CA1222707A - Preparation of hepatitis b virus vaccine - Google Patents
Preparation of hepatitis b virus vaccineInfo
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- CA1222707A CA1222707A CA000436153A CA436153A CA1222707A CA 1222707 A CA1222707 A CA 1222707A CA 000436153 A CA000436153 A CA 000436153A CA 436153 A CA436153 A CA 436153A CA 1222707 A CA1222707 A CA 1222707A
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- C12N2730/00—Reverse transcribing DNA viruses
- C12N2730/00011—Details
- C12N2730/10011—Hepadnaviridae
- C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
- C12N2730/10122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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Abstract
TITLE
Preparation or hepatitis B Virus Vaccine ABSTRACT
A DNA fragment which can code for HBsAg, derived from Dane particle DNA, is fused to the regulatory region of the yeast arg3 gene and cloned in a yeast vector. The recombinant vector is used to transform competent yeast cells wherein the HBsAg gene is expressed.
Preparation or hepatitis B Virus Vaccine ABSTRACT
A DNA fragment which can code for HBsAg, derived from Dane particle DNA, is fused to the regulatory region of the yeast arg3 gene and cloned in a yeast vector. The recombinant vector is used to transform competent yeast cells wherein the HBsAg gene is expressed.
Description
o~ :
10 ~
TITLE
Preparation of hepatitis B Virus Vaccine FIELD OF THE_I~VENTIOM
The invention relates to cloning of a gene which codes for hepatitis B surface antigen in yeast by use of 20 recombinant DNA ~echniques and preparation of a hepatitis B
virus vaccine from antigen produced by the yeast.
BACKGROUND OF INVENTION
Infection with he~alitis B vi L US ( HBV) is a serious, widespread health problemO Infection can be manifested in 25 acute or chronic phases. The number of cases of acute hepatitis in the United States is estimated to be at least 100,000 per year with a fatality rate of l to 2 per cent~
In the United States, the prevalence of ch~ronic carriers of HBV among healthy adults varies between 0Ol and 1%
30 depending on age and social class. In Sollth America, the prevalence of chronic carriers is about l to 3%; in the U~SoS~Ro and southern Europe, about 3 to 6%; and in Asia and Africa, more than 10%o In developed countries, there exists a need for a 35 vaccine for people at high risk of exposure, such as
10 ~
TITLE
Preparation of hepatitis B Virus Vaccine FIELD OF THE_I~VENTIOM
The invention relates to cloning of a gene which codes for hepatitis B surface antigen in yeast by use of 20 recombinant DNA ~echniques and preparation of a hepatitis B
virus vaccine from antigen produced by the yeast.
BACKGROUND OF INVENTION
Infection with he~alitis B vi L US ( HBV) is a serious, widespread health problemO Infection can be manifested in 25 acute or chronic phases. The number of cases of acute hepatitis in the United States is estimated to be at least 100,000 per year with a fatality rate of l to 2 per cent~
In the United States, the prevalence of ch~ronic carriers of HBV among healthy adults varies between 0Ol and 1%
30 depending on age and social class. In Sollth America, the prevalence of chronic carriers is about l to 3%; in the U~SoS~Ro and southern Europe, about 3 to 6%; and in Asia and Africa, more than 10%o In developed countries, there exists a need for a 35 vaccine for people at high risk of exposure, such as
- 2 ~3L222~
patients and personnel in medical units where blood i8 handled, military personnel, spouses of chronic carriers, travelers to areas of high HBV endemicity, newborns of chronic carriers, homosexuals, prostitutes and drug 5 abusers~ In third world countries, there exists a need for an inexpensive vaccine for mass immunization. Mass immunization programs may ultimately affect not only the incidence of acute hepatitis and the pool of chronic carriers but may also reduce the morbidity and mortality 10 from chronic active hepatitis and hepatocellular carcinoma.
Dane particles, which are believed to be hepatitis B
virions and which are isolatable from infected patients, have a diameter of about 42nm. Each consists of an envelope comprising the hepatitis B surface antigen 15 (HBsAg), a capsid (HBcAg), an endogenous polymerase and a DNA yenome. ~le genome is circular and double-stranded with a single strand region consisting of about 200 bases.
The single strand region can be filled in, ln vitro, by the action of the endogenous polymerase. The longer strand 20 contains approximately 3,200 bases.
It has been difficult to prepare HBV vaccines because it has proven difficult to propagate the virus in tissue culture and because the only known host is man. ~ma~l amounts of authentic HBV antigens have been isolated from 20 infected humans. F-D-C Reports, pp. 3-4, July 19, 1982, carries a report of clinical studies of a recently developed Hepatitis B vaccine.
Valenzuela et al., ~ature, Volume 298~ 347-350 (1982), report synthesis of HBsAg in yeast using an expression 25 vector wherein the HBsAg coding sequence is an 835 bp Taql-Hpal fragment and the promotor is the yeast alcohol dehydrogenase I promotorO Several earlier brief reports noted research preceding this reference. These are Valenzuela et al., Arch. Biol._Med. EXPD (Chile), Volume 30 14(1), 21-22 (1981), which reports expression in yeast of a
patients and personnel in medical units where blood i8 handled, military personnel, spouses of chronic carriers, travelers to areas of high HBV endemicity, newborns of chronic carriers, homosexuals, prostitutes and drug 5 abusers~ In third world countries, there exists a need for an inexpensive vaccine for mass immunization. Mass immunization programs may ultimately affect not only the incidence of acute hepatitis and the pool of chronic carriers but may also reduce the morbidity and mortality 10 from chronic active hepatitis and hepatocellular carcinoma.
Dane particles, which are believed to be hepatitis B
virions and which are isolatable from infected patients, have a diameter of about 42nm. Each consists of an envelope comprising the hepatitis B surface antigen 15 (HBsAg), a capsid (HBcAg), an endogenous polymerase and a DNA yenome. ~le genome is circular and double-stranded with a single strand region consisting of about 200 bases.
The single strand region can be filled in, ln vitro, by the action of the endogenous polymerase. The longer strand 20 contains approximately 3,200 bases.
It has been difficult to prepare HBV vaccines because it has proven difficult to propagate the virus in tissue culture and because the only known host is man. ~ma~l amounts of authentic HBV antigens have been isolated from 20 infected humans. F-D-C Reports, pp. 3-4, July 19, 1982, carries a report of clinical studies of a recently developed Hepatitis B vaccine.
Valenzuela et al., ~ature, Volume 298~ 347-350 (1982), report synthesis of HBsAg in yeast using an expression 25 vector wherein the HBsAg coding sequence is an 835 bp Taql-Hpal fragment and the promotor is the yeast alcohol dehydrogenase I promotorO Several earlier brief reports noted research preceding this reference. These are Valenzuela et al., Arch. Biol._Med. EXPD (Chile), Volume 30 14(1), 21-22 (1981), which reports expression in yeast of a
- 3 ~ Z'~
DNA frayment containing a sequence which codes for a protein similar to HBsAg ligated to a yeast alcohol dehydrogenase promotor region; a report in Scrip ~o. 616, pO 14 (AugO 12, 1981), which states that a team oE U. S.
5 researchers including P. Valenzuela and W. ~. Rutter have announced production in yeast of "the protein-coating surrounding hepatitis B virus;" and Zuckerman, Nature, Volume 295, 98-99 (1982), which reports that W. J. Rutter has reported expression of glycosylated HBsAg in yeast 10 cells.
Antigenic components of HBV, such as ~BsAg, have been prepared in bacteria following insertion of a recombinant DNA molecule containing a gene which codes for the an~igen. Burrell et al., Nature, Volume 279, Number 5708, 15 43-47 (1979), report expression in E. coli strain HB101 of HBV DNA sequences cloned in plasmid pBR322.
Murray et al., European Patent Application 13,828, disclose preparation of a recombinant vector which can code for HBV antigens, including HBsAg, in E.coli strain HBlOlo 20 The vector is prepared from Dane particle DNA and plasmid pBR322. The authors state that useful hosts may include other bacterial hosts, yeasts and other fungi, animal or plant cells and other hosts, although the only demonstrated host is E. coli.
Charnay, et al., Nature, Volume 286, 893-895 (1980), report construction of a bacteriophage carrying a fusion of the B-galactosidase gene and the HBsAg structural geneO
The bacteriophage directs synthesis of a fusion protein comprising antigenic determinants of both HBsAg and 30 B-galactosidase~
Tiollais et al., United Kingdom Patent Application 2,034,323, disclose preparation of a coliphage containing HBV D~A. Fused phage-HBV D~A is transformed into E. coli strain C600.
DNA frayment containing a sequence which codes for a protein similar to HBsAg ligated to a yeast alcohol dehydrogenase promotor region; a report in Scrip ~o. 616, pO 14 (AugO 12, 1981), which states that a team oE U. S.
5 researchers including P. Valenzuela and W. ~. Rutter have announced production in yeast of "the protein-coating surrounding hepatitis B virus;" and Zuckerman, Nature, Volume 295, 98-99 (1982), which reports that W. J. Rutter has reported expression of glycosylated HBsAg in yeast 10 cells.
Antigenic components of HBV, such as ~BsAg, have been prepared in bacteria following insertion of a recombinant DNA molecule containing a gene which codes for the an~igen. Burrell et al., Nature, Volume 279, Number 5708, 15 43-47 (1979), report expression in E. coli strain HB101 of HBV DNA sequences cloned in plasmid pBR322.
Murray et al., European Patent Application 13,828, disclose preparation of a recombinant vector which can code for HBV antigens, including HBsAg, in E.coli strain HBlOlo 20 The vector is prepared from Dane particle DNA and plasmid pBR322. The authors state that useful hosts may include other bacterial hosts, yeasts and other fungi, animal or plant cells and other hosts, although the only demonstrated host is E. coli.
Charnay, et al., Nature, Volume 286, 893-895 (1980), report construction of a bacteriophage carrying a fusion of the B-galactosidase gene and the HBsAg structural geneO
The bacteriophage directs synthesis of a fusion protein comprising antigenic determinants of both HBsAg and 30 B-galactosidase~
Tiollais et al., United Kingdom Patent Application 2,034,323, disclose preparation of a coliphage containing HBV D~A. Fused phage-HBV D~A is transformed into E. coli strain C600.
4 ~2'70~
In United Kingdom Patent Application 2,070,621, first published as PCT application WO81/00577, a plasmid which comprises a part of the HBsAg gene and the promotor and the Z gene of the lactose operon and which can be cloned in E.
In United Kingdom Patent Application 2,070,621, first published as PCT application WO81/00577, a plasmid which comprises a part of the HBsAg gene and the promotor and the Z gene of the lactose operon and which can be cloned in E.
5 coli is disclosed.
Rutter et al., European Patent Application 20,251, disclose recombinant vectors including a recombinant vector comprising plasmid pBR322 and BamHI fragments of HBV DNA, which can be used to transform E. coli. Another vector, 10 comprising a BamHI fragment of HBV D~A and a portion of the tryptophan operon, was used to obtain expression in E. coli strain HB101.
Edman et al., Nature, Volume 291, Number 5815, 503-506 (1981), describe construction of plasmids which direct 15 synthesis of HBcAg and a ~-lactamase-HBsAg fusion protein, under the control of the tryptophan operon regulatory region, in E. coliO
Other references disclosing insertion of HBV DNA into bacteria include Charnay, et al., Pro~. Med VirolO, Volume 20 27~ 88-92 (1981), MacKay et al, Proc. Natl. AcadO Sci.
U.S., Volume 78, Number 7, 4510-4514 (1981); Fritsch et al!, C. R. Acad. Sci. Volume 287, Number 16, 1453 (1978), U. K. Patent Specification 2,034,323 (Derwent ~o. 468;4C).
and Pasek et al., Nature, Volume 282 No. 6, 575 (1979).
HBV D~A has also been cloned in mammalian cells.
These include, human, mouse, and human hepatoma cell lines. For example, Dubois et al., Proc. Natl. Acad. Sci.
U. S., Volume 77, Number 8, 4549-4553 (1980), report transformation of mouse cells with a plasmid containing the 30 HBV genome and expression of HBsAg; Hirschman et al., Proc.
Natl. Acad. Sci. U.S., Volume 77, ~umber 9, 5507-5511 (1980), report production of HBV-like particles by HeLa cells transformed with HBV DNA~
Procedures for preparing HBV vaccine using HBsAg from 35 human blood are reported by Funakoshi et al., Prog. Med.
7~
Virol., Volume 27, 163-167 (1981), and Maupas et al., Prog.
Med. Virol~, Volume 27, 185-201 (1981). The vaccine prepared by Funakoshi et al. contains 40 ~g of purified, formalin-treated HBsAg, phosphate sodium chloride, 20 mg of 5 mannitol; and 0.1% of aluminlum hydroxide as adjuvant. In the latter paper, Maupas et al. report that one dose of vaccine was 1 ml of purified, formalin-treated HBsAg containing 2-10 ~g/ml of protein (Lowry's method) and 0.1%
of aluminum hydroxideO The protocol used in the study 10 reported by Maupas et alO called for three injections at one month intervals with a booster after one year; the authors propose a protocol consisting of two injections of concentrated HBsAg at three month intervals.
Additional references to preparation of HBV vaccines 15 include Maupas et al., Adamowicz et alO, and Funakoshi et al. at pages 3, 37 and 57, respectively, of Hepatitis B
Vaccine INSERM Symposium No. 18, editO by Maupas and Guesry, 1981, Elsevier/North-Holland Biomedical Press.
