAAACCTTCGGATGGAAACTGCACCTGTATTCCCATCCCATCATCCTGG
GCTTTCGGAAAATTCCTATGGGAGTGGGCCTCAGCCCGTTTCTCTTGGCTCA
GTTTACTAGTGCCATTTGTTCAGTGGTTCGTAGGGCTTTCCCCCATTGTTTG
GCTTTCAGTTATATGGATGATGTGGTATTGGGGGCCAAGTCTGTACAGCATC
TTGAGTCCCTTTTTACCGCTGTTACCAATTTTCTTTTGTCTTTGGGCATACA
TT and fragments thereof which encode polypeptides displaying the HBV
antigenicity or HBV specificity of a hepatitis B virus surface antigen; and (b) DNA sequences which are degenerate as a result of the genetic code to any of the foregoing DNA sequences and which encode polypeptides displaying the HBV antigenicity or HBV antigen specificity of a hepatitis B virus surface antigen.
(a) ATGGAGAACATCACATCAGGATTCCTAGGACCCCTGCTCGTGTTACAGGCGG
GGTTTTTCTTGTTGACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTG
GTGGACTTCTCTCAATTTTCTAGGGGGAACTACCGTGTGTCTTGGCCAAAAT
TCGCAGTCCCCAATCTCCAATCACTCACCAACCTCCTGTCCTCCAACTTGTC
CTGGTTATCGCTGGATGTGTCTGCGGCGTTTTATCATCTTCCTCTTCATCCT
GCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTGGACTATCAAGGTATGTTG
CCCGTTTGTCCTCTAATTCCAGGATCATCAACCACCAGCACGGGATCCTGCA
GAACCTGCACGACTCCTGCTCAAGGAATCTCTATGTATCCCTCCTGTTGCTG
TACAAAACCTTCGGATGGAAACTGCACCTGTATTCCCATCCCATCATCCTGG
GCTTTCGGAAAATTCCTATGGGAGTGGGCCTCAGCCCGTTTCTCTTGGCTCA
GTTTACTAGTGCCATTTGTTCAGTGGTTCGTAGGGCTTTCCCCCATTGTTTG
GCTTTCAGTTATATGGATGATGTGGTATTGGGGGCCAAGTCTGTACAGCATC
TTGAGTCCCTTTTTACCGCTGTTACCAATTTTCTTTTGTCTTTGGGCATACA
TT and fragments thereof which encode polypeptides displaying the HBV
antigenicity or HBV specificity of a hepatitis B virus surface antigen; and (b) DNA sequences which are degenerate as a result of the genetic code to any of the foregoing DNA sequences and which encode polypeptides displaying the HBV antigenicity or HBV antigen specificity of a hepatitis B virus surface antigen.
22. The recombinant DNA molecule according to claim 17 or 18, wherein the expression control sequence is selected from the lac system, the trp system, the major operator and promoter regions of phage X and the control region of phage fd coat protein.
23. The recombinant DNA molecule according to claim 17 or 19, wherein the expression control sequence is selected from the lac system, the trp system, the major operator and promoter regions of phage X and the control region of phage fd coat protein.
24. The recombinant DNA molecule according to claim 17 or 18, said recombinant DNA molecule being selected from the recombinant DNA
molecules contained in the transformed E.coli HB101 strains identified by accession numbers NCIB 11548 and 11549.
molecules contained in the transformed E.coli HB101 strains identified by accession numbers NCIB 11548 and 11549.
25. The recombinant DNA molecule according to claim 17 or 19, said recombinant DNA molecule being selected from the recombinant DNA
molecules contained in the transformed E.coli 1{B101 strains identified by accession numbers NCIB 11558, 11559 and 11560.
molecules contained in the transformed E.coli 1{B101 strains identified by accession numbers NCIB 11558, 11559 and 11560.
26. A method for producing a polypeptide displaying HBV antigenicity or HBV
antigen specificity characterized by the step of culturing a host cell transformed with the recombinant DNA molecule according to claim 17.
antigen specificity characterized by the step of culturing a host cell transformed with the recombinant DNA molecule according to claim 17.
27. A method for producing a polypeptide displaying the HBV antigenicity or HBV antigen specificity of a hepatitis B virus core antigen characterized by the step of culturing a host cell transformed with the recombinant DNA
molecule according to claim 18.
molecule according to claim 18.
28. A method for producing a polypeptide displaying the HBV antigenicity or HBV antigen specificity of a hepatitis B virus surface antigen characterized by the step of culturing a host cell transformed with the recombinant DNA
molecule according to claim 19.
molecule according to claim 19.
29. The method according to claim 26 or 27, wherein the polypeptide displaying HBV antigenicity or HBV antigen specificity is selected from polypeptides of the formulae:
MetAspIleAspProTyrLysGluPheGlyAlaThrValGluLeuLeuSer PheLeuProSerAspPhePheProSerValArgAspLeuLeuAspThrAla AlaAlaLeuTyrArgAspAlaLeuGluserProGluHisCysSerProHis HisThrAlaLeuArgGlnAlaIleLeuCysTrpGlyAspLeuMetThrLeu AlaThrTrpValGlyThrAsnLeuGluAspProAlaSerArgAspLeuVal ValSerTyrValAsnThrAsnValGlyLeuLysPheArgGlnLeuLeuTrp PheHisIleSerCysLeuThrPheGlyArgGluThrValLeuGluTyrLeu ValSerPheGlyValTrpIleArgThrProProAlaTyrArgProProAsn AlaProIleLeuSerThrLeuProGluThrThrValValArgArgArgGly ArgserProArgArgArgThrProserProArgArgArgArgserGlnser ProArgArgArgArgserGlnserArgGluserGlnCys and fragments thereof displaying the HBV antigenicity or HBV antigen specificity of a hepatitis B virus core antigen.
MetAspIleAspProTyrLysGluPheGlyAlaThrValGluLeuLeuSer PheLeuProSerAspPhePheProSerValArgAspLeuLeuAspThrAla AlaAlaLeuTyrArgAspAlaLeuGluserProGluHisCysSerProHis HisThrAlaLeuArgGlnAlaIleLeuCysTrpGlyAspLeuMetThrLeu AlaThrTrpValGlyThrAsnLeuGluAspProAlaSerArgAspLeuVal ValSerTyrValAsnThrAsnValGlyLeuLysPheArgGlnLeuLeuTrp PheHisIleSerCysLeuThrPheGlyArgGluThrValLeuGluTyrLeu ValSerPheGlyValTrpIleArgThrProProAlaTyrArgProProAsn AlaProIleLeuSerThrLeuProGluThrThrValValArgArgArgGly ArgserProArgArgArgThrProserProArgArgArgArgserGlnser ProArgArgArgArgserGlnserArgGluserGlnCys and fragments thereof displaying the HBV antigenicity or HBV antigen specificity of a hepatitis B virus core antigen.
