CA1203763A - Hydrid protein coded by a dna fragment - Google Patents

Abstract

The invention relates to a hybrid protein capable of being encoded by the DNA fragment inserted into the vector.

Description

r) 376 ~
i "NUCLEOTIDE SEQUENCE ENCODING THE SURFACE ANTIGEN OF
HEPATITIS B VIRUS, VECTOR CONTAINING SAID SEQUENCE
NUCLEOTITIC, PROCESS FOR PROVIDING SAME AND ANTIGEN
GOT "

The invention relates to a nucleic acid comprising a nucleotide sequence capable of encoding a immunogenic peptide sequence corresponding to the antigen viral hepatitis B virus, and poly-5 peptides and peptides obtained.
It also relates to a method allowing to obtain such a nucleic acid.
Hepatitis B is a common viral disease especially in tropical Africa, in Asia of 10 South-East and the Far East.
The etiological agent is a virus (HBV) or par-Dane's ticule, comprising an envelope (antigen Australia or HBs antigen), a capsid (HBc antigen), an endogenous polymerase and a circular DNA molecule 15 partially single-stranded: the longest strand of this DNA molecule contains nearly 3,200 nucleotides (SUMMERS
J., O'CONNELL A. and MILLMAN I. (1975 ~ Proc. Nat. Acad.
Sci. USA 7 ~, 4,597-4,601).
Endogenous DNA polymerase can be used 20 to repair the shortest strand in vitro (TA LANDERS, HB GREENBERT and J. S ~ ROBINSON, J. VIROL., 23, 1977, p. 368-376).
The electrophoretic analysis of proteins the envelope showed the presence of 2 to 7 polypeptides 25 of which the main ones are called: polyp ~ ptide I and poly-peptide II (PETERSON DL ~ ROBERTS IM and VYAS GN
(1977) Pr ~. Nat ~ Acad. Sci., USA, 74, 1530 ~ 1534, and PETERSON DL, CHIEN DY, VYAS GN, NITECKI D. and BOND H. (1978) In Viral Hepatitis, ed. G. VYAS, S. COHEN
30 and R. SCHMID, The Franklin Institute Press, Philadelphia, 56g-573).

~. ~

~ 21a 3 ~ 63 Polypeptide I has a molecular weight of 22,000 at 26,000 daltons. Polypeptide II is glycosilated and has a molecular weight from 28,000 to 30,000 daltons. The composition amino acids of these two polypeptides is very S similar, The sequences that form, respectively, their first 15 amino acids (from the ex-N-terminal) and their last 3 amino acids are identical, so the assumption was ~ or-mule that polypeptide II might not differ of polypeptide I only by glycosilation.
The study of the virus is extremely dif-difficult since no system is available cell culture allowing the spread of the virus.
This difficulty has already been partially overcome, more particularly with regard to the ayw serotype.
~ The entire DNA (~ enome) of the virus has been identified and cloned, nota ~ ment in E. coli, after its prior insertion-ble in the unique EcoRI site of a vector ~ gt.WES. ~ B, depending on the technique by FRITSCH A., POURCEL C., CHARNAY P. and TIOLLAIS P. (1978) CR Acad. de Paris, 287, 1 4S3-1 456).
At this ~ our, the sequence of polypeptides I
and II themselves, the localization in the viral DNA of the sequence encoding these peptides have not been performed.
The object of the invention is to obtain a sequence 25 this much smaller DNA than the viral DNA itself, containing the sequence capable of encoding the peptide sequence endowed with immunogenic properties allowing, when is introduced into the body of a living host, duire the latter's manufacture of antibodies susceptible 30 wounds to protect later this same host with regard to viral hepatitis B virus, especially when it this is in a virulent state.
The invention follows not only from the analysis complete nucleotide of the Dane particle genome ~ L ~ d ~ a 6 ~