Yeasts have been used as host organisms for certain 20 other DNA sequences. For example, Fraser et al., UO K3 Patent Application 2,068,969, disclose preparation of chicken ovalbumin in yeast; Scrip NoO 640, pO 11 (NovO 4, 1981) contains a report that a type of interferon is being prapared in yeast. In European Patent 11,562 (Derwent NoO
25 38762C) are reported hybrid yeast plasmids containing the ura3 yeast gene in the 2 ~ plasmid.
SU~ARY OF THE INVENTION
The invention relates to the preparation of a 30 recombinant DNA molecule comprising a nucleotide sequence which can code for HBsAg and a regulatory region derived from the yeast ~ 3 gene which can effect transcription of the H~sAg sequence in the yeast, Saccharomyces cerevisiae.
The molecule includes vectors in which the HBsAg sequence 35 and the regulatory region have been inserted, which vectors '~2~Z~
can be used to prepare a yeast vector or to maintain the HBsAg and regulatory re~ions. Microorganisms containing the recombinant D~A molecule, such as microorganisms transformed with plasmids of the invention, are included 5 within the invention. The invention also includes a plasmid having the ~3 regulatory region and a restriction site proximate thereto for insertion of a coding sequence, such as an HBsAg sequence, such that the protein synthesi~ed by expression of the coding sequence is devoid 10 of extraneous amino acid residues.
The invention also includes the preparation of a vaccine for stimulating protection against HBV infection in humans comprising a vaccinal amount of HBsAg prepared in accordance with this invention and a suitable carrierO
Further, the invention includes processes for preparing the recombinant DNA molecule and microorganisms containing the molecules as well as processes for preparing HBsAg and HBsAg containing vaccine.
BRIEF DES RIPTION OF T~E FIGURES
Figure 1 is a restriction endonuclease cleavage map of pRIT10601.
Figure 2 is a restriction endonuclease cleavage map of pRIT10616.
Figure 3 is a restriction endonuclease cleavage map of 25 pMC200.
Figure 4 is the nucleotide sequence of a portion of the 3300 bp HindIII yeast DN~ insert i~ pMC200 ~hich portion contains HincII, BglII and EcoRI sites.
Figure S is a flow sheet illustrating preparation of 30 pRIT10671 and pRITl06730 Figure 6 is a flow sheet illustrating preparation of pRIT10749~
Figure 7 is a flow sheet illustrating preparation ofpRIT10761 and pRIT10764.
. 7 _ ~Z2~7~7 DETAILED DESCRIPTION OF THE INVENTION
The recombinant DNA molecule is prepared by fusing a nucleotide sequence which includes a structural gene for HBsAg with the yeast ~3 regulatory region, which 5 regulatory region can direct transcription of the HBsAg sequence in yeast, thereby effecting expression thereof.
By "recombinant DNA molecule" is meant a DNA fragment containing the HBsAg coding sequence and the regulatory region as well as other DNA molecules containing the 10 fragment, such as plasmid or phage vectors.
By "regulatory region" is meant a sequence which contains a promotor region and other sequences necessary for transcription. The yeast arg3 regulatory region is especially advantageous because it can be a powerful 15 promotor for expression of an HBsAg coding sequenceO
By "HBsAg" is meant a protein which is structurally identical to authentic HBsAg or has substantially the same antigenic determinants as authentic HBsAg, that is, is capable of stimulating production of antibodies which 20 specifically recognize and react with authentic HBsAg o~ is specîfically recognized by anti-HBsAg antibodies.
The HBsAg coding sequence can be isolated from DNA
extracted from Dane particles in lnfected huma~ serum by filling in the single strand region with a DNA polymerase, 25 preerably the endogenous polymerase, followed by digestion with a restriction endonuclease. The choice of endonuclease will depend, in part, on the particular Dane particles. For example, as illustrated in the Examples below, the HBsAg coding sequence of HBV DNA of certain Dane 30 particles of the adw serotype can be isolated on a single BamHI fragment; the HBsAg coding sequence of HBV DNA of certain Dane particles of the ayw serotype can be isolated on a HhaI fragment. HBV DNA of Dane particles of the same 7~7 ~,'..
serotype may also exhibit different patterns of restriction sites, Restriction of D~A to prepare DNA fragments used in the invention, ligation of such fragments to prepare 5 recombinant D~A molecules used in the invention and insertion into microorganisms are carried out by Xnown techniques such as techniques disclosed in the previously and subsequently cited references. Conditions are selected to avoid denaturation of the DNA and enzymes~ For example, 10 the pH is buffered to remain at about 7.0 to 11.0 and the temperature is kept below about 60 CO Preferably restriction is carried out at a temperature of about 30 to 40C and ligation is carried out at about O to 10C.
Restriction enzymes and ligases used in carrying out this 15 invention are commercially available and should be used in accordance with instructions included therewith. T4 Dl~A
~igase is the preferred ligase.
Fusing of the HBsAg sequence to the regulatory region can be accomplished by use of intermediate vectors as 20 illustrated in the Examples, below. Alternatively, the HBsAg sequence can be inserted directly into a vector which contains the regulatory region~ A vector is DNA which can carry and maintain the D~A fragment of the invention, including, for example, phages and plasmids. Techniques 25 for cloning DNA fragments in phages are disclosed, for example, by Charnay et al. Nature, Volume 286, 893-895 (1980) and Tiollais, United Kingdom Patent Application 2,034,3230 Preferably, the HBsAg sequence is positioned relative to the regulatory region such that the HBsAg 30 synthesized by expression of the HBsAg sequence is devoid of extraneous amino acid residues.
A regulatory region which has been found to be especially useful is derived from the yeast arg3 gene which codes for ornithine carbamoyltransferase (OCT). Use of the 35 arg3 regulatory region is advantageous because its activity . g is subject to both specific and general control mechanismsO It has been cloned in Eo coli on plasmid pYeura3arg3 as reported by Crabeel et al., Proc. Natl.
~cad. Sci. U.S.A., Volume 78, 5026 (1981)o Preferred hosts 5 are _. cerevisiae strains in which the arginine biosynthetic pathway is derepressed, such as strain lc1697do Use of such strains results in increased expression from the ~3 promotor as compared to the other strain which was used in the Examples, strain DC50 The preferred vector ~or cloning the fused DNA
fragment into yeast is the plasmid YEpl3, which is capable of replication and maintenance in both E. coli and S0 cerevisiae and is, therefore, known as a shuttle vectorO
-Several other yeast vectors are known and availableO The 15 HBsAg and regulatory regions can be inserted into a yeast vector sequentially or, as illustrated in the Examples, below, simultaneously. Transformation with plasmid vectors will normally result in incorporation of the DNA molecule of the invention as a plasmid. However, other reactions, 20 such as recombination events, can result in incorporation of the DNA molecule into chromosomal DNA.
Vaccines for stimulating protection against HBV
infection in humans comprising HBsAg produced by yeast in accordance with the invention and a suitable carrier can be 25 prepared by known techniques. Use of an adjuvant~ such as aluminum hydroxide, is desirable. The HBsAg so produced can also be combined with other immunogens to prepare combination vaccines The HBV or combination vaccines can be administered, for example, by the subcutaneous, 30 intravenous or intramuscular route. The DNA fragment of the invention and the HBsAg produced thereby, can also be used as a probe for detection of HBV in biological samples by DNA hydridization techniques and various immunoassays.
EXAMPLES
__ In the following examples of the invention, which are illustrative and not limiting, all percentages are by weight and all temperatures are in degrees CelsiusO
Example 1 Preparation of Intermediate Plasmid, RIT10601, bv Combinin~ HBV DNA with pBR322 p ~ _ __ _ HBs~g positive serum of ~y~ serotype was defibrinated by addition of CaC12 to a final concentration of 0.28%
10 and centrifuged for 2 h at 27,000 rpm in a SW 27 rotor on 10-20% sucrose gradients made up in a buffer (pH 7O5) containing 10 mM tromethamine-HCl, 1 M NaCl and 1 mM ~DTAo A transparent pellet containing Dane particles was resuspended in the same buffer and centrifuged on buffered 15 20~ sucrose layered on a 65% sucrose cushion. An opalescent band at the cushion interface was recovered and centri~uged on a similar 20-65~ gradient at 200,000 x C for 4 h to pellet the Dane particles.
A. Single strand regions of the HBV genome within the 20 Dane .particles were repai.red using the endogenous DNA
polymerase by resuspending the Dane particles in a reaction mixture (pH 8.0) containing 50 mM tromethamine-HCl, 10 mM
MgC12, 500 mM NaCl, 0.5 mM dithiothreitol, 50 mM eacn of dATP, dCTP and dGTP and 8 ~uM of P-dTTP (350 Ci/mmole) 25 and incubating the resuspended particles for 5 h at 37O
The resuspension was diluted to pH 7O5 with a buffer containing 10 mM tromethamine-HCl, 10 mM EDTA, 100 mM NaCL
and 0.02~ sodium dodecyl sulfate (pH 7.5) and incubated with 0.5 mg/ml of pronase for 1 h at 37 followed by 30 phenol extraction and ethanol precipitationO
Digestion of the DNA with BamHI restriction endonuclease, yielded two radioactive fragments with sizes of about 1450 bp and 1600 bp as judged by agarose gel electrophoresis and autoradiography of the gel.
2~7 B. About 30 ng of Dane particle DNA was repaired with the endogenous DNA polymerase in the presence o non-labelled dTTP, and was extracted and recovered, as described above. The DNA was mixed with 100 ng of plasmid 5 pBR322 which had been previously digested with BamHI
restriction endonuclease and treated with alkaline phosphatase. Plasmid pBR322 is commonly used in recombinant DNA procedures and is on deposit, without restriction on availability, in the American Type Culture 10 Collection under accession number 37017. The mixture was extracted with phenol, precipitated with ethanol, centrifuged, dried, resuspended in 12 ul of a mixture (pH
7.5) containing 50 mM tromethamine-HCl, 1 mM ATP, 10 mM
MgC12, 10 mM dithiothreitol, 50/ug/ml gelatin and 2 15 units/ml T4 DNA ligase. The suspension was incubated for 4 h at 10 and then held on ice for 18 h.
The ligated DNA mixture was used to transform competent cells of E~ coli K12 strain- C600 prepared according to the procedure of Cohen et al., Proc; ~atl.
20 Acad. Sci. U. S., Volume 69, 2110 (1972~. Transformants were selected on a solid medium containing ampicillin (200 ,uglml). Isolated colonies were screened for loss of tetracycline resistance, indicative of insertion o~ a foreign D~A fragment in the BamHI site of pBR322. One such 25 transformant clone was found to contain a plasmid, pRIT10601, which on digestion with BamHI endonuclease gave a pBR322 fragment of 4360 bp and HBV DNA fragments of 1600 bp and 1450 bp~ A culture of E. coli K12 strain C600(pRIT10601) was deposited in accordance with 30 regulations of the E-~ropean Patent Convention (EPC) and the Budapest Treaty in the American Type Culture Collection, Rockville, Maryland U.S.A. on June 2, 1982 under accession number ATCC 39132. A restriction endonuclease cleavage map of plasmid pRIT10601 is shown in Figure 1.
'7~7 C. The sizes of fragments generated by digestion of the Dane particle DNA and pRIT10601 with various restriction enzymes were compared as follows. Dane particle DNA, that is, HBV DNA, was labelled with 32p by 5 the endogeneous polymerase reaction described above or by treating purlfied DNA with DNA polymerase I from E. coli The labelled HBV DNA was mixed with pRIT10601 and the mixture was treated with a restriction endonuclease and electrophoresed on an agarose gel. The gel was stained 10 with ethidium bromide and photographed under UV light to loca~e the DNA fragments and was then dried and autoradiographed to locate the radioactive HBV DNA
fragments. The following labelled HBV fragments were found to match exactly the size of pRIT10601 fragments: 1450 and 15 1600 bp BamHI fragments; a 1330 bp HpaI fragment; and a 1130 bp BamHI-XhoI fragment. Labelled Dane particle DNA
also hydridized specifically to both the 1450 and 1600 bp fragments released by BamHI digestion of pRIT10601 following transfer of these fragments from an agarose gel 20 onto a nitrocellulose filter by the technique of Southern, J. Mol. Biol., Volume 98, 503 (1975).
These results show that the cloned DNA insert on pRIT10601 represents the HBV genome and that the relative orientation of the two BamHI fragments on pRIT10601 is the 25 same as in the virion. pRIT10601 was used to prepare pRIT10671 in Example 10, below~
Example 2. Preparation of Intermediate Plasmid, RIT10616 bv Combinina ~BV DNA with ~ACYC184 P ~
Dane particles were isolated from HBsAg positive serum of adw serotype as described above. Restriction endonuclease analysis of P-labelled HBV DNA indicated that the DNA contained one EcoRI site~
HBV D~A, filled in by the endogenous polymerase 3~ reaction using non-labelled nucleotides, was digested with 2~7 EcoRI. The restricted DNA was mixed with plasmid pACYC184 which had been previously digested with EcoRI and treated with alkaline phosphatase. Plasmid pACYC184 is on deposit, without restrictions on availability, in the American Type 5 Culture Collection under accession number 37033. The mixture was liyated with T4 DNA ligase.
The ligated D~A mixture was used to transform competent cells of E. coli K12 strain C600. Transformants were selected on tetracycline (15 ug/ml~ agar medium and 10 screened for loss of chloramphenicol resistance which is indicative of insertion in the EcoRI site of pACYC184. A
transformed colony was found to contain a plasmid, pRIT10616, which consists of pACYC184 with a 3200 bp insert, comprising the HBV DNA, at the EcoRI site. A
15 restriction map of pRIT10616 is shown in Figure 2. Eo coli K12 strain C600 (p~IT10616) was deposited in accordance with EPC regulations and the Budapest Treaty in the American Type Culture Collection on June 2, 1982 under accession number ATCC 39131.