30. The method according to claim 26 or 28, wherein the polypeptide displaying HBV antigenicity or HBV antigen specificity is selected from polypeptides of the formulae:
MetGluAsnIleThrSerGlyPheLeuGlyProLeuLeuValLeuGlnAla GlyPhePheLeuLeuThrArgIleLeuThrIleProGlnSerLeuAspSer TrpTrpThrSerLeuAsnPheLeuGlyGlyThrThrValCysLeuGlyGln AsnserGlnserProIleserAsnHisSerProThrserCysProProThr CysProGlyTyrArgTrpMetCysLeuArgArgPheIleIlePheLeuPhe IleLeuLeuLeuCysLeuIlePheLeuLeuValLeuLeuAspTyrGlnGly MetLeuProValCysProLeuIleProGlySerSerThrThrSerThrGly SerCysArgThrCysThrThrProAlaGlnGlyIleserMetTyrProser CysCysCysThrLysProSerAspGlyAsnCysThrCysIleProIlePro SerSerTrpAlaPheGlyLysPheLeuTrpGluTrpAlaSerAlaArgPhe SerTrpLeuSerLeuLeuValProPheValGlnTrpPheValGlyLeuSer ProIleValTrpLeuSerValIleTrpMetMetTrpTyrTrpGlyProSer LeuTyrSerIleLeuSerProPheLeuProLeuLeuProIlePhePheCys LeuTrpAlaTyrIle and fragments thereof displaying the HBV antigenicity or HBV antigen specificity of a hepatitis B virus surface antigen.
MetGluAsnIleThrSerGlyPheLeuGlyProLeuLeuValLeuGlnAla GlyPhePheLeuLeuThrArgIleLeuThrIleProGlnSerLeuAspSer TrpTrpThrSerLeuAsnPheLeuGlyGlyThrThrValCysLeuGlyGln AsnserGlnserProIleserAsnHisSerProThrserCysProProThr CysProGlyTyrArgTrpMetCysLeuArgArgPheIleIlePheLeuPhe IleLeuLeuLeuCysLeuIlePheLeuLeuValLeuLeuAspTyrGlnGly MetLeuProValCysProLeuIleProGlySerSerThrThrSerThrGly SerCysArgThrCysThrThrProAlaGlnGlyIleserMetTyrProser CysCysCysThrLysProSerAspGlyAsnCysThrCysIleProIlePro SerSerTrpAlaPheGlyLysPheLeuTrpGluTrpAlaSerAlaArgPhe SerTrpLeuSerLeuLeuValProPheValGlnTrpPheValGlyLeuSer ProIleValTrpLeuSerValIleTrpMetMetTrpTyrTrpGlyProSer LeuTyrSerIleLeuSerProPheLeuProLeuLeuProIlePhePheCys LeuTrpAlaTyrIle and fragments thereof displaying the HBV antigenicity or HBV antigen specificity of a hepatitis B virus surface antigen.
31. A host cell transformed with at least one recombinant DNA molecule according to claim 17.
32. A host cell transformed with at least one recombinant DNA molecule according to claim 18.
33. A host cell transformed with at least one recombinant DNA molecule according to claim 19.
34. The host cell according to claim 31 or 32, said host cell being selected from strains of E.coli, Pseudomonas, Bacillus subtilis, other Bacilli, yeast, other fungi or animal or plant cells in culture.
35. The host cell according to claim 31 or 33, said host cell being selected from strains of E.coli, Pseudomonas, Bacillus subtilis, other Bacilli, yeast, other fungi or animal or plant cells in culture.
36. The host cell according to claim 31 or 32, said host cell being selected from the transformed E.coli HB101 strains identified by accession numbers NCIB
11548 and 11549.
11548 and 11549.
37. The host cell according to claim 31 or 33, said host cell being selected from the transformed E.coli HB101 strains identified by accession numbers NCIB
11558, 11559 and 11560.
11558, 11559 and 11560.
38. A polypeptide essentially free of human or primate serum proteins which displays HBV antigenicity or HBV antigen specificity, said polypeptide being produced by a host cell transformed with the recombinant DNA molecule according to claim 17, said polypeptide excluding polypeptides having the same post-translational modifications and secondary, tertiary and quaternary structures as polypeptides produced as a result of HBV infection.
39. A polypeptide essentially free of human or primate serum proteins which displays the HBV antigenicity or HBV antigen specificity of a hepatitis B
virus core antigen, said polypeptide being produced by a host cell transformed with the recombinant DNA molecule according to claim 18, said polypeptide excluding polypeptides having the same post-translational modifications and secondary, tertiary and quaternary structures as polypeptides produced as a result of HBV infection.
virus core antigen, said polypeptide being produced by a host cell transformed with the recombinant DNA molecule according to claim 18, said polypeptide excluding polypeptides having the same post-translational modifications and secondary, tertiary and quaternary structures as polypeptides produced as a result of HBV infection.
40. A polypeptide essentially free of human or primate serum proteins which displays the HBV antigenicity or HBV antigen specificity of a hepatitis B
virus surface antigen, said polypeptide being produced by a host cell transformed with the recombinant DNA molecule according to claim 19, said polypeptide excluding polypeptides having the same post-translational modifications and secondary, tertiary and quaternary structures as polypeptides produced as a result of HBV infection.
virus surface antigen, said polypeptide being produced by a host cell transformed with the recombinant DNA molecule according to claim 19, said polypeptide excluding polypeptides having the same post-translational modifications and secondary, tertiary and quaternary structures as polypeptides produced as a result of HBV infection.
41. The polypeptide according to claim 38 or 39, said polypeptide being selected from polypeptides of the formulae:
MetAspIleAspProTyrLysGluPheGlyAlaThrValGluLeuLeuSer PheLeuProSerAspPhePheProSerValArgAspLeuLeuAspThrAla AlaAlaLeuTyrArgAspAlaLeuGluSerProGluHisCysSerProHis HisThrAlaLeuArgGlnAlaIleLeuCysTrpGlyAspLeuMetThrLeu AlaThrTrpValGlyThrAsnLeuGluAspProAlaSerArgAspLeuVal ValSerTyrValAsnThrAsnValGlyLeuLysPheArgGlnLeuLeuTrp PheHisIleSerCysLeuThrPheGlyArgGluThrValLeuGluTyrLeu ValSerPheGlyValTrpIleArgThrProProAlaTyrArgProProAsn AlaProIleLeuSerThrLeuProGluThrThrValValArgArgArgGly ArgSerProArgArgArgThrProSerProArgArgArgArgSerG1nSer ProArgArgArgArgserGlnserArgGluserGlnCys and fragments thereof displaying the HBV antigenicity or HBV antigen specificity of a hepatitis B virus core antigen.