inventors did, but the idea that they had to identify the coding gene (hereinafter referred to as "S gene") of the above polypeptides, to search in the complete pre-established nucleotide structure of the ge-name of the Dane particle, those of the sequences of nu-cleotides capable of encoding peptide sequences known proximal and terminal of these polypeptides.
It will be recalled that PETERSON and collaborators have reported, especially in the articles whose references rences were recalled more ~ aut, than the proxi-male (first N-terminal amino acid) of the first 15 amino acids can in principle be analyzed as follows:
met glu asn ile thr ser gly phe leu gly pro leu leu val ser and that the terminal sequence of these same polypeptides (last C-terminal amino acid) was:
val tyr ile Figure 1 is a schematic map of the genome of Dane's particle ~ This one has two ~ rins b1 and b2; the shortest of them (~ 2) being normally devoid of the part represented by a line entered interrupted in the drawingO
It is known that this DNA contains only one EcoRI site.
The arrow f1 gives the direction of the numbering nucleos ~ ides of which the longest strand b1 is composed, and the arrow f2 gives the direction of DNA transcription of the virus, in particular by cellular machinery of those Hepatitis B virus invaded . concerns the expression of the S gene.
The EcoRI site can therefore be numbered 0 or, as has now been determined from a more exact ~
for that of the hepatitis B viruses belonging to the sero-ayw type, 3 182.
The circle in full interior line gives the scale in% of DNA length and makes it possible to specify the 12 ~ 3763 positions of some of its parts.
The numbers 3 ', 5' and 5 ', 3' at the ine of the card are aimed at the terminal ends turers of the same numbers in the agreed representations the ends of the nucleic acid chains.
According to the invention, it has been shown that the "S gene" essentially constituted the fra ~ ment of the strand the longest b1 located between positions 73.6 and 95.1 from the schematic map of figure 1. Abbreviations "Start" and "Stop" represent the initiation points and stopping the transcription of the "S gene".
The ~ igure 2 is representative of the part terminal of the above genome, in particular between the positions 60.4 and 100 ~ in% of DNA length). Each of the letters in figure 2 correspond so conventional to one of the 4 basic DNA nucleotides:
A: Adenine G: Guanine T: Thymine C: Cytosine The lower lines, in each pair of li-genes of which figure 2 is made up, correspond to the nucleic acid corresponding to the nucleotide chain b ~.
The analytical technique used to establish the more detailed map as shown in figure 2, will be brief vely recalled below.
Characterization of the nucleotide sequence of the "S gene" as proposed in the context of the present invention, and whose proximal ends p "S" and terminal t "S" are indicated in FIG. 2, results from the observation that:
- the first 14 triplets (in the direction of the reading f2) from the nucleotide numbered 3,030 opposite EcoRI terminal end screws, are respectively 3 ~ 3 able to encode the 14 no-acid priers of the proxi- sequence male of the 15 amino acid prenuers of the above poIypeptides, - the last 4 GTA TAC ATT TAA triplets read on the complementary chain b2 to the transcribed chain bl correspond respectively to the 3 terminal amino acids the above polypeptides and a stop codon:
- this nucleotide sequence (678 nucleotides) does not include a stop codon, at least when adopts the reading framework implying that the first tri-plet "read" on DNA by cellular machinery, ie AUG, (corresponding on the complementary strand to ATG), - the complete translation of the general information tick starting with the initial ATG codon leads to a theoretical polypeptide of 226 amino acids, having a weight molecular strength of 25,422 daltons.
The nucleotide structure of the "S gene" as well as the polypeptide chain resulting from the translation of the "S gene" are shown in Figure 3.
These values are fully consistent with the data.
analytical results which result from electropho-of the polypeptide I on polyacrylamide gels which have already been described by the previous authors (re-References 9-12 according to the bibliography at the end of the description of this patent application).
The difference observed at the level of the 15th amino-acid of the proximal peptide sequence of polypeptide I:
leucine according to the maps of Figures 2 and 3 mentioned above, and not serine according to the above findings so-called authors, can perhaps be attributed to the fact that these authors worked with a different genetic variant rent from that which was the subject of this study. We note that the difference can also be related to substitution of a single nucleotide in the triplet con-identified "rrTA" in the particular "S gene" represented in the cards of Figures 2 and 3, instead of "TCA", one of the ~ ~ 3 triples likely to be translated into serine.
The invention therefore relates more particularly nucleic acid fragments that can be excised of DNA from the Dane particle, these fragments being more particularly characterized in that they contain the part of the "S ~ gene capable of encoding the part of the envelope protein of the virus which is responsible immunological properties of the hepatitis B virus As such, the invention therefore relates to an acid nucleic acid comprising at most of the order of 1000-1 100 nu-cleotids, more particularly characterized in that it is able to encode an immunogenic peptide sequence, it-even able to induce the production of antibodies in vivo hepatitis B virus, this sequence peptide containing essentially the structure represented seen in FIG. 3, or any peptide sequence having equivalent immunogenic properties.
The invention also relates to a vector in view of the expression of said nucleotide sequence in a microorganism or in eukaryotic cells to con-dition that the genetic fusion was carried out in con-serving the reading phase of the "S 'gene ~.
The nucleotide sequences used conform my to the invention present in relation to each other variability leading, when expressed, to formation of determinants varying according to the subtype of hepatitis B virus (subtypes d, w, y, r of group a).
For one of the peptide sequences represented tées in Figure 3, it will be observed that the first amino acid born from the above sequence O methionine, is N-terminal and that the amino acid of the opposite end: isoleucine, is C-terminal ~
The invention also relates, more particularly-ment, the nucleotide sequence shown in Figure 4 encoding the peptide sequence as it results from the ~% ~ 3 ~ 6; ~