Example 3. Preparation of Intermediate Plasmid, pRIT10640,Containing a Nucleotide Sequence Codinq for HBsAg, by Combininq pRIT10616 with pBR313 pRIT10616 was purified by CsCl-ethidium bromide 25 density gradient centrifugation substantially as described by Kahn et al, Methods in Enzymology, Volume 68, 268 (1979).
The DNA was digested with BamHI endonuclease, mixed with BamHI-digested, alkaline phosphatase-treated DNA of plasmid pBR313, ligated and used to transform competent 30 cells of E. coli K12 strain C600 substantially as described in Example 1. Plasmid pBR313 is on deposit, without restrictions on availability, in the American Type Culture Collection under accession number 37018.
Trans~ormants were selected on ampicillin-containing 35 agar and screened for loss of tetracycline resistance, 14 ~:227~7 indicative of insertion at the BamHI site of pBR313. A
transformed colony was found to contain a plasmid, pRIT10640, which consists of pBR313 with a 1350 bp insert at the BamHI site. The insert is a nucleotide sequence 5 which can code for HBsAg. It codes for part of a putative HBs~g precursor protein and the complete surface antigen and includes 565 bp of 3' non-coding sequencesO
c Example 4. Preparation of Intermediate Plasmid pMC200, Containing the arg3 Regulatory Region, by Combining Yeast arg3 Gene with pBR322 A 3300 bp yeast D~A fragment specifying the arg3 gene was obtained by diyestion of pYeura3~3 with HindIIIO
Plasmid p~eura3arg3 has been described by Crabeel et al., 15 Proc. ~atl. Acad. Sci. U.S., Volume 78, 5026 (1981)~ The 3300 bp fragment was cloned into the HindIII site of pBR322 and transformed into _. coli K12 strain MM294, substantially as described above. Transformants were selected on ampicillin medium and screened for tetracycline 20 resistance. A transformed colony was found to contain a plasmid, pMC200, which consists of pBR322 with an insert at the HindIII site. This plasmid contains the arg3 regulatory region which can effect transcription of a HBs~g nucleotide sequence in yeast cells as described in 25 following examples~ E. coli K12 strain MM294(pMC200) was deposited in accordance with EPC regulations and the Budapest Treaty in the American Type Culture Collection on June 2, 1982 under accession number ATCC 39130.
The nucleotide sequence of part of the arg3 gene, 30 including part of the ~-terminal coding sequences for ornithine carbamoyltransferase (OCT) and part of the 5' non-translated leader region has been determined by Huyghen et al., ~rch. Intl. Physical. Biochem., Volume 89, B172 - (1981). Figure 4 illustrates a 210 bp fragment of the 35 known sequence The 210 bp fragment contains unique - 15 ~ 7~7 HincII, BglII and EcoRI sites on the 3300 bp HindIII yeast DNA insert in pMC200; the initiation codon for the OCT
protein coding sequence is believed to be the boxed ATG
codon. Introduction into the HincII, BglII or EcoP~I sites S of the yeast D~A of coding sequences for HBsAg alone or HBsAg precursor plus HBsAg derived from cloned HBV DNA is - expected to result in a gene fusion and production of a hybrid protein transcribed and translated from the ~3 regulatory region provided that the gene fusion is in the 10 correct orientation for continued translation beyond the site of fusion.
Example 5. Preparation of Intermediate Plasmids, ~RIT10671 and pRIT10672, Containing HBsAg and Regulatory Regions, by Combini~gLpRIT10641 with pMC200 pMC200, 5~g, was digested with 6.4 units of BglII for 2.5 h at 37, diluted with an equal volume of a buffer (pH 10.5) containing 0.1 M glycine, 0.01 M MgC12 6H2O
and 0.1 m~ ZnC12, and incubated with 0.5 units of calf 20 intestine alkaline phosphatase for 30 minutes at 37 to remove 5' terminal phosphate residues. The mixture was ex racted twice with buffer-saturated phenol and three times with ether. The DNA was precipitated with ethanol and dissolved in 0.01 M tromethamine buffer (pH 7.5).
pRIT106~0, 25 ~ug, was digested with BamHI to excise the 1350 bp BamHI fragment of HBV DNA. The 1350 bp fragment was purified by preparative agarose gel electrophoresis and electroelution. The eluted D~A was recovered and concentrated by ethanol precipitation and 30 dissolved in 20 ~1 of 0.01 M tromethamine buffer (pH 7.0).
A 0.3 Jug aliquot of the BamHI fragment, which contains the HBsAg coding sequence, was mixed with 0.5 ,ug of the BglII-digested pMC200 ànd the mixture was ligated by incubation with T~ DNA ligase.
- 16 ~ 7~'7 The ligated DNA was used to transform competent cells o~ _. coli K12 strain MM294. Transformants were selected on agar plates containing 200 ~g/ml ampicillin. Twelve resistant colonies were isolated by serial passage on 5 ampicillin agar and plasmids were isolated by the procedure described by Birnboim et al., Nucl. Acid Res. Volume 7, 1513 (1979). Analysis by agarose gel electrophoresis showed that all of the plasmids were restricted by HpaI, indicative of insertion o~ the BamHI fragment and that both 10 orientations of the inserted fragment were present among the twelve transformed colonies. The plasmids were isolated by CsCl-ethidium bromide density gradient centrifugation. One plasmid, pRIT10671, contained the BamHI fragment fused at the BglII site in correct 15 orientation for transcription of the HBsAg coding sequence to generate a 2~6 amino acid fusion protein consisting of 18 N-terminal amino acids of OCT, 42 amino acids of the putative HBsAg precursor protein and the 226 amino acids of HBs~g. The fusion protein is HBsAg as shown in Example 8, 20 below. Transformants containing pRIT10671 are designated _. coli K12 strain MM294(pRIT10671)o pRIT10671 is illustrated in Figure 5O
Use of pRIT10640 as an intermediate plasmid is not essential. For example, the BamHI fragment could have been 25 excised from pRIT10616~
Another plasmid, pRIT10672, contained the BamHI
fragment in the incorrect orientation for transcription of the HBsAg coding sequence. Transformants containing this plasmid are designated Eo coli K12 strain MM294(pRIT10672)~
Example 6. Preparation of Plasmids, pRIT10673 and pRIT10674 Shuttle Vectors, by Combining pRIT10671 and pRIT10672_with Shuttle Vector YEpl3 Vector YEpl3, is an E. coli-S. cerevisiae shuttle vector. It has been described by Broach et al, Gene, 35 Volume 8, 121 (1979). It was supplied b~ J. Hicks, Cold ~ 17 - ~2~27~
Spring Harbor Laboratories, New York, U.S.A. A small HindIII fragment was excised from the plasmid by digestion with HindIII and t~e plasmid was religated to prepare a derivative plasmid, YEpl3 HindIII which contains a single 5 HindIII site. Purified YEpl3 HindIII was digested with HindIII and treated with alkaline phosphatase to inhibit religation. The DNA was recovered by phenol extraction and ethanol precipitation.
Purified pRIT10671 and pRIT10672 were digested with 10 BamHI and treated with alkaline phosphatase to inhibit reformation of the pBR322 moiety. The treated DNA was further digested with ~indIII to liberate a 4650 bp HindIII
fragment, which contains the regulatory region-HBsAg gene fusion, and the samples were extracted with phenol and 15 precipitated with ethanol. The DNA preparations derived from pRIT10671 and pRIT10672~ 0.4 ~g of each, were separately mixed with 0.4 ~ug of the HindIII-digested YEpl3 LIindIII and the mixtures were incubated with T4 DNA ligase A portion of each of the ligated mixtures was used to 20 transform competent cells of E. coli K12 strain MM2940 _ Transformants were selected on ampicillin agarO
Transformant colonies were isolated and examined for their plasmid content by the procedure described by ~irnboim et al., Nucl. Acid. Re_., Volume 7, 1513 (1979). One 25 transformant colony, Eo coli K12 strain MM294(pRIT10673), contained a plasmid, pRIT10673, which contains the HindIII
fragment from pRIT10671 inserted on YEp 13HindIII, in the correct orientation, as illustrated in Figure 6. Another transformant colony, E. coli K12 strain MM294(pRIT10674), _._ 30 contained a plasmid, pRIT10674, which contains the HindIII
fragment from pRIT10672 inserted on YEpl3 HindIII in the incorrect orientation.
~~
~ v a Example 7. Transformation of Yeast with pRIT10673 and pRIT10674 Plasmids pRIT10673 and pRIT10674 were isolated from cleared lysates of E. coli K12 strains MM294(pRIT10673) and 5 MM294(pRIT10674) by CsCl-ethidium bromide density gradient centrifugation.
A S cerevisiae strain DC5 The S. cerevisiae strain DC5 (leu 2-3, leu 2-112, his 3, can 1-11) described by Broach et al., Gene, Volume 8, 10 121 (1979), was obtained from J. HicXs, Cold Spring Harbor Laboratories, New York, U.S.A. and was deposited in accordance with EPC regulations and the Budapest Treaty in the American Type Culture Collection in Rockville, Maryland, U.S.A., on June 2, 1982 under accession number 15 ATCC 20630. Cells of strain DC5 were grown and prepared for transformation by the procedures described by Hinnen et al., Proc. Natl. Acad. Sci. U.S.A., Volume 75, 1929 (1978) except that protoplasting was done in 008 M sorbitol, 0.03 M ~-mercaptoethanol, and 0.1 M potassium phosphate buffer 20 (pH 7.5) using a mixture of ~-glucouronidase (0.24 units/ml final concentration) and arylsulphatase (1.2 units/ml final concentration)O ~east protoplasts were separately incubated with 10 lug of pRIT10673 and 10 ug of pRIT10674 and transformants were selected in regeneration agar 25 lacking leucineO Colonies which grew in the regeneration agar were recovered and streaked on solid medium containing 0.675% Yeast Nitrogen Base medium lacking amino acids, 2~
glucose, 2% agar and 80 jug/ml histidine and were grown at 30 . One colony of strain DC5 had been transformed with 30 pRIT10673 and is designated S. cerevisiae strain DC5(pRIT10673). Another colony had been transformed with pRIT10674 and is designated S. cerevisiae strain DC5(pRIT1067~)o Cultures of strains DC5(pRIT10673) and DC5 (pRIT10674) 35 were grown in Yeast Nitrogen Base medium plus 80 ~g/ml - 19- :~2~ 7 histidine to optical densities of 0.33 to 1.0 at 620 ~m.
The latter strain is useful as a negative control because it contains the HBsAg coding sequence fused in incorrect orientation to the regulatory region. Cells were recovered 5 by centrifugation, washed with phosphate buffered saline (PBS) and resuspended in 5 ml PBS plus 1 mM phenyl methyl sulphonyl fluoride (PMSF) at 20 to 160-fold concentrations. Cells were broken by two passages through a French Press~re Cell at 12,000 psi (83 MPa) and the 10 lysate was centrifuged at 7700 x G for 15 minutes and then for 30 minutes at 30,000 x G. The supernatants were recovered and filtered over a Millex GV membrane.
Supernatants were tested for the presence 3f protein reacting with specific anti-HBsAg antibodies by the 15 Ausria~ radioimmunoassay procedures. Clarified cell extracts of S. cerevisiae strain DC5(pRIT10673) prepared in this manner gave positive reactions in this radioimmunoassay even when tested at 16 to 256-fold dilutions in PBS. In contrast, extracts of strain 20 DC5(pRIT10674) were negative in this assay for the presence of proteins reacting with anti-HBsAg antibodies~
B. S. cerevisiae strain lc1697d _. cerevisiae strain lc1697d was deposited in accordance with EPC regulations and the Budapest Treaty in 25 the American Type Culture Collection, on June 2, 1982 under accession number ATCC 20631. Using the procedure described above, the arginine bradytrophe strain lc1697d 5argJ -, leu 2-1) was transformed with pRIT10673 and pRITl0674. One leucine-independent colony of the bradytrophe strain had 30 been transformed with pRIT10673 and is designated SO
cerevisiae strain lc1697d(pRIT10673). Another leucine-independent colony, which had been transformed with pRIT10674, is designated S. cerevisiae strain lc1697d(pRIT10674).
* Trademark ' ,,~
- 20 ~ 7~7 Cultures of strains lc1697d(pRIT10673) and lc1697d (pRIT10674) were grown in Yeast Nitrogen Base medium supplemented with 20 ~g/ml arginine. The cells were recovered and cell extracts were prepared as described 5 above. Claxified cell extracts of strain lc1697d (pRIT10673) gave positive results in the Ausria~
radioimmunoassay at dilutions of up to 1/2048 whereas extracts of strain lc1697d(pRIT10674) were uniformly negative in this assay.
From these results, it is concluded that yeast cells of strains DC5 and lc1697d transformed with pRIT10673 specifically synthesize HBsAg in the form of a fusion protein having HBsAg determinants.
Exam~le 8. Immunization of Rabbits with ~BsAg from S. cerevisiae strain lc1697d(pRIT10673) S. cerevisiae strain lc1697d(pRIT10673) was grown to an optical density at 620 um of 0.60 and collected by centrifugation. The cells were resuspended at a 40-fold 20 concentration in PBS plus 1 mM PMSF. A clarified cell extract was prepared as described in Example 7. A
clarified cell extract of strain lc1697d~pRIT10674) was similarly prepared. These extracts were used to immunize rabbits. A first group of six rabbits received parenteral 25 injections of 1 ml of extract of strain lc1697d(pRIT10673) mixed with 1 ml of Freund's complete adjuvant on days 0, 9, 15, 30 and 37. A second group of three rabbits received parenteral injections of 1 ml of extract of strain lc1697d(pRIT10674) mixed with 1 ml of Freund's complete 30 adjuvant on the same days. Sera was obtained from both groups on days 0 (pre-immune) 23, 51 and 65 and, from the first group, on day ~4, and tested for the presence of anti-~BsAg antibodies using the Ausab radioimmunoassay.