MetAspIleAspProTyrLysGluPheGlyAlaThrValGluLeuLeuSer PheLeuProSerAspPhePheProSerValArgAspLeuLeuAspThrAla AlaAlaLeuTyrArgAspAlaLeuGluSerProGluHisCysSerProHis HisThrAlaLeuArgGlnAlaIleLeuCysTrpGlyAspLeuMetThrLeu AlaThrTrpValGlyThrAsnLeuGluAspProAlaSerArgAspLeuVal ValSerTyrValAsnThrAsnValGlyLeuLysPheArgGlnLeuLeuTrp PheHisIleSerCysLeuThrPheGlyArgGluThrValLeuGluTyrLeu ValSerPheGlyValTrpIleArgThrProProAlaTyrArgProProAsn AlaProIleLeuSerThrLeuProGluThrThrValValArgArgArgGly ArgSerProArgArgArgThrProSerProArgArgArgArgSerG1nSer ProArgArgArgArgserGlnserArgGluserGlnCys and fragments thereof displaying the HBV antigenicity or HBV antigen specificity of a hepatitis B virus core antigen.
42. The polypeptide according to claim 38=or 40, said polypeptide being selected from polypeptides of the formulae:
MetGluAsnIleThrSerGlyPheLeuGlyProLeuLeuValLeuGlnAla GlyPhePheteuLeuThrArgIleteuThrIleProGlnSerteuAspSer TrpTrpThrSerLeuAsnPheLeuGlyGlyThrThrValCysteuGlyGln AsnSerG1nSerProIleSerAsnHisSerProThrSerCysProProThr CysProGlyTyrArgTrpMetCysteuArgArgPheIleIlePheLeuPhe IleLeuLeuLeuCysLeuIlePheLeuLeuValLeuLeuAspTyrGlnGly MetteuProValCysProLeuIleProGlySerSerThrThrSerThrGly SerCysArgThrCysThrThrProAlaGlnGlyIleSerMetTyrProSer CysCysCysThrLysProSerAspGlyAsnCysThrCysIleProIlePro SerSerTrpAlaPheGlytysPheLeuTrpGluTrpAlaSerAlaArgPhe SerTrpteuSerteuteuValProPheValGlnTrpPheValGlyteuSer ProIleValTrpLeuSerValIleTrpMetMetTrpTyrTrpGlyProSer LeuTyrSerIleLeuSerProPheLeuProLeuLeuProIlePhePheCys LeuTrpAlaTyrI le and fragments thereof displaying the HBV antigenicity or HBV antigen specificity of a hepatitis B virus surface antigen.
MetGluAsnIleThrSerGlyPheLeuGlyProLeuLeuValLeuGlnAla GlyPhePheteuLeuThrArgIleteuThrIleProGlnSerteuAspSer TrpTrpThrSerLeuAsnPheLeuGlyGlyThrThrValCysteuGlyGln AsnSerG1nSerProIleSerAsnHisSerProThrSerCysProProThr CysProGlyTyrArgTrpMetCysteuArgArgPheIleIlePheLeuPhe IleLeuLeuLeuCysLeuIlePheLeuLeuValLeuLeuAspTyrGlnGly MetteuProValCysProLeuIleProGlySerSerThrThrSerThrGly SerCysArgThrCysThrThrProAlaGlnGlyIleSerMetTyrProSer CysCysCysThrLysProSerAspGlyAsnCysThrCysIleProIlePro SerSerTrpAlaPheGlytysPheLeuTrpGluTrpAlaSerAlaArgPhe SerTrpteuSerteuteuValProPheValGlnTrpPheValGlyteuSer ProIleValTrpLeuSerValIleTrpMetMetTrpTyrTrpGlyProSer LeuTyrSerIleLeuSerProPheLeuProLeuLeuProIlePhePheCys LeuTrpAlaTyrI le and fragments thereof displaying the HBV antigenicity or HBV antigen specificity of a hepatitis B virus surface antigen.
43. A polypeptide essentially free of human or primate serum proteins which displays the HBV antigenicity or HBV antigen specificity of a hepatitis B
virus core antigen, said polypeptide being selected from polypeptides of the formulae:
MetAspIleAspProTyrLysGluPheGlyAlaThrValGluteuLeuSer PheLeuProSerAspPhePheProSerValArgAspLeuLeuAspThrAla AlaAlaLeuTyrArgAspAlateuGluSerProGluHisCysSerProHis HisThrAlateuArgGlnAlaIleLeuCysTrpGlyAspLeuMetThrLeu AlaThrTrpValGlyThrAsnLeuGluAspProAlaSerArgAspLeuVal ValSerTyrValAsnThrAsnValGlyteuLysPheArgGlnLeuLeuTrp PheHisIleSerCysteuThrPheGlyArgGluThrValLeuGluTyrLeu ValSerPheGlyValTrpIleArgThrProProAlaTyrArgProProAsn AlaPro I leLeuSerThrLeuProGluThrThrValValArgArgArgGly ArgSerProArgArgArgThrProSerProArgArgArgArgSerGlnSer ProArgArgArgArgSerGlnSerArgGluSerGlnCys andfragments thereof displaying the HBV antigenicity or HBV antigen specificity of a hepatitis B virus core antigen, said polypeptide excluding polypeptides having the same post-translational modifications and secondary, tertiary and quaternary structures as polypeptides produced as a result of HBV infection.
virus core antigen, said polypeptide being selected from polypeptides of the formulae:
MetAspIleAspProTyrLysGluPheGlyAlaThrValGluteuLeuSer PheLeuProSerAspPhePheProSerValArgAspLeuLeuAspThrAla AlaAlaLeuTyrArgAspAlateuGluSerProGluHisCysSerProHis HisThrAlateuArgGlnAlaIleLeuCysTrpGlyAspLeuMetThrLeu AlaThrTrpValGlyThrAsnLeuGluAspProAlaSerArgAspLeuVal ValSerTyrValAsnThrAsnValGlyteuLysPheArgGlnLeuLeuTrp PheHisIleSerCysteuThrPheGlyArgGluThrValLeuGluTyrLeu ValSerPheGlyValTrpIleArgThrProProAlaTyrArgProProAsn AlaPro I leLeuSerThrLeuProGluThrThrValValArgArgArgGly ArgSerProArgArgArgThrProSerProArgArgArgArgSerGlnSer ProArgArgArgArgSerGlnSerArgGluSerGlnCys andfragments thereof displaying the HBV antigenicity or HBV antigen specificity of a hepatitis B virus core antigen, said polypeptide excluding polypeptides having the same post-translational modifications and secondary, tertiary and quaternary structures as polypeptides produced as a result of HBV infection.