Figure 5 or any analogous peptide sequence endowed with equivalent immunogenic properties.
It goes without saying that by "equid peptide sequence valente ", mentioned above, you must hear any peptide sequence in which certain parts may not be strictly identical to the corresponding parts ~ of the peptide sequence represented in the Figures 3 and 5, these variations possibly being due to local tations not affecting immunogenicity general protein or structural changes due to the different serotypes under which the proteins of the genre in question are likely to arise (notably serotypes adw, adr and a ~ r).
The invention relates more particularly to nucleotide sequence containing the peptide sequence as shown in Figure 6 or any se-analogue peptide sequence with immunogenic properties equivalent.
The invention relates more particularly still the following peptide sequences:
Alanine ~ lutamine ~ lycine-Threonine-S ~ kinP
Threonine-Alanine ~ Glutamine-Glycine-Threonine-Se'rine Threonine-Threonine-Alanine-Glutamine-Glycine-Threonine-Serine In the first peptide mentioned above, the end alanine is N-terminal and the serine end is C-tenminal.
In the second and third peptides above-indi ~ ués, the threonine end is N-terminal and the extreme serine mite is C-terminal.
By way of example, one can in particular prepare the pentapeptide starting from the C-terminal serine to which we hang the threonine by the method of Castro described in T ~ trahédron Letters, 1975, n14, page 1219-1222. Then the aci-amines glycine, glutamine, alanine are added by the method called repeated mixed anhydride (rema method) described by Beierman in Chemistry and ~ iology oE Peptides, E ~. J. ~ eienhofer, Ann. Arbor 37 ~ 3 Science Publ., Ann. Arb. Mich. 351 (1972).
The invention also relates to the products both of the fixation of the pentapeptide on a carrier molecule getting larger, especially of the polypeptide or protein type, the compositions containing this pentapeptide in fixing products, no-tamr ~ nt in combination with a pharmaceutically acceptable vehicle, and more particularly vaccines against hepatitis B. These vehicles pharmaceuticals are appropriate, in a conventional manner, to the mcde of ad-chosen administration, no oral, parenteral, rectal or by nebulization on mucous membranes, especially nasal.
The hexapeptide and the polypeptide has 7 amino acids can be synthesized by synthesis techniques classic peptide.
According to the present invention, these peptides are held to be the anti ~ enic site of the polypeptides of larger size which question higher and responsible the immunizing power of the viral envelope (Journal of Biol. Stand. 1976, 4, 295-304, RAO and VYAS "Biochemical Characterization of Hepatitis B Surface Anti ~ en in Relation to Serologic Activity ").
The invention also relates ~ t still the frag-DNA elements likely to encode: The production of such pentapeptide, hexapeptide and polypeptide with 7 amino acids.
It's about :
- for pentapeptide, in particular polynucleo-formula formula:
5 'GCT CAA GGA ACC TCT 3' 3 'GGA GTT CCT TGG AGA 5' - for the hexapeptide, in particular polynucleoti-of formula:
5 'ACT GCT CAA GGA ACC TCT 3' 3 'TGA CGA GTT CCT TGG AGA 5' - for the polypeptide with 7 amino acids of the poly-nucleotide of formula:
5 'ACT ACT GCT CAA GGA ACC TCT 3' 76 ~