The results of these assays, which are given in Table 1, 35 show that four of six rabbits receiving injectio~s of -- 21 ~ 7~7 extract from strain lc1697d(pRIT10673) produced antibodies direeted against HBsAg. None of the three control rabbits reeeiving injections of extract from strain le1697d(pRIT10674) showed evidenee of produetion of S anti HBsAg antibodies. From these results, it is eoneluded that extracts of strain lc1697d(pRIT10673) eontain H~sAg, which ean be used in a vaecine to stimulate protection against HBV infection in humans without serious side effectsO
TABLE I
PROD~CTION OF HBsAg A~'TIBODIES BY RABBITS I~ IZED ~'ITH CELL-FREE EXTP~hCTS
()F S. CERE~1ISIAE STRAI~S lc1697d(pRIT10673) and lc1697d(pRIT10674) SOVRCE OF EXTRACT R~BBIT ANTI-HBsAg SERUI~ TITRE ~Y Ausa~) ASSAY
NU~5:BER DAY DAY DAY DAY DAY
Strain lc1697d 1 neg Deg 1/16 1/16 1/64 -(pRIT10673) 2 neg 1/1024 1/8192 1/8192 1/40g6-neg 1/64 1/16 1/16 1/16 4 neg 1/64 1/2048 1/1024 1/2048 r.eg /~. 024 1/2~6 1/1024 i'2048
Rutter et al., European Patent Application 20,251, disclose recombinant vectors including a recombinant vector comprising plasmid pBR322 and BamHI fragments of HBV DNA, which can be used to transform E. coli. Another vector, 10 comprising a BamHI fragment of HBV D~A and a portion of the tryptophan operon, was used to obtain expression in E. coli strain HB101.
Edman et al., Nature, Volume 291, Number 5815, 503-506 (1981), describe construction of plasmids which direct 15 synthesis of HBcAg and a ~-lactamase-HBsAg fusion protein, under the control of the tryptophan operon regulatory region, in E. coliO
Other references disclosing insertion of HBV DNA into bacteria include Charnay, et al., Pro~. Med VirolO, Volume 20 27~ 88-92 (1981), MacKay et al, Proc. Natl. AcadO Sci.
U.S., Volume 78, Number 7, 4510-4514 (1981); Fritsch et al!, C. R. Acad. Sci. Volume 287, Number 16, 1453 (1978), U. K. Patent Specification 2,034,323 (Derwent ~o. 468;4C).
and Pasek et al., Nature, Volume 282 No. 6, 575 (1979).
HBV D~A has also been cloned in mammalian cells.
These include, human, mouse, and human hepatoma cell lines. For example, Dubois et al., Proc. Natl. Acad. Sci.
U. S., Volume 77, Number 8, 4549-4553 (1980), report transformation of mouse cells with a plasmid containing the 30 HBV genome and expression of HBsAg; Hirschman et al., Proc.
Natl. Acad. Sci. U.S., Volume 77, ~umber 9, 5507-5511 (1980), report production of HBV-like particles by HeLa cells transformed with HBV DNA~
Procedures for preparing HBV vaccine using HBsAg from 35 human blood are reported by Funakoshi et al., Prog. Med.
7~
Virol., Volume 27, 163-167 (1981), and Maupas et al., Prog.
Med. Virol~, Volume 27, 185-201 (1981). The vaccine prepared by Funakoshi et al. contains 40 ~g of purified, formalin-treated HBsAg, phosphate sodium chloride, 20 mg of 5 mannitol; and 0.1% of aluminlum hydroxide as adjuvant. In the latter paper, Maupas et al. report that one dose of vaccine was 1 ml of purified, formalin-treated HBsAg containing 2-10 ~g/ml of protein (Lowry's method) and 0.1%
of aluminum hydroxideO The protocol used in the study 10 reported by Maupas et alO called for three injections at one month intervals with a booster after one year; the authors propose a protocol consisting of two injections of concentrated HBsAg at three month intervals.
Additional references to preparation of HBV vaccines 15 include Maupas et al., Adamowicz et alO, and Funakoshi et al. at pages 3, 37 and 57, respectively, of Hepatitis B
Vaccine INSERM Symposium No. 18, editO by Maupas and Guesry, 1981, Elsevier/North-Holland Biomedical Press.
Yeasts have been used as host organisms for certain 20 other DNA sequences. For example, Fraser et al., UO K3 Patent Application 2,068,969, disclose preparation of chicken ovalbumin in yeast; Scrip NoO 640, pO 11 (NovO 4, 1981) contains a report that a type of interferon is being prapared in yeast. In European Patent 11,562 (Derwent NoO
25 38762C) are reported hybrid yeast plasmids containing the ura3 yeast gene in the 2 ~ plasmid.
SU~ARY OF THE INVENTION
The invention relates to the preparation of a 30 recombinant DNA molecule comprising a nucleotide sequence which can code for HBsAg and a regulatory region derived from the yeast ~ 3 gene which can effect transcription of the H~sAg sequence in the yeast, Saccharomyces cerevisiae.
The molecule includes vectors in which the HBsAg sequence 35 and the regulatory region have been inserted, which vectors '~2~Z~
can be used to prepare a yeast vector or to maintain the HBsAg and regulatory re~ions. Microorganisms containing the recombinant D~A molecule, such as microorganisms transformed with plasmids of the invention, are included 5 within the invention. The invention also includes a plasmid having the ~3 regulatory region and a restriction site proximate thereto for insertion of a coding sequence, such as an HBsAg sequence, such that the protein synthesi~ed by expression of the coding sequence is devoid 10 of extraneous amino acid residues.
The invention also includes the preparation of a vaccine for stimulating protection against HBV infection in humans comprising a vaccinal amount of HBsAg prepared in accordance with this invention and a suitable carrierO
Further, the invention includes processes for preparing the recombinant DNA molecule and microorganisms containing the molecules as well as processes for preparing HBsAg and HBsAg containing vaccine.
BRIEF DES RIPTION OF T~E FIGURES
Figure 1 is a restriction endonuclease cleavage map of pRIT10601.
Figure 2 is a restriction endonuclease cleavage map of pRIT10616.
Figure 3 is a restriction endonuclease cleavage map of 25 pMC200.
Figure 4 is the nucleotide sequence of a portion of the 3300 bp HindIII yeast DN~ insert i~ pMC200 ~hich portion contains HincII, BglII and EcoRI sites.
Figure S is a flow sheet illustrating preparation of 30 pRIT10671 and pRITl06730 Figure 6 is a flow sheet illustrating preparation of pRIT10749~
Figure 7 is a flow sheet illustrating preparation ofpRIT10761 and pRIT10764.
. 7 _ ~Z2~7~7 DETAILED DESCRIPTION OF THE INVENTION
The recombinant DNA molecule is prepared by fusing a nucleotide sequence which includes a structural gene for HBsAg with the yeast ~3 regulatory region, which 5 regulatory region can direct transcription of the HBsAg sequence in yeast, thereby effecting expression thereof.
By "recombinant DNA molecule" is meant a DNA fragment containing the HBsAg coding sequence and the regulatory region as well as other DNA molecules containing the 10 fragment, such as plasmid or phage vectors.
By "regulatory region" is meant a sequence which contains a promotor region and other sequences necessary for transcription. The yeast arg3 regulatory region is especially advantageous because it can be a powerful 15 promotor for expression of an HBsAg coding sequenceO
By "HBsAg" is meant a protein which is structurally identical to authentic HBsAg or has substantially the same antigenic determinants as authentic HBsAg, that is, is capable of stimulating production of antibodies which 20 specifically recognize and react with authentic HBsAg o~ is specîfically recognized by anti-HBsAg antibodies.
The HBsAg coding sequence can be isolated from DNA
extracted from Dane particles in lnfected huma~ serum by filling in the single strand region with a DNA polymerase, 25 preerably the endogenous polymerase, followed by digestion with a restriction endonuclease. The choice of endonuclease will depend, in part, on the particular Dane particles. For example, as illustrated in the Examples below, the HBsAg coding sequence of HBV DNA of certain Dane 30 particles of the adw serotype can be isolated on a single BamHI fragment; the HBsAg coding sequence of HBV DNA of certain Dane particles of the ayw serotype can be isolated on a HhaI fragment. HBV DNA of Dane particles of the same 7~7 ~,'..
serotype may also exhibit different patterns of restriction sites, Restriction of D~A to prepare DNA fragments used in the invention, ligation of such fragments to prepare 5 recombinant D~A molecules used in the invention and insertion into microorganisms are carried out by Xnown techniques such as techniques disclosed in the previously and subsequently cited references. Conditions are selected to avoid denaturation of the DNA and enzymes~ For example, 10 the pH is buffered to remain at about 7.0 to 11.0 and the temperature is kept below about 60 CO Preferably restriction is carried out at a temperature of about 30 to 40C and ligation is carried out at about O to 10C.
Restriction enzymes and ligases used in carrying out this 15 invention are commercially available and should be used in accordance with instructions included therewith. T4 Dl~A
~igase is the preferred ligase.
Fusing of the HBsAg sequence to the regulatory region can be accomplished by use of intermediate vectors as 20 illustrated in the Examples, below. Alternatively, the HBsAg sequence can be inserted directly into a vector which contains the regulatory region~ A vector is DNA which can carry and maintain the D~A fragment of the invention, including, for example, phages and plasmids. Techniques 25 for cloning DNA fragments in phages are disclosed, for example, by Charnay et al. Nature, Volume 286, 893-895 (1980) and Tiollais, United Kingdom Patent Application 2,034,3230 Preferably, the HBsAg sequence is positioned relative to the regulatory region such that the HBsAg 30 synthesized by expression of the HBsAg sequence is devoid of extraneous amino acid residues.
A regulatory region which has been found to be especially useful is derived from the yeast arg3 gene which codes for ornithine carbamoyltransferase (OCT). Use of the 35 arg3 regulatory region is advantageous because its activity . g is subject to both specific and general control mechanismsO It has been cloned in Eo coli on plasmid pYeura3arg3 as reported by Crabeel et al., Proc. Natl.
~cad. Sci. U.S.A., Volume 78, 5026 (1981)o Preferred hosts 5 are _. cerevisiae strains in which the arginine biosynthetic pathway is derepressed, such as strain lc1697do Use of such strains results in increased expression from the ~3 promotor as compared to the other strain which was used in the Examples, strain DC50 The preferred vector ~or cloning the fused DNA
fragment into yeast is the plasmid YEpl3, which is capable of replication and maintenance in both E. coli and S0 cerevisiae and is, therefore, known as a shuttle vectorO
-Several other yeast vectors are known and availableO The 15 HBsAg and regulatory regions can be inserted into a yeast vector sequentially or, as illustrated in the Examples, below, simultaneously. Transformation with plasmid vectors will normally result in incorporation of the DNA molecule of the invention as a plasmid. However, other reactions, 20 such as recombination events, can result in incorporation of the DNA molecule into chromosomal DNA.
Vaccines for stimulating protection against HBV
infection in humans comprising HBsAg produced by yeast in accordance with the invention and a suitable carrier can be 25 prepared by known techniques. Use of an adjuvant~ such as aluminum hydroxide, is desirable. The HBsAg so produced can also be combined with other immunogens to prepare combination vaccines The HBV or combination vaccines can be administered, for example, by the subcutaneous, 30 intravenous or intramuscular route. The DNA fragment of the invention and the HBsAg produced thereby, can also be used as a probe for detection of HBV in biological samples by DNA hydridization techniques and various immunoassays.
EXAMPLES
__ In the following examples of the invention, which are illustrative and not limiting, all percentages are by weight and all temperatures are in degrees CelsiusO
Example 1 Preparation of Intermediate Plasmid, RIT10601, bv Combinin~ HBV DNA with pBR322 p ~ _ __ _ HBs~g positive serum of ~y~ serotype was defibrinated by addition of CaC12 to a final concentration of 0.28%
10 and centrifuged for 2 h at 27,000 rpm in a SW 27 rotor on 10-20% sucrose gradients made up in a buffer (pH 7O5) containing 10 mM tromethamine-HCl, 1 M NaCl and 1 mM ~DTAo A transparent pellet containing Dane particles was resuspended in the same buffer and centrifuged on buffered 15 20~ sucrose layered on a 65% sucrose cushion. An opalescent band at the cushion interface was recovered and centri~uged on a similar 20-65~ gradient at 200,000 x C for 4 h to pellet the Dane particles.
A. Single strand regions of the HBV genome within the 20 Dane .particles were repai.red using the endogenous DNA
polymerase by resuspending the Dane particles in a reaction mixture (pH 8.0) containing 50 mM tromethamine-HCl, 10 mM
MgC12, 500 mM NaCl, 0.5 mM dithiothreitol, 50 mM eacn of dATP, dCTP and dGTP and 8 ~uM of P-dTTP (350 Ci/mmole) 25 and incubating the resuspended particles for 5 h at 37O
The resuspension was diluted to pH 7O5 with a buffer containing 10 mM tromethamine-HCl, 10 mM EDTA, 100 mM NaCL
and 0.02~ sodium dodecyl sulfate (pH 7.5) and incubated with 0.5 mg/ml of pronase for 1 h at 37 followed by 30 phenol extraction and ethanol precipitationO
Digestion of the DNA with BamHI restriction endonuclease, yielded two radioactive fragments with sizes of about 1450 bp and 1600 bp as judged by agarose gel electrophoresis and autoradiography of the gel.