44. A polypeptide essentially free of human or primate serum proteins which displays the HBV antigenicity or HBV antigen specificity of a hepatitis B
virus surface antigen, said polypeptide being selected from polypeptides of the formulae:
MetGluAsnIleThrSerGlyPheLeuGlyProLeuLeuValLeuGlnAla GlyPhePheLeuLeuThrArgIleLeuThrIleProGlnSerLeuAspSer TrpTrpThrSerLeuAsnPheLeuGlyGlyThrThrValCysLeuGlyGln AsnSerG1nSerProIleSerAsnHisSerProThrSerCysProProThr CysProGlyTyrArgTrpMetCysLeuArgArgPheIleIlePheLeuPhe IleLeuLeuLeuCysLeuIlePheLeuLeuValLeuLeuAspTyrGlnGly MetLeuProValCysProLeuIleProGlySerSerThrThrSerThrGly SerCysArgThrCysThrThrProAlaGlnGlyIleSerMetTyrProSer CysCysCysThrLysProSerAspGlyAsnCysThrCysIleProIlePro SerSerTrpAlaPheGlyLysPheLeuTrpGluTrpAlaSerAlaArgPhe SerTrpLeuSerLeuLeuValProPheValGlnTrpPheValGlyLeuSer ProIleValTrpLeuSerValIleTrpMetMetTrpTyrTrpGlyProSer LeuTyrSerIleLeuSesProPheLeuProLeuLeuProIlePhePheCys LeuTrpAlaTyrI le and fragments thereof displaying the HBV antigenicity or HBV antigen specificity of a hepatitis B virus surface antigen, said polypeptide excluding polypeptides having the same post-translational modifications and secondary, tertiary and quaternary structures as polypeptides produced as a result of HBV infection.
virus surface antigen, said polypeptide being selected from polypeptides of the formulae:
MetGluAsnIleThrSerGlyPheLeuGlyProLeuLeuValLeuGlnAla GlyPhePheLeuLeuThrArgIleLeuThrIleProGlnSerLeuAspSer TrpTrpThrSerLeuAsnPheLeuGlyGlyThrThrValCysLeuGlyGln AsnSerG1nSerProIleSerAsnHisSerProThrSerCysProProThr CysProGlyTyrArgTrpMetCysLeuArgArgPheIleIlePheLeuPhe IleLeuLeuLeuCysLeuIlePheLeuLeuValLeuLeuAspTyrGlnGly MetLeuProValCysProLeuIleProGlySerSerThrThrSerThrGly SerCysArgThrCysThrThrProAlaGlnGlyIleSerMetTyrProSer CysCysCysThrLysProSerAspGlyAsnCysThrCysIleProIlePro SerSerTrpAlaPheGlyLysPheLeuTrpGluTrpAlaSerAlaArgPhe SerTrpLeuSerLeuLeuValProPheValGlnTrpPheValGlyLeuSer ProIleValTrpLeuSerValIleTrpMetMetTrpTyrTrpGlyProSer LeuTyrSerIleLeuSesProPheLeuProLeuLeuProIlePhePheCys LeuTrpAlaTyrI le and fragments thereof displaying the HBV antigenicity or HBV antigen specificity of a hepatitis B virus surface antigen, said polypeptide excluding polypeptides having the same post-translational modifications and secondary, tertiary and quaternary structures as polypeptides produced as a result of HBV infection.
45. A DNA molecule coding for a polypeptide displaying HBV antigenicity or HBV antigen specificity, said DNA molecule being produced by the step of culturing a host cell transformed with the recombinant DNA molecule according to claim 17.
46. A DNA molecule coding for a polypeptide displaying the HBV antigenicity or HBV antigen specificity of a hepatitis B virus core antigen, said DNA
molecule being produced by the step of culturing a host cell transformed with the recombinant DNA molecule according to claim 18.
molecule being produced by the step of culturing a host cell transformed with the recombinant DNA molecule according to claim 18.
47. A DNA molecule coding for a polypeptide displaying the HBV antigenicity or HBV antigen specificity of a hepatitis B virus surface antigen, said DNA
molecule being produced by the step of culturing a host cell transformed with the recombinant DNA molecule according to claim 19.
molecule being produced by the step of culturing a host cell transformed with the recombinant DNA molecule according to claim 19.
48. A pharmaceutically acceptable composition for stimulating the production of antibodies to hepatitis B virus infection, said composition being characterized by at least one polypeptide according to claim 38 and a pharmaceutically acceptable carrier.
49. A pharmaceutically acceptable composition for stimulating the production of antibodies to hepatitis B virus infection, said composition being characterized by at least one polypeptide according to claim 39 and a pharmaceutically acceptable carrier.
50. A pharmaceutically acceptable composition for stimulating the production of antibodies to hepatitis B virus infection, said composition being characterized by at least one polypeptide according to claim 40 and a pharmaceutically acceptable carrier.
51. Use of a polypeptide displaying HBV antigenicity or HBV antigen specificity to detect HBV infection in blood serum, said polypeptide being essentially free of human or primate serum proteins and being produced by a host cell transformed with the recombinant DNA molecule according to claim 17.
52. Use of a polypeptide displaying the HBV antigenicity or HBV antigen specificity of a hepatitis B virus core antigen to detect HBV infection in blood serum, said polypeptide being essentially free of human or primate serum proteins and being produced by a host cell transformed with the recombinant DNA molecule according to claim 18.
53. Use of a polypeptide displaying the HBV antigenicity or HBV antigen specificity of a hepatitis B virus surface antigen to detect HBV infection in blood serum, said polypeptide being essentially free of human or primate serum proteins and being produced by a host cell transformed with the recombinant DNA molecule according to claim 19.
54. Use of a DNA molecule to detect HBV infection in blood serum, said DNA
molecule coding for a polypeptide essentially free of human or primate serum proteins which displays HBV antigenicity or HBV antigen specificity and being produced by a host cell transformed with the recombinant DNA
molecule according to claim 17.
molecule coding for a polypeptide essentially free of human or primate serum proteins which displays HBV antigenicity or HBV antigen specificity and being produced by a host cell transformed with the recombinant DNA
molecule according to claim 17.
55. Use of a DNA molecule to detect HBV infection in blood serum, said DNA
molecule coding for a polypeptide essentially free of human or primate serum proteins which displays the HBV antigenicity or HBV antigen specificity of a hepatitis B virus core antigen and being produced by a host cell transformed with the recombinant DNA molecule according to claim 18.
molecule coding for a polypeptide essentially free of human or primate serum proteins which displays the HBV antigenicity or HBV antigen specificity of a hepatitis B virus core antigen and being produced by a host cell transformed with the recombinant DNA molecule according to claim 18.