3 'TGA TGA CGA GTT CCT TGG AGA 5' or in each of the three cases, complementary polynucleotide silent relating to the three respective polynucleotides preceding teeth or any polynucleotide in which each triplets can be replaced by any similar triplet able to code the production of the same amino acid.
T, I nucleic acid according to the invention can also be characterized in that it comprises at least one of two strands mutually complementary to a sequence DNA as shown in Figure 4 t in which the numbers corresponding to the posi-tions of the first nucleotides of each of the suc-stops of 10 nucleotides shown opposite the po-Eco ~ I sition not shown in the figure: it goes from these numbers do not occur at the level of the - CaraCtériSatiQn of the nucleotide sequence of the genus in question). This DNA fragment is delimited by two sites HincII.
It will be appreciated that this nucleotide sequence corresponds to genetic information whose translation leads to the peptide sequence shown in FIG. 5.
The invention naturally relates to the sequences single-strand or double-strand equivalent nucleotides, including the strand having the structure which follows from ~ 5 the succession of the lower lines of figure 4, DNA
with corresponding double strand, or the corresponding messenger RNAs layering, in particular that represented by the sequences complementary to nucleotides formed by the lines lower of the pairs of lines of figure 4 (direction of arrow f2).
Likewise, the scope of the invention nucleotide chains which differ from the previous ones tes by certain triples O ~ l small sequences of triplets, insofar as these nucleotide sequences remain able to encode a polypeptide conserving the activities ~ Z03 ~ 6; ~