2~7 B. About 30 ng of Dane particle DNA was repaired with the endogenous DNA polymerase in the presence o non-labelled dTTP, and was extracted and recovered, as described above. The DNA was mixed with 100 ng of plasmid 5 pBR322 which had been previously digested with BamHI
restriction endonuclease and treated with alkaline phosphatase. Plasmid pBR322 is commonly used in recombinant DNA procedures and is on deposit, without restriction on availability, in the American Type Culture 10 Collection under accession number 37017. The mixture was extracted with phenol, precipitated with ethanol, centrifuged, dried, resuspended in 12 ul of a mixture (pH
7.5) containing 50 mM tromethamine-HCl, 1 mM ATP, 10 mM
MgC12, 10 mM dithiothreitol, 50/ug/ml gelatin and 2 15 units/ml T4 DNA ligase. The suspension was incubated for 4 h at 10 and then held on ice for 18 h.
The ligated DNA mixture was used to transform competent cells of E~ coli K12 strain- C600 prepared according to the procedure of Cohen et al., Proc; ~atl.
20 Acad. Sci. U. S., Volume 69, 2110 (1972~. Transformants were selected on a solid medium containing ampicillin (200 ,uglml). Isolated colonies were screened for loss of tetracycline resistance, indicative of insertion o~ a foreign D~A fragment in the BamHI site of pBR322. One such 25 transformant clone was found to contain a plasmid, pRIT10601, which on digestion with BamHI endonuclease gave a pBR322 fragment of 4360 bp and HBV DNA fragments of 1600 bp and 1450 bp~ A culture of E. coli K12 strain C600(pRIT10601) was deposited in accordance with 30 regulations of the E-~ropean Patent Convention (EPC) and the Budapest Treaty in the American Type Culture Collection, Rockville, Maryland U.S.A. on June 2, 1982 under accession number ATCC 39132. A restriction endonuclease cleavage map of plasmid pRIT10601 is shown in Figure 1.
'7~7 C. The sizes of fragments generated by digestion of the Dane particle DNA and pRIT10601 with various restriction enzymes were compared as follows. Dane particle DNA, that is, HBV DNA, was labelled with 32p by 5 the endogeneous polymerase reaction described above or by treating purlfied DNA with DNA polymerase I from E. coli The labelled HBV DNA was mixed with pRIT10601 and the mixture was treated with a restriction endonuclease and electrophoresed on an agarose gel. The gel was stained 10 with ethidium bromide and photographed under UV light to loca~e the DNA fragments and was then dried and autoradiographed to locate the radioactive HBV DNA
fragments. The following labelled HBV fragments were found to match exactly the size of pRIT10601 fragments: 1450 and 15 1600 bp BamHI fragments; a 1330 bp HpaI fragment; and a 1130 bp BamHI-XhoI fragment. Labelled Dane particle DNA
also hydridized specifically to both the 1450 and 1600 bp fragments released by BamHI digestion of pRIT10601 following transfer of these fragments from an agarose gel 20 onto a nitrocellulose filter by the technique of Southern, J. Mol. Biol., Volume 98, 503 (1975).
These results show that the cloned DNA insert on pRIT10601 represents the HBV genome and that the relative orientation of the two BamHI fragments on pRIT10601 is the 25 same as in the virion. pRIT10601 was used to prepare pRIT10671 in Example 10, below~
Example 2. Preparation of Intermediate Plasmid, RIT10616 bv Combinina ~BV DNA with ~ACYC184 P ~
Dane particles were isolated from HBsAg positive serum of adw serotype as described above. Restriction endonuclease analysis of P-labelled HBV DNA indicated that the DNA contained one EcoRI site~
HBV D~A, filled in by the endogenous polymerase 3~ reaction using non-labelled nucleotides, was digested with 2~7 EcoRI. The restricted DNA was mixed with plasmid pACYC184 which had been previously digested with EcoRI and treated with alkaline phosphatase. Plasmid pACYC184 is on deposit, without restrictions on availability, in the American Type 5 Culture Collection under accession number 37033. The mixture was liyated with T4 DNA ligase.
The ligated D~A mixture was used to transform competent cells of E. coli K12 strain C600. Transformants were selected on tetracycline (15 ug/ml~ agar medium and 10 screened for loss of chloramphenicol resistance which is indicative of insertion in the EcoRI site of pACYC184. A
transformed colony was found to contain a plasmid, pRIT10616, which consists of pACYC184 with a 3200 bp insert, comprising the HBV DNA, at the EcoRI site. A
15 restriction map of pRIT10616 is shown in Figure 2. Eo coli K12 strain C600 (p~IT10616) was deposited in accordance with EPC regulations and the Budapest Treaty in the American Type Culture Collection on June 2, 1982 under accession number ATCC 39131.
Example 3. Preparation of Intermediate Plasmid, pRIT10640,Containing a Nucleotide Sequence Codinq for HBsAg, by Combininq pRIT10616 with pBR313 pRIT10616 was purified by CsCl-ethidium bromide 25 density gradient centrifugation substantially as described by Kahn et al, Methods in Enzymology, Volume 68, 268 (1979).
The DNA was digested with BamHI endonuclease, mixed with BamHI-digested, alkaline phosphatase-treated DNA of plasmid pBR313, ligated and used to transform competent 30 cells of E. coli K12 strain C600 substantially as described in Example 1. Plasmid pBR313 is on deposit, without restrictions on availability, in the American Type Culture Collection under accession number 37018.
Trans~ormants were selected on ampicillin-containing 35 agar and screened for loss of tetracycline resistance, 14 ~:227~7 indicative of insertion at the BamHI site of pBR313. A
transformed colony was found to contain a plasmid, pRIT10640, which consists of pBR313 with a 1350 bp insert at the BamHI site. The insert is a nucleotide sequence 5 which can code for HBsAg. It codes for part of a putative HBs~g precursor protein and the complete surface antigen and includes 565 bp of 3' non-coding sequencesO
c Example 4. Preparation of Intermediate Plasmid pMC200, Containing the arg3 Regulatory Region, by Combining Yeast arg3 Gene with pBR322 A 3300 bp yeast D~A fragment specifying the arg3 gene was obtained by diyestion of pYeura3~3 with HindIIIO
Plasmid p~eura3arg3 has been described by Crabeel et al., 15 Proc. ~atl. Acad. Sci. U.S., Volume 78, 5026 (1981)~ The 3300 bp fragment was cloned into the HindIII site of pBR322 and transformed into _. coli K12 strain MM294, substantially as described above. Transformants were selected on ampicillin medium and screened for tetracycline 20 resistance. A transformed colony was found to contain a plasmid, pMC200, which consists of pBR322 with an insert at the HindIII site. This plasmid contains the arg3 regulatory region which can effect transcription of a HBs~g nucleotide sequence in yeast cells as described in 25 following examples~ E. coli K12 strain MM294(pMC200) was deposited in accordance with EPC regulations and the Budapest Treaty in the American Type Culture Collection on June 2, 1982 under accession number ATCC 39130.
The nucleotide sequence of part of the arg3 gene, 30 including part of the ~-terminal coding sequences for ornithine carbamoyltransferase (OCT) and part of the 5' non-translated leader region has been determined by Huyghen et al., ~rch. Intl. Physical. Biochem., Volume 89, B172 - (1981). Figure 4 illustrates a 210 bp fragment of the 35 known sequence The 210 bp fragment contains unique - 15 ~ 7~7 HincII, BglII and EcoRI sites on the 3300 bp HindIII yeast DNA insert in pMC200; the initiation codon for the OCT
protein coding sequence is believed to be the boxed ATG
codon. Introduction into the HincII, BglII or EcoP~I sites S of the yeast D~A of coding sequences for HBsAg alone or HBsAg precursor plus HBsAg derived from cloned HBV DNA is - expected to result in a gene fusion and production of a hybrid protein transcribed and translated from the ~3 regulatory region provided that the gene fusion is in the 10 correct orientation for continued translation beyond the site of fusion.
Example 5. Preparation of Intermediate Plasmids, ~RIT10671 and pRIT10672, Containing HBsAg and Regulatory Regions, by Combini~gLpRIT10641 with pMC200 pMC200, 5~g, was digested with 6.4 units of BglII for 2.5 h at 37, diluted with an equal volume of a buffer (pH 10.5) containing 0.1 M glycine, 0.01 M MgC12 6H2O
and 0.1 m~ ZnC12, and incubated with 0.5 units of calf 20 intestine alkaline phosphatase for 30 minutes at 37 to remove 5' terminal phosphate residues. The mixture was ex racted twice with buffer-saturated phenol and three times with ether. The DNA was precipitated with ethanol and dissolved in 0.01 M tromethamine buffer (pH 7.5).
pRIT106~0, 25 ~ug, was digested with BamHI to excise the 1350 bp BamHI fragment of HBV DNA. The 1350 bp fragment was purified by preparative agarose gel electrophoresis and electroelution. The eluted D~A was recovered and concentrated by ethanol precipitation and 30 dissolved in 20 ~1 of 0.01 M tromethamine buffer (pH 7.0).
A 0.3 Jug aliquot of the BamHI fragment, which contains the HBsAg coding sequence, was mixed with 0.5 ,ug of the BglII-digested pMC200 ànd the mixture was ligated by incubation with T~ DNA ligase.
- 16 ~ 7~'7 The ligated DNA was used to transform competent cells o~ _. coli K12 strain MM294. Transformants were selected on agar plates containing 200 ~g/ml ampicillin. Twelve resistant colonies were isolated by serial passage on 5 ampicillin agar and plasmids were isolated by the procedure described by Birnboim et al., Nucl. Acid Res. Volume 7, 1513 (1979). Analysis by agarose gel electrophoresis showed that all of the plasmids were restricted by HpaI, indicative of insertion o~ the BamHI fragment and that both 10 orientations of the inserted fragment were present among the twelve transformed colonies. The plasmids were isolated by CsCl-ethidium bromide density gradient centrifugation. One plasmid, pRIT10671, contained the BamHI fragment fused at the BglII site in correct 15 orientation for transcription of the HBsAg coding sequence to generate a 2~6 amino acid fusion protein consisting of 18 N-terminal amino acids of OCT, 42 amino acids of the putative HBsAg precursor protein and the 226 amino acids of HBs~g. The fusion protein is HBsAg as shown in Example 8, 20 below. Transformants containing pRIT10671 are designated _. coli K12 strain MM294(pRIT10671)o pRIT10671 is illustrated in Figure 5O
Use of pRIT10640 as an intermediate plasmid is not essential. For example, the BamHI fragment could have been 25 excised from pRIT10616~
Another plasmid, pRIT10672, contained the BamHI
fragment in the incorrect orientation for transcription of the HBsAg coding sequence. Transformants containing this plasmid are designated Eo coli K12 strain MM294(pRIT10672)~
Example 6. Preparation of Plasmids, pRIT10673 and pRIT10674 Shuttle Vectors, by Combining pRIT10671 and pRIT10672_with Shuttle Vector YEpl3 Vector YEpl3, is an E. coli-S. cerevisiae shuttle vector. It has been described by Broach et al, Gene, 35 Volume 8, 121 (1979). It was supplied b~ J. Hicks, Cold ~ 17 - ~2~27~
Spring Harbor Laboratories, New York, U.S.A. A small HindIII fragment was excised from the plasmid by digestion with HindIII and t~e plasmid was religated to prepare a derivative plasmid, YEpl3 HindIII which contains a single 5 HindIII site. Purified YEpl3 HindIII was digested with HindIII and treated with alkaline phosphatase to inhibit religation. The DNA was recovered by phenol extraction and ethanol precipitation.
Purified pRIT10671 and pRIT10672 were digested with 10 BamHI and treated with alkaline phosphatase to inhibit reformation of the pBR322 moiety. The treated DNA was further digested with ~indIII to liberate a 4650 bp HindIII
fragment, which contains the regulatory region-HBsAg gene fusion, and the samples were extracted with phenol and 15 precipitated with ethanol. The DNA preparations derived from pRIT10671 and pRIT10672~ 0.4 ~g of each, were separately mixed with 0.4 ~ug of the HindIII-digested YEpl3 LIindIII and the mixtures were incubated with T4 DNA ligase A portion of each of the ligated mixtures was used to 20 transform competent cells of E. coli K12 strain MM2940 _ Transformants were selected on ampicillin agarO
Transformant colonies were isolated and examined for their plasmid content by the procedure described by ~irnboim et al., Nucl. Acid. Re_., Volume 7, 1513 (1979). One 25 transformant colony, Eo coli K12 strain MM294(pRIT10673), contained a plasmid, pRIT10673, which contains the HindIII
fragment from pRIT10671 inserted on YEp 13HindIII, in the correct orientation, as illustrated in Figure 6. Another transformant colony, E. coli K12 strain MM294(pRIT10674), _._ 30 contained a plasmid, pRIT10674, which contains the HindIII
fragment from pRIT10672 inserted on YEpl3 HindIII in the incorrect orientation.
~~
~ v a Example 7. Transformation of Yeast with pRIT10673 and pRIT10674 Plasmids pRIT10673 and pRIT10674 were isolated from cleared lysates of E. coli K12 strains MM294(pRIT10673) and 5 MM294(pRIT10674) by CsCl-ethidium bromide density gradient centrifugation.