56. Use of a DNA molecule to detect HBV infection in blood serum, said DNA
molecule coding for a polypeptide essentially free of human or primate serum proteins which displays the HBV antigenicity or HBV antigen specificity of a hepatitis B virus surface antigen and being produced by a host cell transformed with the recombinant DNA molecule according to claim 19.
molecule coding for a polypeptide essentially free of human or primate serum proteins which displays the HBV antigenicity or HBV antigen specificity of a hepatitis B virus surface antigen and being produced by a host cell transformed with the recombinant DNA molecule according to claim 19.
57. A hepatitis B virus diagnostic kit comprising a polypeptide essentially free of human or primate serum proteins which displays HBV antigenicity or HBV
antigen specificity and instruction for use, said polypeptide being produced by a host cell transformed with the recombinant DNA molecule according to claim 17, said polypeptide excluding polypeptides having the same post-translational modifications and secondary, tertiary and quaternary structures as polypeptides produced as a result of HBV infection.
antigen specificity and instruction for use, said polypeptide being produced by a host cell transformed with the recombinant DNA molecule according to claim 17, said polypeptide excluding polypeptides having the same post-translational modifications and secondary, tertiary and quaternary structures as polypeptides produced as a result of HBV infection.
58. A hepatitis B virus diagnostic kit comprising a polypeptide essentially free of human or primate serum proteins which displays the HBV antigenicity or HBV antigen specificity of a hepatitis B virus core antigen and instruction for use, said polypeptide being produced by a host cell transformed with the recombinant DNA molecule according to claim 18, said polypeptide excluding polypeptides having the same post-translational modifications and secondary, tertiary and quaternary structures as polypeptides produced as a result of HBV
infection.
infection.
59. A hepatitis B virus diagnostic kit comprising a polypeptide essentially free of human or primate serum proteins which displays the HBV antigenicity or HBV antigen specificity of a hepatitis B virus surface antigen and instruction for use, said polypeptide being produced by a host cell transformed with the recombinant DNA molecule according to claim 19, said polypeptide excluding polypeptides having the same post-translational modifications and secondary, tertiary and quaternary structures as polypeptides produced as a result of HBV
infection.
infection.
60. The hepatitis B virus diagnostic kit according to claim 57 or 58, wherein said polypeptide is selected from polypeptides of the formulae:
MetAspIleAspProTyrLysGluPheGlyAlaThrValGluLeuLeuSer PheLeuProSerAspPhePheProSerValArgAspLeuLeuAspThrAla AlaAlaLeuTyrArgAspAlaLeuGluSerProGluHisCysSerProHis HisThrAlaLeuArgGlnAlaIleLeuCysTrpGlyAspLeuMetThrLeu AlaThrTrpValGlyThrAsnLeuGluAspProAlaSerArgAspLeuVal ValSerTyrValAsnThrAsnValGlyLeuLysPheArgGlnLeuLeuTrp PheHisIleSerCysLeuThrPheGlyArgGluThrValLeuGluTyrLeu ValSerPheGlyValTrpIleArgThrProProAlaTyrArgProProAsn AlaProIleLeuSerThrLeuProGluThrThrValValArgArgArgGly ArgSerProArgArgArgThrProSerProArgArgArgArgSerGlnSer ProArgArgArgArgSerGlnSerArgGluSerGlnCys andfragments thereof displaying the HBV antigenicity or HBV antigen specificity of a hepatitis B virus core antigen.
MetAspIleAspProTyrLysGluPheGlyAlaThrValGluLeuLeuSer PheLeuProSerAspPhePheProSerValArgAspLeuLeuAspThrAla AlaAlaLeuTyrArgAspAlaLeuGluSerProGluHisCysSerProHis HisThrAlaLeuArgGlnAlaIleLeuCysTrpGlyAspLeuMetThrLeu AlaThrTrpValGlyThrAsnLeuGluAspProAlaSerArgAspLeuVal ValSerTyrValAsnThrAsnValGlyLeuLysPheArgGlnLeuLeuTrp PheHisIleSerCysLeuThrPheGlyArgGluThrValLeuGluTyrLeu ValSerPheGlyValTrpIleArgThrProProAlaTyrArgProProAsn AlaProIleLeuSerThrLeuProGluThrThrValValArgArgArgGly ArgSerProArgArgArgThrProSerProArgArgArgArgSerGlnSer ProArgArgArgArgSerGlnSerArgGluSerGlnCys andfragments thereof displaying the HBV antigenicity or HBV antigen specificity of a hepatitis B virus core antigen.
61. The hepatitis B virus diagnostic kit according to claim 57 or 59, wherein said polypeptide is selected from polypeptides of the formulae:
MetGluAsnIleThrSerGlyPheLeuGlyProLeuLeuValLeuGlnAla GlyPhePheLeuLeuThrArgIleLeuThrIleProGlnSerLeuAspSer TrpTrpThrSerLeuAsnPheLeuGlyGlyThrThrValCysLeuGlyGln AsnSerGlnSerProIleSerAsnHisSerProThrSerCysProProThr CysProGlyTyrArgTrpMetCysLeuArgArgPheIleIlePheLeuPhe IleLeuLeuLeuCysLeuIlePheLeuLeuValLeuLeuAspTyrGlnGly MetLeuProValCysProLeuIleProGlySerSerThrThrSerThrGly SerCysArgThrCysThrThrProAlaGlnGlyIleSerMetTyrProSer CysCysCysThrLysProSerAspGlyAsnCysThrCysIleProIlePro SerSerTrpAlaPheGlyLysPheLeuTrpGluTrpAlaSerAlaArgPhe SerTrpLeuSerLeuLeuValProPheValG1nTrpneValGlyLeuSer ProIleValTrpLeuSerValIleTrpMetMetTrpTyrTrpGlyProSer LeuTyrSerIleLeuSerProPheLeuProLeuLeuProIlePhePheCys LeuTrpAlaTyr I le and fragments thereof displaying the HBV antigenicity or HBV antigen specificity of a hepatitis B virus surface antigen.