immunogens characteristic of hepatitis vi viruses rale B. Generally speaking, these are nu-cleotides which, if necessary, after denaturation of DNA
double stranded to produce single nucleic acids corresponding strand, remain susceptible to hybridize on at least about 90 X of their length with one of the strands of the DNA of FIG. 4.
Preferred nucleic acids according to the invention are still the ones who can be circumcised from DNA from viral hepatitis and ~ ui, when they are double ble strand, are characterized by the existence of one of their ends of a HincII, HhaI, AvaI or EcoRI end and at their other end by an AvaIII, HincII or HhaI.
The positions of these various ends with respect to EcoRT site screws are shown schematically in ~ igure 2.
The nucleic acid according to the invention is intended incorporation into a vector allowing its expression-sion in bacteria and in eukaryotic cells, especially for the production of a protein or a peptide capable of inducing in the organism of a host living producing antibodies active against the virus viral hepatitis B. The resulting protein or peptide both of the translation of the nucleotide sequence according to '-the invention can be used as a vaccine agent or as an agent for diagnosis.
The nucleic acid according to the invention can also be used as a probe to detect the presence or not in blood or test serum samples, Dane particle, HBs antigen or fragments of this one, etc. (by classical DNA hybridization technique-DNA).
Other characteristics of the invention result from will be further from the following brief description of the techniques analysis, identification and obtaining ques DNA fragments according to the invention. Reference seranaturally made to the drawings whose figures have d ~
have been taken into account in the above. The figures fres or numbers in parentheses correspond to references references of the bibliography annexed to this ~ descrip-tion.
`The invention also relates to particular vectors linkers allowing expression of nucleotide sequences described above, especially in the form of a protein hybrid in which a protein eragment presenting the immunological characteristics of HBsA ~ adjoins a molecule carrier conferring on all immunogenic properties or immunoreactive, likely to induce production protective antibodies against viral infection in the host organism in which this protein has previously been introduced.
In particular, the invention relates to a vector -phage or plasmid - containing at least part of the o peron lactose, more particularly the promoter and the Z gene of this operon, this vector being characterized in that that it is modified for insertion, in phase, into a site appropriate for the Z gene, such as the ~ coRI site of any one conquers DNA fragments from the main patent, including those containing most of the "S gene". She con-also identifies those of these modified vectors, in the-which at least part of the DNA DNA coding for most of the ~ -galactosidase would be replaced ; created by a DNA fragment able to code for any other non-immunogenic carrier molecule, or whose possible immunological properties, if these exist, do not do not work with those of the peptide part presenting the immunological properties of HBsAg, for example essential that which extends in the direction of the read ture from its HhaI site ~
The invention is also more particularly ~ L2 ~ 3763 relating to a hybrid protein characterized in that it contains a polypeptide sequence having the properties HBsAg-specific immunological tees, adjoining a polypeptide sequence consisting of the major part of ~ -galactosidase, which ~ plays the role of carrier protein.
The invention does not extend only to this mo-the particular hybrid school, whose essential role is to constitute a model of a protein constructed according to the techniques of genetic engineering and endowed with properties immunogenic and immunoreactive characteristics of the anti HBsAg gene, but also to any other hybrid protein in which all or part of 1 ~ part ~ -galactosidase can be replaced by any other non-carrier molecule in ~ unogenic, or whose possible immunological properties that, if these exist, do not interfere with those of the peptide part having the immuno-HBsA ~ logic.
~ 'other features of the invention appear-will arise again during the description of pre-fairies, in combination with the drawings in which:
- the figures la and lh illustrate schematically -the successive stages in the production of a vector of the plasmid type incorporating a fragment of HBV VNA, FIGS. 2a to 2c schematically illustrate the initial structures of the vector used (Figure 2a) final of the modified vector obtained (Figure 2b) and that of the hybrid protein resulting from the expression of this vector altered in E. coli (Figure 2c).
A ~ SE ~ NUCLEOTIDIOUES UENCES
Products and methods used - The enæymes and the chemicals used ________ ____ _____________ ______________ _ Restriction enzymes used: BamHI, HhaI, HincII, HaeIII, XbaI, MboI, HinfI, HpaII, XhoI, are those manufactured by BIOLABS. We used DNA-polymer shave I from BOEHRINGER. Bacterial alkaline phosphatase 3 ~ 6 ~