A S cerevisiae strain DC5 The S. cerevisiae strain DC5 (leu 2-3, leu 2-112, his 3, can 1-11) described by Broach et al., Gene, Volume 8, 10 121 (1979), was obtained from J. HicXs, Cold Spring Harbor Laboratories, New York, U.S.A. and was deposited in accordance with EPC regulations and the Budapest Treaty in the American Type Culture Collection in Rockville, Maryland, U.S.A., on June 2, 1982 under accession number 15 ATCC 20630. Cells of strain DC5 were grown and prepared for transformation by the procedures described by Hinnen et al., Proc. Natl. Acad. Sci. U.S.A., Volume 75, 1929 (1978) except that protoplasting was done in 008 M sorbitol, 0.03 M ~-mercaptoethanol, and 0.1 M potassium phosphate buffer 20 (pH 7.5) using a mixture of ~-glucouronidase (0.24 units/ml final concentration) and arylsulphatase (1.2 units/ml final concentration)O ~east protoplasts were separately incubated with 10 lug of pRIT10673 and 10 ug of pRIT10674 and transformants were selected in regeneration agar 25 lacking leucineO Colonies which grew in the regeneration agar were recovered and streaked on solid medium containing 0.675% Yeast Nitrogen Base medium lacking amino acids, 2~
glucose, 2% agar and 80 jug/ml histidine and were grown at 30 . One colony of strain DC5 had been transformed with 30 pRIT10673 and is designated S. cerevisiae strain DC5(pRIT10673). Another colony had been transformed with pRIT10674 and is designated S. cerevisiae strain DC5(pRIT1067~)o Cultures of strains DC5(pRIT10673) and DC5 (pRIT10674) 35 were grown in Yeast Nitrogen Base medium plus 80 ~g/ml - 19- :~2~ 7 histidine to optical densities of 0.33 to 1.0 at 620 ~m.
The latter strain is useful as a negative control because it contains the HBsAg coding sequence fused in incorrect orientation to the regulatory region. Cells were recovered 5 by centrifugation, washed with phosphate buffered saline (PBS) and resuspended in 5 ml PBS plus 1 mM phenyl methyl sulphonyl fluoride (PMSF) at 20 to 160-fold concentrations. Cells were broken by two passages through a French Press~re Cell at 12,000 psi (83 MPa) and the 10 lysate was centrifuged at 7700 x G for 15 minutes and then for 30 minutes at 30,000 x G. The supernatants were recovered and filtered over a Millex GV membrane.
Supernatants were tested for the presence 3f protein reacting with specific anti-HBsAg antibodies by the 15 Ausria~ radioimmunoassay procedures. Clarified cell extracts of S. cerevisiae strain DC5(pRIT10673) prepared in this manner gave positive reactions in this radioimmunoassay even when tested at 16 to 256-fold dilutions in PBS. In contrast, extracts of strain 20 DC5(pRIT10674) were negative in this assay for the presence of proteins reacting with anti-HBsAg antibodies~
B. S. cerevisiae strain lc1697d _. cerevisiae strain lc1697d was deposited in accordance with EPC regulations and the Budapest Treaty in 25 the American Type Culture Collection, on June 2, 1982 under accession number ATCC 20631. Using the procedure described above, the arginine bradytrophe strain lc1697d 5argJ -, leu 2-1) was transformed with pRIT10673 and pRITl0674. One leucine-independent colony of the bradytrophe strain had 30 been transformed with pRIT10673 and is designated SO
cerevisiae strain lc1697d(pRIT10673). Another leucine-independent colony, which had been transformed with pRIT10674, is designated S. cerevisiae strain lc1697d(pRIT10674).
* Trademark ' ,,~
- 20 ~ 7~7 Cultures of strains lc1697d(pRIT10673) and lc1697d (pRIT10674) were grown in Yeast Nitrogen Base medium supplemented with 20 ~g/ml arginine. The cells were recovered and cell extracts were prepared as described 5 above. Claxified cell extracts of strain lc1697d (pRIT10673) gave positive results in the Ausria~
radioimmunoassay at dilutions of up to 1/2048 whereas extracts of strain lc1697d(pRIT10674) were uniformly negative in this assay.
From these results, it is concluded that yeast cells of strains DC5 and lc1697d transformed with pRIT10673 specifically synthesize HBsAg in the form of a fusion protein having HBsAg determinants.
Exam~le 8. Immunization of Rabbits with ~BsAg from S. cerevisiae strain lc1697d(pRIT10673) S. cerevisiae strain lc1697d(pRIT10673) was grown to an optical density at 620 um of 0.60 and collected by centrifugation. The cells were resuspended at a 40-fold 20 concentration in PBS plus 1 mM PMSF. A clarified cell extract was prepared as described in Example 7. A
clarified cell extract of strain lc1697d~pRIT10674) was similarly prepared. These extracts were used to immunize rabbits. A first group of six rabbits received parenteral 25 injections of 1 ml of extract of strain lc1697d(pRIT10673) mixed with 1 ml of Freund's complete adjuvant on days 0, 9, 15, 30 and 37. A second group of three rabbits received parenteral injections of 1 ml of extract of strain lc1697d(pRIT10674) mixed with 1 ml of Freund's complete 30 adjuvant on the same days. Sera was obtained from both groups on days 0 (pre-immune) 23, 51 and 65 and, from the first group, on day ~4, and tested for the presence of anti-~BsAg antibodies using the Ausab radioimmunoassay.
The results of these assays, which are given in Table 1, 35 show that four of six rabbits receiving injectio~s of -- 21 ~ 7~7 extract from strain lc1697d(pRIT10673) produced antibodies direeted against HBsAg. None of the three control rabbits reeeiving injections of extract from strain le1697d(pRIT10674) showed evidenee of produetion of S anti HBsAg antibodies. From these results, it is eoneluded that extracts of strain lc1697d(pRIT10673) eontain H~sAg, which ean be used in a vaecine to stimulate protection against HBV infection in humans without serious side effectsO
TABLE I
PROD~CTION OF HBsAg A~'TIBODIES BY RABBITS I~ IZED ~'ITH CELL-FREE EXTP~hCTS
()F S. CERE~1ISIAE STRAI~S lc1697d(pRIT10673) and lc1697d(pRIT10674) SOVRCE OF EXTRACT R~BBIT ANTI-HBsAg SERUI~ TITRE ~Y Ausa~) ASSAY
NU~5:BER DAY DAY DAY DAY DAY
Strain lc1697d 1 neg Deg 1/16 1/16 1/64 -(pRIT10673) 2 neg 1/1024 1/8192 1/8192 1/40g6-neg 1/64 1/16 1/16 1/16 4 neg 1/64 1/2048 1/1024 1/2048 r.eg /~. 024 1/2~6 1/1024 i'2048
6 neg 1/16 1/256 1/1024 112048 S;rain lc1697d -(p~IT10674) 7 neg neg --- neg ---8 neg 1/1 --- neg neg 9 ne~ neg --- neg neg ~'ighest dilution of serum gi~in~ a positive result in the Ausab~)~s Y
~2~7~3~
Example 9. Preparation of Plasmid, pRIq'10749, Containinq the arg3 Regulatory Reqion, from pMC200 Purified pMC200 was digested with ~incII and electrophoresed on a 10% acrylamide gel. A 1940 bp 5 fragment containing the ary3 regulatory region was recovered from the gel by electroelution and ethanol precipitation. About ~ ~g of the fragment was incubated with 0.2 units of Bal31 exonuclease, for 1 to 3 seconds at in 100 ~1 of buffer (pH 8.0) containing 12.5 mM
10 MgC12, 12.5 mM CaC12, 200 mM NaCl, 1 mM EDTA and 200 mM
tromethamine-HCl. The treated DNA was extracted with phenol and precipitated with ethanol. A 1 jug aliquot was incubated with T4 DN~ polymerase in the presence of deoxynucleotide triphosphates to repair any single strand 15 extremities and was then digested with EcoRI endonucleaseO
This procedure produced DNA fragments of varying lengths due to the resection with Bal31. Each fragment had a fixed EcoRI extension at one end with the othe~ extremity being blunt ended and located at some distance from the original 20 HincII site and the OCT protein initiation codon.
Fragments of about 1480 bp containing the arg3 regulatory region were isolated by electrophoresis on a 7.5~
acrylamide gel followed by electroelution and ethanol precipitation. This cut-back reyulatory region is 25 preferred because it can promote transcription leading to synthesis of a protein devoid o~ OCT amino acid sequences, as described in Example 10, below.
In a second series of reactions, pMC200 was digested with XbaI and the 5' single strand extremities were filled 30 in or rendered blunt-ended, by incubation with T4 DNA
polymerase and deoxynucleotide triphosphates. This DNA was digested with EcoRI and a large EcoRI-T4/Xbal fragment of about 5700 bp, containing yeast D~A sequences located 3' to the XbaI site, plus pBR32~ sequences, was purified by 35 electrophoresis, electroelution and ethanol precipitation.
- 23 ~ lZ ~ 2 ~ ~ ~
This fragment was mixed with and ligated to the 1480 bp promotor-containing fragments.
The ligation mixture was used to transform competent cells of E. coli K12 strain MM294. Transformants were 5 selected on ampicillin agar~ Plasmid DNA was isolated from transformant colonies by the procedure described by Birnboim et al., Nucl. Acid. Res., ~olume 7, 1513, (1979) and were screened for presence of an XbaI site by digestion with XbaIO An XbaI site was present only in those plasmids 10 in which the filled-in XbaI site on a fragment from the second series of reactions was ligated to another fragment from the first series which terminated in a 3' T residue so as to restore an XbaI recognition sequence, TCTAGA, in a new location. A plasmid possessing an XbaI site was 15 identifed as pRIT10749. A flow sheet illustrating these manipulations is provided in Figure 6. E. coli K12 strain MM294 (pRIT10749) was deposited in accordance with EPC
regulations and the Budapest Treaty in the American Type Culture Collection, Rockville, Maryland, U.S.A~ on June 2, 20 1982 under accession number ATCC 39133.
A sample of pRIT10749 was digested with a combination of BstEII and XbaI~ The size of the deleted arg3 regulatory region was estimated to be about 210 bp by comparison with pMC200 which had been similarly digested 25 W7 th BstEII and XbaI and by reference to the known molecular weights of fragments of phage ~ x17~ D~A
described by Fuchs et al. Gene, Volume 4, 1, (1978).
To determine the DNA sequence of the BstEII-XbaI
fragment of pRIT10749, plasmid D~A was digested with 30 BstEII, labelled with ~- P-ATP by exchange kination and digested with BamHI endonuclease to liberate a 2000 bp fragment which was purified by electroelution and ethanol precipitation. D~A sequencing of the labelled fragment was performed according to the chemical modification method 35 described by Maxam et al., Methods in_En~ymology, Volume - 2~ 7 65, 499 (19~0). A part of this sequence was determined to be CCCATCAACTTGTACACTCGT _AGA. The underscored nucleotides were derived from the 5700 bp EcoRI-T4/Xbal fragment, Comparison to the appropriate region of the D~A sequence 5 shown in Figure 4 indicates that the restored XbaI site is located in the 5' untranslated leader region nine nucleotides upstream of the original HincII site in the pMC200 yeast DNA insert.
Example 10. Preparation of Plasmids, pRIT10759 and pRIT10761, Containing HBsAg and Regulatory Regions, by Combining pRIT10749_with pRIT10601 pRIT10601 was digested with HhaI endonuclease, and electorphoresed on a 7.5% acrylamide gel. A 1100 bp 15 fragment was recovered by electroelution and ethanol precipitation~ The DNA sequence of part of this fragment contains sequences corresponding to the known N-terminal amino acid sequence of HBsAg of ayw serotype as described by Peterson et al., Viral Hepatitis, G. No Vyas, S. N.
20 Cohen and R. Schmid, Eds., Franklin Institute Press, Philadelphia, UoS~A~ ~ 1978, p.569. The fragment contains 6 nucleotides located 5' to the HBsAg initiation codon, the complete HBsAg coding sequence and about 390 3' non-translated nucleotidesO
DNA of plasmid pRIT10749 was digested with XbaI and about 400 ng of this DNA was mixed with about 280 ng of the purified HhaI fragment. The mixture was incubated with 0.5 units of T4 DNA polymerase for 30 minutes at 37 in 20 ~1 buffer (pH 7.5) containing 20 mM tromethamine-HCl, 10 mM
30 M~C12, 1 mM dithiothreitol, and 33 mM each of dATP, dCTP, dGTP and dTTP. To this reaction mixture was added 2.5 Jul of 1 mM ATP, 2.5 ~1 of 10 mM EDTA and 2 ~ul (2 units) of T4 DNA ligase and the mixture was incubated for 16 h at 15 .
The ligated mixture was used to transform competent 35 cells of Eo coli K12 strain MM294 with selection being made - 25 - ~222~7 for ampicillin resistant transformants. Plasmid preparations were made from several of these transformants and analyzed by restriction endonuclease digestion and gel electrophoresis. The results showed that the blunt-ended 5 HBV fr~gment had been inserted in both possible orientations into the XbaI-digested pRIT10749. One such plasmid, pRIT10761 contained the 1100 bp insert in the correct orientation for transcription of the HBsAg sequence from the ~3 promotor and gave a 271 bp fragment after 10 sequential digestion with BstEII and XbaI. It is illustrated in Figure 7. A second plasmid, pRIT10759 contained the insert in the incorrect orientation and gave rise to an estimated 1175 bp fragment on digestion with a combination of BstEII and XbaI endcnucleases. The 15 nucleotide sequence at the arg3 regulatory region-HBsAg insert junction was determined for pRIT10761, by sequencing the 271 bp BstEII-XbaI fragment using the chemical modification methods of Maxam et al., Methods in Enzymologx, Volume 65, 499 (1980), after exchange kination 20 labelling of the XbaI terminus with y - P-ATPo The sequence determined for the ar~3 regulatory region - HBsAg junction is TACACTCGTCTACTGAACATGo It can be seen that the XbaI restriction site was not completely repaired by the T4 DNA polymerase and that one guanine residue was lost. The 25 ar~3 non-translated leader region is immediately followed by 6 nucleotides of HBV origin, CTGAAC, followed by the HBsAg initiation codon, ATG, and the coding sequence of the HBsAg structural geneO The HBsAg initiation codon is the first initiation codon encountered downstream of the ~3 30 promotor. Therefore, translation of the mRNA transcribed from the DNA will initiate at that codon and HBsAg synthesized will be devoid of extraneous amino acid residues. The HBsAg synthesized is not a fusion protein;
it is substantially structurally identical to authentic 35 HBsAg.