MetGluAsnIleThrSerGlyPheLeuGlyProLeuLeuValLeuGlnAla GlyPhePheLeuLeuThrArgIleLeuThrIleProGlnSerLeuAspSer TrpTrpThrSerLeuAsnPheLeuGlyGlyThrThrValCysLeuGlyGln AsnSerGlnSerProIleSerAsnHisSerProThrSerCysProProThr CysProGlyTyrArgTrpMetCysLeuArgArgPheIleIlePheLeuPhe IleLeuLeuLeuCysLeuIlePheLeuLeuValLeuLeuAspTyrGlnGly MetLeuProValCysProLeuIleProGlySerSerThrThrSerThrGly SerCysArgThrCysThrThrProAlaGlnGlyIleSerMetTyrProSer CysCysCysThrLysProSerAspGlyAsnCysThrCysIleProIlePro SerSerTrpAlaPheGlyLysPheLeuTrpGluTrpAlaSerAlaArgPhe SerTrpLeuSerLeuLeuValProPheValG1nTrpneValGlyLeuSer ProIleValTrpLeuSerValIleTrpMetMetTrpTyrTrpGlyProSer LeuTyrSerIleLeuSerProPheLeuProLeuLeuProIlePhePheCys LeuTrpAlaTyr I le and fragments thereof displaying the HBV antigenicity or HBV antigen specificity of a hepatitis B virus surface antigen.
62. A hepatitis B virus diagnostic kit comprising a DNA molecule coding for a polypeptide essentially free of human or primate serum proteins which displays HBV antigenicity or HBV antigen specificity and instruction for use, said DNA molecule being produced by a host cell transformed with the recombinant DNA molecule according to claim 17.
63. A hepatitis B virus diagnostic kit comprising a DNA molecule coding for a polypeptide essentially free of human or primate serum proteins which displays the HBV antigenicity or HBV antigen specificity of a hepatitis B
virus core antigen and instruction for use, said DNA molecule being produced by a host cell transformed with the recombinant DNA molecule according to claim 18.
virus core antigen and instruction for use, said DNA molecule being produced by a host cell transformed with the recombinant DNA molecule according to claim 18.
64. A hepatitis B virus diagnostic kit comprising a DNA molecule coding for a polypeptide essentially free of human or primate serum proteins which displays the HBV antigenicity or HBV antigen specificity of a hepatitis B
virus surface antigen and instruction for use, said DNA molecule being produced by a host cell transformed with the recombinant DNA molecule according to claim 19.
virus surface antigen and instruction for use, said DNA molecule being produced by a host cell transformed with the recombinant DNA molecule according to claim 19.
65. A replicable expression vector suitable for transforming bacterial cells, comprising DNA encoding a hepatitis B antigen operatively linked to a bacterial expression control sequence.
66. Bacterial cells transformed with a replicable expression vector, comprising DNA encoding a hepatitis B antigen operatively linked to a bacterial expression control sequence, said bacterial cells capable of expressing said hepatitis B antigen when cultured under appropriate conditions.
67. A method of producing a recombinant non-glycosylated hepatitis B
antigen (HBAg) from bacterial cells comprising:
- constructing a recombinant DNA which contains a segment encoding a HBAg operatively linked to an expression control sequence;
- introducing said recombinant DNA into an expressible cloning vector;
- transforming bacterial cells with said expressible cloning vector;
- culturing said bacterial cells under conditions appropriate for the expression of the HBAg; and - recovering said HBAg from the cell culture.
antigen (HBAg) from bacterial cells comprising:
- constructing a recombinant DNA which contains a segment encoding a HBAg operatively linked to an expression control sequence;
- introducing said recombinant DNA into an expressible cloning vector;
- transforming bacterial cells with said expressible cloning vector;
- culturing said bacterial cells under conditions appropriate for the expression of the HBAg; and - recovering said HBAg from the cell culture.
68. A recombinant non-glycosylated hepatitis B antigen peptide.
69. Use of a recombinant non-glycosylated hepatitis B antigen peptide for the detection of hepatitis B virus infection in mammals.
70. Use of a recombinant non-glycosylated hepatitis B antigen peptide for the preparation of a vaccine against hepatitis B virus in mammals.
71. Use of a recombinant non-glycosylated hepatitis B antigen peptide for the stimulation of antibody production in a mammal.
72. A recombinant DNA encoding a hepatitis B surface antigen.
73. A replicable expression vector suitable for transforming bacterial cells, comprising DNA encoding a hepatitis B surface antigen operatively linked to a bacterial expression control sequence.
74. Bacterial cells transformed with a replicable expression vector, comprising DNA encoding a hepatitis B surface antigen operatively linked to a bacterial expwssion control sequence, said bacterial cells capable of expressing said hepatitis B surface antigen when cultured under appropriate conditions.
75. A method of producing a recombinant non-glycosylated hepatitis B
surface antigen (HBsAg) from bacterial cells comprising:
- constructing a recombinant DNA which contains a segment encoding an HBsAg operatively linked to an expression control sequence;
- introducing said recombinant DNA into an expressible cloning vector;
- transforming bacterial cells with said expressible cloning vector;
- culturing said bacterial cells under conditions appropriate for the expression of the HBsAg; and - recovering said HBsAg from the cell culture.
surface antigen (HBsAg) from bacterial cells comprising:
- constructing a recombinant DNA which contains a segment encoding an HBsAg operatively linked to an expression control sequence;
- introducing said recombinant DNA into an expressible cloning vector;
- transforming bacterial cells with said expressible cloning vector;
- culturing said bacterial cells under conditions appropriate for the expression of the HBsAg; and - recovering said HBsAg from the cell culture.
76. A recombinant, non-glycosylated hepatitis B surface antigen peptide.
77. Use of a recombinant non-glycosylated hepatitis B surface antigen peptide for the detection of hepatitis B virus infection in mammals.
78. Use of a recombinant non-glycosylated hepatitis B surface antigen peptide for the preparation of a vaccine against hepatitis B virus in mammals.
79. Use of a recombinant non-glycosylated hepatitis B surface antigen peptide for the stimulation of antibody production in a mammal.
80. A recombinant DNA encoding a hepatitis B core antigen.
81. A replicable expression vector suitable for transforming bacterial cells, comprising DNA encoding a hepatitis B core antigen operatively linked to a bacterial expression control sequence.
82. Bacterial cells transformed with a replicable expression vector, comprising DNA encoding a hepatitis B core antigen operatively linked to a bacterial expression control sequence, said bacterial cells capable of expressing said hepatitis B core antigen when cultured under appropriate conditions.
83. A method of producing a recombinant non-glycosylated hepatitis B core antigen (HBcAg) from bacterial cells comprising:
- constructing a recombinant DNA which contains a segment encoding a HBcAg operatively linked to an expression control sequence;
- introducing said recombinant DNA into an expressible cloning vector;
- transforming bacterial cells with said expressible cloning vector;
- culturing said bacterial cells under conditions appropriate for the expression of the HBcAg; and - recovering said HBcAg from the cell culture.
- constructing a recombinant DNA which contains a segment encoding a HBcAg operatively linked to an expression control sequence;
- introducing said recombinant DNA into an expressible cloning vector;
- transforming bacterial cells with said expressible cloning vector;
- culturing said bacterial cells under conditions appropriate for the expression of the HBcAg; and - recovering said HBcAg from the cell culture.