and the polynucleotide kinase were provided by PL BIOCHEMICALS. The chemical agents were the following before:
. Dimethyl sulfate (ALDRICH), . Hydrazine (EASTMAN KODAK), Acrylamide and bis-acrylamide (2 times crystal-read - SERVA), . Dideoxy nucleotide triphosphates and deoxynucleotide triphosphates ~ PL
~ IOCHEMICALS), . IMERCK3 piperidine redistilled under vacuum.
- Preparation of HBV DNA
_ _ _ _ _ _ _ _. _ _ _ _ _ _ _ _ _ _ _ _ The entire HBV genome ~ subtype aYw) has been clo-born in E. coli by bringing into play the unique site of ECoRI triction of the vector ~ gt.WES. ~ B (14). The cloned DNA is hereinafter called "Eco HBV DNA".
The recombinant bacteriophage was amplified by petri dish on Agar and extracted the DNA
in a manner known per se ~ After digestion of DNA by the EcoRI restriction zyme, the Eco sequence was purified H ~ V DNA by ultracentrifugation, in a gradient of su-crose, using the technique described in the bi-bliographic (16, 17).
- Preparation of fragment: s of DNA labeled 5.32 p ____________________________________________ 10 to 20 picomoles of Eco HBV DNA have been completed hydrolyzed by the various restriction enzymes tion, under the conditions recommended by the ~ abricant.
The DNA fragments have been dephosphorylated by phos-alkaline phatase, which was then inactivated by alkaline treatment. DNA was then precipitated with ethanol, according to the technique described in the article (18). After redissolution in a sper-based buffer midin, DNAs have been tagged at their 5 'ends with an ATP {~ 32p} (3,000 Ci / mM manufactured by NEW ENGLAND
NUCLEAR) and with polynucleotide-Xinase (according to ~ v ~ ~ ~

technique referred to in article (19).
The restriction DNA fragments were separated res by electrophoresis on polyacrylamide gel, then eluted. The marked ends have been segregated gatio ~ by electrophoresis on polyacrylamide gel defaçon known per se, after restriction with another en-zyme or by denaturing DNA fragments of the genus in question.
- Determination of the structure of the sequences ___________________________________________ of nucleotide DNA
_____________________ The primary structure of the DNA fragments â dou-wheat strand or single strand was determined essentially according to the technique described by MAXAM and GILBERT (19).
The terrestrial inhibitor method has also been used.
chalnes minerals described by SANGER et al. (20) and adapt-ted by MAAT and SMITH t21), with regard to fragments with double strand marked at one of their 5 'ends.
Chemical and enzymatic reaction products that were analyzed by electrophoresis in gels of acrylamide sequence at 8.16 or 25 X, 1 mm thick.
Techniques and results of the analysis In order to determine if the HBV genome is capa-ble to encode polypeptides I and II, all fragments HaeIII (HaeIII restriction sites of the HBV genome represented felt in Figure l by small arrows) have been marked at their 5 'ends. Substantial parts of their primary structures were determined by the MAXAM and GILBERT method. Nucleotide sequences likely to encode proxi- amino acid sequences males and terminals of polypeptides I and II have been lo-tagged in the HaeIII E and HaeIII F fragments, previously located on the genome restriction map H ~ V (according to the technique described in reference (17) o It is these nucleotide sequences that have been considered as consisting of the ends of the "S gene"