- 26 - ~2~2~7 Example 11. Preparation of Pl3~ IT10764 and pRIT10765 by Combinin~l~RIT10761 with YEpl3 HindIII~
pRIT10761 and pRIT10759 were separately digested with PstI and BamHI to destroy the pB~322 replicon moiety and S then with HindIII to release the DNA fragment carrying the inserted HBV DNA and the regulatory region. The DNA's were extracted with phenol, precipitated with ethanol and dissolved in bu~fer (pH 7.5) containing 0.01 M
tromethamine-HCl and 0.001 M EDTA. A 0.6 ~g portion of each 10 DNA preparation was se~arately mixed with 0.3 ~ug HindIII-digested, alkaline phosphatase treated DN~ of YEpl3 HindIII prepared as described above and the mixture was ligated.
The ligated mixture was used to transform competent 15 cells of E. coli K12 strain MM294 with selection being made for ampicillin resistant transformants~ One transformant was shown to contain a plasmid, pRIT10764, consisting of the HindIII fragment from pRIT10761 inserted on YEp 13HindIII. A further transformant was shown to contain a 20 plasmid, pRIT10765, consisting of the ~indIII fragment from pRIT10759 inserted on YEp 13HindIII~ A flow sheet illustrating the preparation of pRIT10761 and pRIT10764 is provided in Figure 7.
.
Exam le 12 - Transformation of Yeast with P
pRIT10764 and pRIT10765 pRIT10764 and pRIT10765 were isolated from cultures of the transformants prepared in Example 11 by CsCl-ethidium bromide density gradient centrifugation and 10Jug of each 30 was used to transform cells of yeast strain lc1697d as described in Example 7 with selection being made for leucine-independent transformants on regeneration agar.
One leucine-independent colony axising from these transformations is designated strain lc1697d(pRITl0764)o 35 Another transformant is designated S. cerevisiae strain - 27 - ~2~X7~
lc1697d(pRIT10765). Cultures of these strains were grown at 30 in Yeast Nitrogen Base medium supplemented with 2%
glucose and 20 jug/ml arginine to an optical density at 620 ~m of 0.80 to 0088. Cells were harvested and cell-free 5 extracts were prepared and assayed for HBsAg by the Ausria ~ radioimmunoassay as described in Example 7.
Extracts of lc1697d(pRIT10764) gave positive results in this assay at dilutions in PBS of up to 1/1024 whereas undiluted extracts of strain lc1697d(pRIT10765) were 10 uniformly negative in this assay. From the genetic and radiomimmunoassay evidence, it is concluded that cells of S, cerevisiae strain lc1697d(pRIT10764) synthesize HBsAg which can be used in a vaccine for stimulating protection against HBV infection in humans without serious side 15 effectsO
~2~7~3~
Example 9. Preparation of Plasmid, pRIq'10749, Containinq the arg3 Regulatory Reqion, from pMC200 Purified pMC200 was digested with ~incII and electrophoresed on a 10% acrylamide gel. A 1940 bp 5 fragment containing the ary3 regulatory region was recovered from the gel by electroelution and ethanol precipitation. About ~ ~g of the fragment was incubated with 0.2 units of Bal31 exonuclease, for 1 to 3 seconds at in 100 ~1 of buffer (pH 8.0) containing 12.5 mM
10 MgC12, 12.5 mM CaC12, 200 mM NaCl, 1 mM EDTA and 200 mM
tromethamine-HCl. The treated DNA was extracted with phenol and precipitated with ethanol. A 1 jug aliquot was incubated with T4 DN~ polymerase in the presence of deoxynucleotide triphosphates to repair any single strand 15 extremities and was then digested with EcoRI endonucleaseO
This procedure produced DNA fragments of varying lengths due to the resection with Bal31. Each fragment had a fixed EcoRI extension at one end with the othe~ extremity being blunt ended and located at some distance from the original 20 HincII site and the OCT protein initiation codon.
Fragments of about 1480 bp containing the arg3 regulatory region were isolated by electrophoresis on a 7.5~
acrylamide gel followed by electroelution and ethanol precipitation. This cut-back reyulatory region is 25 preferred because it can promote transcription leading to synthesis of a protein devoid o~ OCT amino acid sequences, as described in Example 10, below.
In a second series of reactions, pMC200 was digested with XbaI and the 5' single strand extremities were filled 30 in or rendered blunt-ended, by incubation with T4 DNA
polymerase and deoxynucleotide triphosphates. This DNA was digested with EcoRI and a large EcoRI-T4/Xbal fragment of about 5700 bp, containing yeast D~A sequences located 3' to the XbaI site, plus pBR32~ sequences, was purified by 35 electrophoresis, electroelution and ethanol precipitation.
- 23 ~ lZ ~ 2 ~ ~ ~
This fragment was mixed with and ligated to the 1480 bp promotor-containing fragments.
The ligation mixture was used to transform competent cells of E. coli K12 strain MM294. Transformants were 5 selected on ampicillin agar~ Plasmid DNA was isolated from transformant colonies by the procedure described by Birnboim et al., Nucl. Acid. Res., ~olume 7, 1513, (1979) and were screened for presence of an XbaI site by digestion with XbaIO An XbaI site was present only in those plasmids 10 in which the filled-in XbaI site on a fragment from the second series of reactions was ligated to another fragment from the first series which terminated in a 3' T residue so as to restore an XbaI recognition sequence, TCTAGA, in a new location. A plasmid possessing an XbaI site was 15 identifed as pRIT10749. A flow sheet illustrating these manipulations is provided in Figure 6. E. coli K12 strain MM294 (pRIT10749) was deposited in accordance with EPC
regulations and the Budapest Treaty in the American Type Culture Collection, Rockville, Maryland, U.S.A~ on June 2, 20 1982 under accession number ATCC 39133.
A sample of pRIT10749 was digested with a combination of BstEII and XbaI~ The size of the deleted arg3 regulatory region was estimated to be about 210 bp by comparison with pMC200 which had been similarly digested 25 W7 th BstEII and XbaI and by reference to the known molecular weights of fragments of phage ~ x17~ D~A
described by Fuchs et al. Gene, Volume 4, 1, (1978).
To determine the DNA sequence of the BstEII-XbaI
fragment of pRIT10749, plasmid D~A was digested with 30 BstEII, labelled with ~- P-ATP by exchange kination and digested with BamHI endonuclease to liberate a 2000 bp fragment which was purified by electroelution and ethanol precipitation. D~A sequencing of the labelled fragment was performed according to the chemical modification method 35 described by Maxam et al., Methods in_En~ymology, Volume - 2~ 7 65, 499 (19~0). A part of this sequence was determined to be CCCATCAACTTGTACACTCGT _AGA. The underscored nucleotides were derived from the 5700 bp EcoRI-T4/Xbal fragment, Comparison to the appropriate region of the D~A sequence 5 shown in Figure 4 indicates that the restored XbaI site is located in the 5' untranslated leader region nine nucleotides upstream of the original HincII site in the pMC200 yeast DNA insert.
Example 10. Preparation of Plasmids, pRIT10759 and pRIT10761, Containing HBsAg and Regulatory Regions, by Combining pRIT10749_with pRIT10601 pRIT10601 was digested with HhaI endonuclease, and electorphoresed on a 7.5% acrylamide gel. A 1100 bp 15 fragment was recovered by electroelution and ethanol precipitation~ The DNA sequence of part of this fragment contains sequences corresponding to the known N-terminal amino acid sequence of HBsAg of ayw serotype as described by Peterson et al., Viral Hepatitis, G. No Vyas, S. N.
20 Cohen and R. Schmid, Eds., Franklin Institute Press, Philadelphia, UoS~A~ ~ 1978, p.569. The fragment contains 6 nucleotides located 5' to the HBsAg initiation codon, the complete HBsAg coding sequence and about 390 3' non-translated nucleotidesO
DNA of plasmid pRIT10749 was digested with XbaI and about 400 ng of this DNA was mixed with about 280 ng of the purified HhaI fragment. The mixture was incubated with 0.5 units of T4 DNA polymerase for 30 minutes at 37 in 20 ~1 buffer (pH 7.5) containing 20 mM tromethamine-HCl, 10 mM
30 M~C12, 1 mM dithiothreitol, and 33 mM each of dATP, dCTP, dGTP and dTTP. To this reaction mixture was added 2.5 Jul of 1 mM ATP, 2.5 ~1 of 10 mM EDTA and 2 ~ul (2 units) of T4 DNA ligase and the mixture was incubated for 16 h at 15 .
The ligated mixture was used to transform competent 35 cells of Eo coli K12 strain MM294 with selection being made - 25 - ~222~7 for ampicillin resistant transformants. Plasmid preparations were made from several of these transformants and analyzed by restriction endonuclease digestion and gel electrophoresis. The results showed that the blunt-ended 5 HBV fr~gment had been inserted in both possible orientations into the XbaI-digested pRIT10749. One such plasmid, pRIT10761 contained the 1100 bp insert in the correct orientation for transcription of the HBsAg sequence from the ~3 promotor and gave a 271 bp fragment after 10 sequential digestion with BstEII and XbaI. It is illustrated in Figure 7. A second plasmid, pRIT10759 contained the insert in the incorrect orientation and gave rise to an estimated 1175 bp fragment on digestion with a combination of BstEII and XbaI endcnucleases. The 15 nucleotide sequence at the arg3 regulatory region-HBsAg insert junction was determined for pRIT10761, by sequencing the 271 bp BstEII-XbaI fragment using the chemical modification methods of Maxam et al., Methods in Enzymologx, Volume 65, 499 (1980), after exchange kination 20 labelling of the XbaI terminus with y - P-ATPo The sequence determined for the ar~3 regulatory region - HBsAg junction is TACACTCGTCTACTGAACATGo It can be seen that the XbaI restriction site was not completely repaired by the T4 DNA polymerase and that one guanine residue was lost. The 25 ar~3 non-translated leader region is immediately followed by 6 nucleotides of HBV origin, CTGAAC, followed by the HBsAg initiation codon, ATG, and the coding sequence of the HBsAg structural geneO The HBsAg initiation codon is the first initiation codon encountered downstream of the ~3 30 promotor. Therefore, translation of the mRNA transcribed from the DNA will initiate at that codon and HBsAg synthesized will be devoid of extraneous amino acid residues. The HBsAg synthesized is not a fusion protein;
it is substantially structurally identical to authentic 35 HBsAg.
- 26 - ~2~2~7 Example 11. Preparation of Pl3~ IT10764 and pRIT10765 by Combinin~l~RIT10761 with YEpl3 HindIII~
pRIT10761 and pRIT10759 were separately digested with PstI and BamHI to destroy the pB~322 replicon moiety and S then with HindIII to release the DNA fragment carrying the inserted HBV DNA and the regulatory region. The DNA's were extracted with phenol, precipitated with ethanol and dissolved in bu~fer (pH 7.5) containing 0.01 M
tromethamine-HCl and 0.001 M EDTA. A 0.6 ~g portion of each 10 DNA preparation was se~arately mixed with 0.3 ~ug HindIII-digested, alkaline phosphatase treated DN~ of YEpl3 HindIII prepared as described above and the mixture was ligated.
The ligated mixture was used to transform competent 15 cells of E. coli K12 strain MM294 with selection being made for ampicillin resistant transformants~ One transformant was shown to contain a plasmid, pRIT10764, consisting of the HindIII fragment from pRIT10761 inserted on YEp 13HindIII. A further transformant was shown to contain a 20 plasmid, pRIT10765, consisting of the ~indIII fragment from pRIT10759 inserted on YEp 13HindIII~ A flow sheet illustrating the preparation of pRIT10761 and pRIT10764 is provided in Figure 7.
.
Exam le 12 - Transformation of Yeast with P
pRIT10764 and pRIT10765 pRIT10764 and pRIT10765 were isolated from cultures of the transformants prepared in Example 11 by CsCl-ethidium bromide density gradient centrifugation and 10Jug of each 30 was used to transform cells of yeast strain lc1697d as described in Example 7 with selection being made for leucine-independent transformants on regeneration agar.
One leucine-independent colony axising from these transformations is designated strain lc1697d(pRITl0764)o 35 Another transformant is designated S. cerevisiae strain - 27 - ~2~X7~
lc1697d(pRIT10765). Cultures of these strains were grown at 30 in Yeast Nitrogen Base medium supplemented with 2%
glucose and 20 jug/ml arginine to an optical density at 620 ~m of 0.80 to 0088. Cells were harvested and cell-free 5 extracts were prepared and assayed for HBsAg by the Ausria ~ radioimmunoassay as described in Example 7.
Extracts of lc1697d(pRIT10764) gave positive results in this assay at dilutions in PBS of up to 1/1024 whereas undiluted extracts of strain lc1697d(pRIT10765) were 10 uniformly negative in this assay. From the genetic and radiomimmunoassay evidence, it is concluded that cells of S, cerevisiae strain lc1697d(pRIT10764) synthesize HBsAg which can be used in a vaccine for stimulating protection against HBV infection in humans without serious side 15 effectsO
Claims (43)
1. A process for preparing a recombinant DNA molecule containing a nucleotide sequence which can code for HBsAg and which can be expressed in yeast which comprises fusing a nucleotide sequence which can code for HBsAg with a regulatory region derived from the yeast arg3 gene.