84. A recombinant non-glycosylated hepatitis B core antigen peptide.
85. Use of a recombinant non-glycosylated hepatitis B core antigen peptide for the detection of hepatitis B virus infection in mammals.
86. Use of a recombinant non-glycosylated hepatitis B core antigen peptide for the preparation of a vaccine against hepatitis B virus in mammals.
87. Use of a recombinant non-glycosylated hepatitis B core antigen peptide for the stimulation of antibody production in a mammal.
88. A replicable expression vector suitable for transforming eukaryotic cells, comprising DNA encoding a hepatitis B antigen operatively linked to a eukaryotic expression control sequence.
89. Eukaryotic cells transformed with a replicable expression vector, comprising DNA encoding a hepatitis B antigen operatively linked to a eukaryotic expression control sequence, said cells capable of expressing said hepatitis B
antigen when cultured under appropriate conditions.
antigen when cultured under appropriate conditions.
90. A method of producing a recombinant hepatitis B antigen (HBAg) from eukaryotic cells comprising:
- constructing a recombinant DNA which contains a segment encoding a HBAg operatively linked to an expression control sequence;
- introducing said recombinant DNA into an expressible cloning vector;
- transforming eukaryotic cells with said expressible cloning vector;
- culturing said cells under conditions appropriate for the expression of the HBAg; and - recovering said HBAg from the cell culture.
- constructing a recombinant DNA which contains a segment encoding a HBAg operatively linked to an expression control sequence;
- introducing said recombinant DNA into an expressible cloning vector;
- transforming eukaryotic cells with said expressible cloning vector;
- culturing said cells under conditions appropriate for the expression of the HBAg; and - recovering said HBAg from the cell culture.
91. A replicable expression vector suitable for transforming yeast cells, comprising DNA encoding a hepatitis B antigen operatively linked to a yeast-compatible expression control sequence.
92. Yeast cells transformed with a replicable expression vector, comprising DNA encoding a hepatitis B antigen operatively linked to a yeast-compatible expression control sequence, said yeast cells capable of expressing said hepatitis B antigen when cultured under appropriate conditions.
93. A method of producing a recombinant hepatitis B antigen (HBAg) from yeast .cells comprising:
- constructing a recombinant DNA which contains a segment encoding an HBAg operatively linked to a yeast-compatible expression control sequence;
- introducing said recombinant DNA into an expressible cloning vector;
- transforming yeast cells with said expressible cloning vector;
- culturing said cells under conditions appropriate for the expression of the HBAg; and - recovering said HBAg from the cell culture.
- constructing a recombinant DNA which contains a segment encoding an HBAg operatively linked to a yeast-compatible expression control sequence;
- introducing said recombinant DNA into an expressible cloning vector;
- transforming yeast cells with said expressible cloning vector;
- culturing said cells under conditions appropriate for the expression of the HBAg; and - recovering said HBAg from the cell culture.
94. A recombinant hepatitis B antigen peptide having a yeast pattern of post-translational modification.
95. Use of a recombinant hepatitis B antigen peptide having a yeast pattern of post-translational modification for the detection of hepatitis B virus infection in mammals.
96. Use of a recombinant hepatitis B antigen peptide having a yeast pattern of post-translational modification for the preparation of a vaccine against hepatitis B virus in mammals.
97. Use of a recombinant hepatitis B antigen peptide having a yeast pattern of pos't-translational modification for the preparation of antibodies against hepatitis B virus.
98. A replicable expression vector suitable for transforming yeast cells, comprising DNA encoding a hepatitis B surface antigen operatively linked to a yeast-compatible expression control sequence.
99. Yeast cells transformed with a replicable expression vector, comprising DNA encoding a hepatitis B surface antigen operatively linked to a yeast-compatible expression control sequence, said yeast cells capable of expressing said hepatitis B surface antigen when cultured under appropriate conditions.
100. A method of producing a recombinant hepatitis B surface antigen (HBsAg) from yeast cells comprising:
- constructing a recombinant DNA which contains a segment encoding an HBsAg operatively linked to a yeast-compatible expression control sequence;
- introducing said recombinant DNA into an expressible cloning vector;
- transforming yeast cells with said expressible cloning vector;
- culturing said cells under conditions appropriate for the expression of the HBsAg; and - recovering said HBsAg from the cell culture,
- constructing a recombinant DNA which contains a segment encoding an HBsAg operatively linked to a yeast-compatible expression control sequence;
- introducing said recombinant DNA into an expressible cloning vector;
- transforming yeast cells with said expressible cloning vector;
- culturing said cells under conditions appropriate for the expression of the HBsAg; and - recovering said HBsAg from the cell culture,
101. A recombinant hepatitis B surface antigen peptide having a yeast pattern of post-translational modification.
102. Use of a recombinant hepatitis B surface antigen peptide having a yeast pattern of post-translational modification for the detection of hepatitis B virus infection in mammals.
103. Use of a recombinant hepatitis B surface antigen peptide having a yeast pattern of post-translational modification for the preparation of a vaccine against hepatitis B virus in mammals.
104. Use of a recombinant hepatitis B surface antigen peptide having a yeast pattern of post-translational modification for the preparation of antibodies against hepatitis B virus.
105. A replicable expression vector suitable for transforming mammalian cells, comprising DNA encoding a hepatitis B antigen operatively linked to a mammalian-compatible expression control sequence.
106. Mammalian cells transformed with a replicable expression vector, comprising DNA encoding a hepatitis B antigen operatively linked to a mammalian-compatible expression control sequence, said cells capable of expressing said hepatitis B antigen when cultured under appropriate conditions.
107. A method of producing a recombinant hepatitis B antigen (HBAg) from mammalian cells comprising:
- constructing a recombinant DNA which contains a segment encoding an HBAg operatively linked to a mammalian-compatible expression control sequence;
- introducing said recombinant DNA into an expressible cloning vector;
- transforming mammalian cells with said expressible cloning vector;
- culturing said cells under conditions appropriate for the expression of the HBAg; and - recovering said HBAg from the cell culture.
- constructing a recombinant DNA which contains a segment encoding an HBAg operatively linked to a mammalian-compatible expression control sequence;
- introducing said recombinant DNA into an expressible cloning vector;
- transforming mammalian cells with said expressible cloning vector;
- culturing said cells under conditions appropriate for the expression of the HBAg; and - recovering said HBAg from the cell culture.
108. A composition comprising a pharmaceutically acceptable carrier and a recombinant hepatitis B antigen peptide having a mammalian pattern of post-translational modification, said composition totally lacking primate serum proteins or Dane particles.