15 themselves occupying positions 73.6 and 95.1 opposite vis the EcoRI restriction site tfigure 1) for reasons already given.
The nucleotide sequence between these two posi-tions has been analyzed using chi-known mics, in particular by the method of degradation hydrazine dimethyl sulfate chemical and the method of chain endings. Among the reactions, various chemical options offered by MAX ~ M and GILBERT to partial depurination with formic acid and piperidine methods, which give bands of equal intensity on autoradiograms for frag-with a guanine and an adenine. We also The reactions to hydrazine followed by a piperidine cleavage, to obtain equal bands intensity, for cytosine and thymidine nucleotides:
the electrophoretic fractionation of the products of these two reactions gives a stain in all bases either of the gel columns used. This procedure facilitates the reading of the autoradiogram of the gel.
The reaction to hydrazine in the presence of sodium chloride dium specific for cytosine makes it possible to distinguish this thymidine nucleotide and the reaction to dimethyl sulfate followed by cleavage by specific piperidine of guanine makes it possible to distinguish this latter nucleoti-adenine.
In order to ensure the greatest possible If desired, separate nucleotide sequences forming different mutually overlapping fragments-have been produced by hydrolysis of Eco HBV DNA by various restriction enzymes:
BamHi, HinfI, HpaII, HaeIII and HinclI.
In this way the analysis of each of the sites of restriction used as prime starting points fragments studied was confirmed by analysis of ~ 2 ~ 3 ~ 63 separate fragments in which restriction sites the first fragments were between new ends of these distinct fragments.
The "S gene" shown in Figure 3, which includes menced by the initiation codon ATG, comprises 227 triplets, including a TAA stop codon. The 3 codons corresponding to 3 amino acids from the carboxy terminus of the poly-corresponding peptide are located in the same frame of reading, immediately before the TAA stop codon. Mon of the other two reading frames (respectively offset vis-à-vis the previous 1 and 2 nucleotides) is equal-lacks a stop codon, but encodes a protein quite different from polypeptidesI and II above-mentioned. The third reading frame includes 10 co-stop donations (5 TAG, 4 TGA, 1 ~ AA). On the other strand 15 of DNA, the three reading frames are found respectively-closed by 11, 11, and 6 c ~ stop donations distributed along the DNA sequence.
As already indicated above, the translation complete genetic information starting with the ATG initiation codon leads to a theoretical polypeptide than 226 amino acids corresponding to a molecular weight area of 25,422 daltonsO
It is interesting to note that the sequence nucleotide corresponding to the "S gene" should normal-should be read entirely during translation.
Are also parts of the invention of the chai-nes of nucleotides of the type of the "S ~ gene" described above, which has small additional sequences that can contain up to a hundred nucleotides or which otherwise can be without it, without being altered the corresponding genetic information (22, 23).
The various fragments of the invention which have been defined above can be obtained from the DNA sequence called Eco HBV DNA, using 6 ;; ~ `

corresponding restriction enzymes and techniques known fractionation of DNA fragments, in particular on polyacrylamide gel and using their migrations over distances which are a function of their molecular weights. So we can for example get the fragment, one end of which is delimited by one EcoRI site and the other by an AvaIII site by operating Eco HBV DNA restriction by the enzyme AvaIII, the frag-ment sought consisting of the smallest fragment obtained (a single AvaIII site in Eco HBV DNA).
The fragment delimited by opposite ends EcoRI and HhaI is obtained by hydrolysis of Eco HBV DNA by EcoRI first, then by partial hydrolysis by the enzyme HhaI restriction. We then recover among the pro-restriction products that which contains the AvaIII site.
These restriction techniques have obviously only been proposed as an example, it being understood that the specialist is able to determine the orders restriction enzyme treatment to isolate, from Eco ~ BV DNA in particular, the fragments having the useful restriction ends.
For all intents and purposes, it will be recalled that these restriction operations can be performed within 10 mM Tris buffer; pH 7.8; 6 mM MgC12; ~ -mercapto-6 mM ethanol, the same medium additionally containing and pre-50 mM NaCl ference when EcoRI is used.
As has already been said, the invention relates to the use of the DNA fragments described as sound-allowing the diagnosis of the presence in a serum Dane particles or particles derived from the pre-cedent, carrying a DNA capable of coding a pro-immunogenic tein characteristic of hepatitis B.
The DNA according to the invention can also be in-embodied in a vector allowing, provided that the incorporation poration was carried out in phase, the expression of this ~ 3 ~ 63 18 DNA in a bacteria or other microorganism, or in eukaryotic cells.
B - VECTORS CONTAINING ~ NE NUCLEOTIDIOUS SEQUENCE OF
THE HBs ANTIGEN
Construction of a recombinant bacteriophaqe ~ lac HBs-1 Products at the different stages of this construction are referred to in Figures la and lh.
They are also targeted by the numbers la to lh.
The positions of the 10 "S gene" and certain restriction enzyme sites.
After treatment of DNA ~ HBV with the enzyme HhaI restriction, we separate a DNA fragment (lb) from ~
with 1,084 base pairs, and more specifically the entire "S gene", by agarose gel electrophoresis 15 and electro-elution (Figure lb). We prepare, from this sub-fragment, previously treated with endonuclease Sl, a ~ ous-fragment ~ lc) ~ figure lc), resulting from the al-along the sub-fragment ~ lb) at its ends, by DNA elements called "EcoRI linXers" from ~ ormule:
5 'GGAATTCC
CCTTAAGG 3 ' The fragment obtained is, after formation of the EcoRI cohesive ends, cloned into the plasmid pBR322.
The plasmid obtained hereinafter called pBRHBs (fig.
25 gure ld), contains only one XbaI restriction site located near the head of the "bene S".
By digestion of the recombinant plasmid pBRHBs with a mixture of EcoRI and XbaI enzymes a DNA fragment comprising approximately 980 pairs 30 bases and containing most of the "S gene"
(figure le). This fragment is separated and purified by elec-trophoresis on an agarose gel. The fragment obtained is again treated with endonuclease Sl, then again provided with EcoRI ends by means of the above "EcoRI
35 linkers'l, then subjected to endonuclease treatment ~ 219: ~ 76 ~