2. The process of claim 1 wherein the HBsAg sequence is fused in a position relative to the regulatory region such that the HBsAg synthesized by expression of the HBsAg sequence is devoid of extraneous amino acid residues.
3. The process of claim 1 in which the regulatory region is derived by digestion of pYEura3arg3 with a restriction endonuclease.
4. The process of claim 1 in which the regulatory region is derived from digestion of pYEura3arg3 with HindIII
restriction endonuclease to prepare a 3300 bp yeast DNA
fragment specifying the arg3 gene.
restriction endonuclease to prepare a 3300 bp yeast DNA
fragment specifying the arg3 gene.
5. The process of claim 1 which comprises inserting the HBsAg sequence and the regulatory region into a vector.
6. The process of claim 1 which comprises treating HBV DNA
with BamHI restriction endonuclease to prepare the HBsAg sequence.
with BamHI restriction endonuclease to prepare the HBsAg sequence.
7. The process of claim 5 which comprises inserting a BamHI fragment from HBV DNA extracted from Dane particles of adw serotype into the BglII site of a HindIII fragment of the regulatory region previously inserted into a vector.
8. The process of claim 5 which comprises inserting a HhaI
fragment derived from a fused pair of BamHI fragments of HBV DNA extracted from Dane particles of ayw serotype into the XbaI site of the vector which is a 1480 bp fragment of pMC200 containing the regulatory region and having an EcoRI
extension at one extremity and a terminating in a 3' T
residue at the other extremity which is blunt-ended, fused to a 5700 bp Xbal-EcoRI fragment of pMC200 after DNA
polymerase repair of the Xbal site in the 5700 bp fragment.
fragment derived from a fused pair of BamHI fragments of HBV DNA extracted from Dane particles of ayw serotype into the XbaI site of the vector which is a 1480 bp fragment of pMC200 containing the regulatory region and having an EcoRI
extension at one extremity and a terminating in a 3' T
residue at the other extremity which is blunt-ended, fused to a 5700 bp Xbal-EcoRI fragment of pMC200 after DNA
polymerase repair of the Xbal site in the 5700 bp fragment.
9. The process of claim 6 which comprises fusing a BamHI
fragment of HBV DNA extracted from Dane particles of adw serotype to the regulatory region.
fragment of HBV DNA extracted from Dane particles of adw serotype to the regulatory region.
10. The process of claim 6 which comprise fusing a HhaI
fragment derived from a fused pair of BamHI fragments of HBV DNA extracted from Dane particles of ayw serotype to the regulatory region.
fragment derived from a fused pair of BamHI fragments of HBV DNA extracted from Dane particles of ayw serotype to the regulatory region.
11. The process of claim 7 wherein the HindIII fragment is previously inserted into pBR322.
12. The process of claim 7 which further comprises releasing the HindIII fragment containing the HBsAg insert and the regulatory region by treatment with HindIII and inserting the HindIII fragment into a yeast vector.
13. The process of claim 8 which comprises treating the fused 1480 bp-5700 bp fragments with HindIII to excise a HindIII fragment carrying the HBsAg sequence and the regulatory region and inserting the HindIII fragment into a yeast vector.
14. The process of claim 9 which comprises inserting the BamHI fragment into the BglII site of the regulatory region.
15. The process of claim 10 which comprises fusing the HhaI fragment to the regulatory region at the Xbal site of a 1480 bp fragment of pMC200 containing the regulatory region and having an EcoRI extension at one extremity and terminating in a 3 ' T residue at the other extremity fused to a 5700 bp Xbal-EcoRI fragment of pMC200 after polymerase repair of the Xbal site in the 5700 bp fragment.
16. The process of claim 11 which further comprises releasing the HindIII fragment containing the HBsAg sequence and the regulatory region by treatment with HindIII and inserting the HindIII fragment into a yeast vector.
17. The process of claim 13 wherein the yeast vector is YEp13 which has been previously modified by excision of a HindIII fragment.
18. The process of claim 16 wherein the yeast vector is YEp13 which has been previously modified by excision of a HindIII fragment.
19. A process for preparing HBsAg which comprises inserting into yeast cells a recombinant DNA molecule comprising a nucleotide sequence which can code for HBsAg and a regulatory region derived from the yeast arg3 gene which can effect transcription of the HBsAg sequence in yeast, culturing the yeast and collecting the HBsAg which is produced.
20. A process for preparing HBsAg according to claim 19 wherein the HBsAg sequence is positioned relative to the regulatory region such that the HBsAg synthesized by expression of the HBsAg sequence is substantially devoid of extraneous amino acid residues.
21. A process for preparing HBsAg according to claim 19 wherein the HBsAg sequence is a BamHI fragment of HBV DNA
extracted from Dane particles of adw serotype and inserted in the BglII site of the yeast regulatory region.
extracted from Dane particles of adw serotype and inserted in the BglII site of the yeast regulatory region.
22. A process for preparing HBsAg according to claim 19 wherein the HBsAg sequence is a HhaI fragment derived from a fused pair of BamHI fragments of HBV DNA extracted from Dane particles of ayw serotype and inserted in the XbaI site of a molecule having a 1480 bp fragment of pMC200 containing the regulatory region and having an EcoRI extension at one extremity which is blunt-ended fused to a 5700 bp XbaI-EcoRI fragment of pMC200 after polymerase repair of the XbaI site in the 5700 bp fragment.
23. The process of claim 19 wherein the yeast is S.
cerevisiae strain DC5 or 1c1697d.
cerevisiae strain DC5 or 1c1697d.
24. The process of claim 20 wherein the yeast is S.
cerevisiae strain DC5 or 1c1697d.
cerevisiae strain DC5 or 1c1697d.
25. The process of claim 21 wherein the yeast is S. cere-visiae strain DC5 or 1c1697d.
26. The process of claim 21 wherein the arginine biosyn-thesis pathway is derepressed in the yeast.
27. The process of claim 22 wherein the arginine biosyn-thesis pathway is derepressed in the yeast.
28. The process of claim 22 wherein the yeast is S. cere-visiae strain 1c1697d.
29. A process for preparing a vector comprising the yeast regulatory region and a restriction site proximate thereto such that a protein synthesized by expression of a coding sequence inserted in the restriction site is -devoid of extraneous aminoacid residues, which comprises fusing the regulatory region, which is a 1480 bp fragment of pMC200 having an EcoRI extension at one extremity and ter-minating in a 3' T residue at the other extremity which is blunt-ended. to a 5700 bp Xbal-EcoRI fragment of pMC200 after polymerase repair of the Xbal site of the 5700 bp fragment.
30. A process for preparing an hepatitis B virus vaccine wherein HBsAg is prepared by a process according to any of claims 19 to 21.
31. A process for preparing an hepatitis B virus vaccine wherein HBsAg is prepared by the process of claim 22.
32. A process for preparing an hepatitis B virus vaccine wherein HBsAg is prepared by the process of claim 19 wherein the yeast is S. cerevisiae strain DC5 or Ic1697d.
33. A process for preparing an hepatitis B virus vaccine wherein HBsAg is prepared by a process according to claim 19 wherein the HBsAg sequence is positioned relative to the regulatory region such that the HBsAg synthesized by expression of the HBsAg sequence is substantially devoid of extraneous amino acid residues and wherein the yeast is S.
cerevisiae strain DC5 or Ic1697d.
cerevisiae strain DC5 or Ic1697d.
34. A process for preparing an hepatitis B virus vaccine wherein HBsAg is prepared by a process according to claim .
19 wherein the HBsAg sequence is a BamHI fragment of HBV
DNA extracted from Dane particles of adw serotype and inserted in the BgIII site of the yeast regulatory region and wherein the yeast is S. cerevisiae strain DC5 or 1c1697d.
19 wherein the HBsAg sequence is a BamHI fragment of HBV
DNA extracted from Dane particles of adw serotype and inserted in the BgIII site of the yeast regulatory region and wherein the yeast is S. cerevisiae strain DC5 or 1c1697d.
35. A process for preparing an hepatitis B virus vaccine wherein HBsAg is prepared by a process according to claim 19 wherein the HBsAg sequence is a BamHI fragment of HBV DNA extracted from Dane particle of adw serotype and inserted in the BgIII site of the yeast regulatory region and wherein the arginine biosynthesis pathway is derepres-sed in the yeast.
36. A process for preparing an hepatitis B virus vaccine wherein HBsAg is prepared by a process according to claim 19 wherein the HBsAg sequence is a HhaI fragment derived from a fused pair of BamHI fragments of HBV DNA extracted from Dane particles of ayw serotype and inserted in the XbaI site of a molecule having a 1480 bp fragment of pMC200 containing the regulatory region and having an EcoRI extension at one extremity and a terminating in a 3'T residue at the other extremity which is blunt-ended fused to a 5700 bp XbaI-EcoRI fragment of pMC200 after polymerase repair of the XbaI site in the 5700 bp fragment and wherein the arginine biosynthesis pathway is derepressed in the yeast.
37. The process of claim 36 wherein the yeast is S. cere-visiae strain 1c1697d.
38. The process of claim 29 which further comprises releasing a HindIII fragment containing the arg3 regulatory region by treatment with HindIII and inserting the HindIII fragment into pBR322.
39. The process of claim 29 which further comprises releasing a HindIII fragment containing the arg3 regulatory region by treatment with HindIII and inserting the HindIII fragment into a yeast vector.
40. The process of claim 38 which further comprises inserting a coding sequence at the restriction site proximate to the arg3 regulatory region.
41. The process of claim 40 which further comprises releasing the HindIII fragment containing the arg3 regulatory region and inserted coding sequence by treatment with HindIII and inserting the HindIII fragment into a yeast vector.
42. The process of claim 39 wherein the yeast vector is Yep13 which has been previously modified by excision of a HindIII fragment.
43. The process of claim 41 wherein the yeast vector is Yep13 which has been previously modified by excision of a HindIII fragment.
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US41578982A | 1982-09-08 | 1982-09-08 | |
US415,789 | 1982-09-08 |
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JP (5) | JPS5971694A (en) |
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JPS5936699A (en) * | 1982-08-20 | 1984-02-28 | Chemo Sero Therapeut Res Inst | Shuttle vector |
JPS5931799A (en) * | 1982-08-16 | 1984-02-20 | Science & Tech Agency | Recombinant plasmid and preparation of transformed yeast and hepatitis virus b surface antigen using the same |
WO1986001534A1 (en) * | 1984-09-04 | 1986-03-13 | Takeda Chemical Industries, Ltd. | Recombinant dna and its use |
WO1986003411A1 (en) * | 1984-12-12 | 1986-06-19 | Takeda Chemical Industries, Ltd. | Process for preparing novel protein |
WO1986007384A1 (en) * | 1985-06-03 | 1986-12-18 | Takeda Chemical Industries, Ltd. | Process for preparing hapatitis b virus surface antigen p31 |
US4683294A (en) * | 1985-04-03 | 1987-07-28 | Smith Kline Rit, S.A. | Process for the extraction and purification of proteins from culture media producing them |
DE3677851D1 (en) * | 1985-08-05 | 1991-04-11 | Merck & Co Inc | METHOD FOR INCREASING THE PRODUCTION OF RECOMBINANT PROTEIN IN YEARS OF THE GENERATION SACCHAROMYCES. |
US4895800A (en) * | 1985-11-26 | 1990-01-23 | Phillips Petroleum Company | Yeast production of hepatitis B surface antigen |
ZA88488B (en) * | 1987-01-30 | 1988-10-26 | Smith Kline Rit | Hepatitis b virus surface antigens and hybrid antigens containing them |
DE3883596T2 (en) * | 1987-03-09 | 1994-02-17 | Merck & Co Inc | Purification of recombined hepatitis B surface antigen. |
NZ229260A (en) * | 1988-06-03 | 1994-02-25 | Merck & Co Inc | Hepatitis b virus, expression cassette for pre-s domain, host cells and |
US4962656A (en) * | 1989-06-30 | 1990-10-16 | The United States Of America As Represented By The United States Department Of Energy | Control and monitoring method and system for electromagnetic forming process |
LT3988B (en) | 1992-02-17 | 1996-06-25 | Fermentas Biotech Inst | Recombinant plasmides pfs19, pfps2-48 and pjlfds1 codingsynthesis of human hepatite b of surfice virus antigenes, methods fof producing thereof |
TW340132B (en) * | 1994-10-20 | 1998-09-11 | Ibm | Structure for use as an electrical interconnection means and process for preparing the same |
JP4028850B2 (en) * | 2004-02-04 | 2007-12-26 | 株式会社名機製作所 | Mold for molding disk substrate |
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ES487106A0 (en) * | 1978-12-22 | 1981-05-16 | Biogen Nv | A METHOD FOR PRODUCING AT LEAST ONE POLYPEPTIDE SHOWING HBV ANTIGENICITY |
NZ199722A (en) * | 1981-02-25 | 1985-12-13 | Genentech Inc | Dna transfer vector for expression of exogenous polypeptide in yeast;transformed yeast strain |
GR76274B (en) * | 1981-08-04 | 1984-08-04 | Univ California | |
NZ201705A (en) * | 1981-08-31 | 1986-03-14 | Genentech Inc | Recombinant dna method for production of hepatitis b surface antigen in yeast |
GB2125047B (en) * | 1982-08-09 | 1986-02-19 | Ciba Geigy Ag | Yeast hybrid vectors and their use for the production of polypeptides |
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