109. The use of a composition for the preparation of antibodies against hepatitis B
virus, said composition comprising a pharmaceutically acceptable carrier and a recombinant hepatitis B antigen peptide having a mammalian pattern of post-translational modification, said composition totally lacking primate serum proteins or Dane particles.
virus, said composition comprising a pharmaceutically acceptable carrier and a recombinant hepatitis B antigen peptide having a mammalian pattern of post-translational modification, said composition totally lacking primate serum proteins or Dane particles.
110. A replicable expression vector suitable for transforming mammalian cells, comprising DNA encoding a hepatitis B surface antigen operatively linked to a mammalian-compatible expression control sequence.
111. Mammalian cells transformed with a replicable expression vector, comprising DNA encoding a hepatitis B surface antigen operatively linked to a mammalian-compatible expression control sequence, said cells capable of expressing said hepatitis B surface antigen when cultured under appropriate conditions.
112. A method of producing a recombinant hepatitis B surface antigen (HBsAg) from mammalian cells comprising:
- constructing a recombinant DNA which contains a segment encoding an HBsAg operatively linked to a mammalian-compatible expression control sequence;
- introducing said recombinant DNA into an expressible cloning vector;
- transforming mammalian cells with said expressible cloning vector;
- culturing said cells under conditions appropriate for the expression of the HBsAg; and - recovering said HBsAg from the cell culture.
- constructing a recombinant DNA which contains a segment encoding an HBsAg operatively linked to a mammalian-compatible expression control sequence;
- introducing said recombinant DNA into an expressible cloning vector;
- transforming mammalian cells with said expressible cloning vector;
- culturing said cells under conditions appropriate for the expression of the HBsAg; and - recovering said HBsAg from the cell culture.
113. A composition comprising a pharmaceutically acceptable carrier and a recombinant hepatitis B surface antigen peptide having a mammalian pattern of post-translational modification, said composition totally lacking primate serum proteins, Dane particles, or hepatitis B core antigen.
114. The use of a composition for the preparation of antibodies against hepatitis B
virus, said composition comprising a pharmaceutically acceptable carrier and a recombinant hepatitis B surface antigen peptide having a mammalian pattern of post-translational modification, said composition totally lacking primate serum proteins, Dane particles, or hepatitis B core antigen.
virus, said composition comprising a pharmaceutically acceptable carrier and a recombinant hepatitis B surface antigen peptide having a mammalian pattern of post-translational modification, said composition totally lacking primate serum proteins, Dane particles, or hepatitis B core antigen.
115. A method of producing recombinant hepatitis B antigen (HBAg) from cultured cells comprising:
- constructing a recombinant DNA which contains a segment encoding a HBAg operatively linked to an expression control sequence;
- introducing said recombinant DNA into an expressible cloning vector;
- transforming cells with said expressible cloning vector;
- culturing said cells under conditions appropriate for the expression of the HBAg; and - recovering said HBAg from the cell culture.
- constructing a recombinant DNA which contains a segment encoding a HBAg operatively linked to an expression control sequence;
- introducing said recombinant DNA into an expressible cloning vector;
- transforming cells with said expressible cloning vector;
- culturing said cells under conditions appropriate for the expression of the HBAg; and - recovering said HBAg from the cell culture.
116. A method of producing Hepatitis B surface antigen (HBsAg) from a bacterial culture comprising:
- constructing a recombinant DNA comprising a segment encoding HBsAg under the control of appropriate promoter and termination sequences;
- introducing said recombinant DNA into a replicable expression vector;
- transforming a culture of E. coli strain HB101 with said replicable expression vector;
- culturing said transformed culture of E. coli under conditions appropriate for expression of HBsAg; and - recovering said HBsAg from the culture.
- constructing a recombinant DNA comprising a segment encoding HBsAg under the control of appropriate promoter and termination sequences;
- introducing said recombinant DNA into a replicable expression vector;
- transforming a culture of E. coli strain HB101 with said replicable expression vector;
- culturing said transformed culture of E. coli under conditions appropriate for expression of HBsAg; and - recovering said HBsAg from the culture.
117. A culture of E. coli transformed with a replicable expression vector capable of transforming bacterial cells, said vector comprising a segment of DNA
encoding Hepatitis B surface antigen (HBsAg) under the control of appropriate promoter and termination sequences, said culture being derived from E. coli strain HB101 and capable of expressing HBsAg.
encoding Hepatitis B surface antigen (HBsAg) under the control of appropriate promoter and termination sequences, said culture being derived from E. coli strain HB101 and capable of expressing HBsAg.
118. A method of producing recombinant hepatitis B antigen (HBAg) from cultured cells comprising:
- culturing cells transformed with an expressible cloning vector comprising a recombinant DNA which contains a segment encoding a HBAg operatively linked to an expression control sequence under conditions appropriate for the expression of the HBAg; and - recovering said HBAg from the cell culture.
- culturing cells transformed with an expressible cloning vector comprising a recombinant DNA which contains a segment encoding a HBAg operatively linked to an expression control sequence under conditions appropriate for the expression of the HBAg; and - recovering said HBAg from the cell culture.
119. A method of producing a recombinant hepatitis B surface antigen (HBsAg) from cultured cells comprising:
- culturing cells transformed with an expressible cloning vector comprising a recombinant DNA which contains a segment encoding a HBsAg operatively linked to an expression control sequence under conditions appropriate for the expression of the HBsAg; and - recovering said HBsAg from the cell culture.
- culturing cells transformed with an expressible cloning vector comprising a recombinant DNA which contains a segment encoding a HBsAg operatively linked to an expression control sequence under conditions appropriate for the expression of the HBsAg; and - recovering said HBsAg from the cell culture.
120. A recombinant polypeptide consisting essentially of the full-length hepatitis B
surface antigen (HBsAg) of 226 amino acids and having a yeast pattern of post-translational modification.
surface antigen (HBsAg) of 226 amino acids and having a yeast pattern of post-translational modification.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA342497A CA1341644C (en) | 1978-12-22 | 1979-12-21 | Recombinant dna molecules and their method of production |
Applications Claiming Priority (7)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB7849907 | 1978-12-22 | ||
| GB49907/78 | 1978-12-22 | ||
| GB7850039 | 1978-12-27 | ||
| GB50039/78 | 1978-12-27 | ||
| GB7937910 | 1979-11-01 | ||
| GB79/37910 | 1979-11-01 | ||
| CA342497A CA1341644C (en) | 1978-12-22 | 1979-12-21 | Recombinant dna molecules and their method of production |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1341644C true CA1341644C (en) | 2023-03-07 |
Family
ID=85476377
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA342497A Active CA1341644C (en) | 1978-12-22 | 1979-12-21 | Recombinant dna molecules and their method of production |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1341644C (en) |
-
1979
- 1979-12-21 CA CA342497A patent/CA1341644C/en active Active
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