EcoRI to reform the corresponding cohesive ends dantes ~ The fragment of figure le which includes approximately 980 base pairs are then inserted by in vitro fusion in the EcoRI site of the plasmid pBR322, to form the plasmid pXbaHBs (Figure lf). This plasmid is cloned with as usual, like the plasmid pBR322.
Several clones were obtained.
It is extracted and purified, after treatment with EcoRI of the DNAs of three of these clones, pXbaHBs-1, pXbaHBs-2 pXbaHBs-3 (figure lg), the fragments called below - "HBs fragments" tfigure lh).
The nucleotide sequences of the ends of above fragments ~ normally obtained inside the "S gene") are determined using the procedure described by MAXAM and GILBERT (Proc. Nat. Acad. Sci. USA
79, 560-564 (1977). These determinations revealed that the terminal end nucleotide sequences, cor-corresponding to the "S gene" were not identical in the three clones (figure lg), the differences are really probably due to heterogeneities produced during digestion by the endonuclease S1.
The two fragments from pYbaHBs-1 and p ~ baBHs-2 are inserted by in vitro fusion into the genome bacteriophage ~ plac 5-1 (21), which has only one EcbRI site located near the end of the lac Z gene.
Because of the lac Z gene reading frame, as it can be deduced from the amino acid sequence of the ~ -galactosidase (23), we see -and experience will confirm that the insertion of the HBs fragment of pXbaHBs-1 in the EcoRI site of the lac Z gene of ~ plac 5-1 must lead re the conservation of the adequate reading phase of the "S gene". On the contrary, the insertion of the HBs fragment of pXbaHBs-2 should turn out not to be able to be inserted in the previous vector with conservation of the frame of 3S appropriate reading. It was nevertheless used as witness in later experiences.
These operations were carried out with courses in known techniques. In particular the frag-pXbaHBs-1 HBs "elements, pXbaHBs-2 were inserted using 5 of a ligase in the DNA of ~ plac 5-1 which had previously lable was cleaved by EcoRI. Mixtures of fragments of DNA obtained was then used to transfect the E. coli strain C600RecBC rk mk. Clones of bacteria riophage become lac ~ due to the insertion of fragments 10 HBs in the EcoRI sites of the lacZ gene are amplified and purified according to the method described in (21).
The DNA of the various haceriophages has been extracts and orientations of inserted DNA fragments determined by electrophoretic analysis of their frag-Restriction elements 9amHI. We can thus determine that two lines called ~ lac ~ Bs-l and ~ lacHBs-2 corres-laying on the plasmid pXbaHBs-1 and pXbaHBs-2 contained a HBs fragment correctly oriented.
Figure 2a is a schematic map of the vector 20 teur ~ plac 5-1 before its modification by the HBs-1 fragment, from pXbaHBs-1.
Figure 2c is a schematic map of a part of this same vector showing: the intro-picks up its Z gene by insertion into its EcoRI site of 2S abovementioned HBs-1.
Figure 2c schematically shows the structures of the po-The polypeptide hybrid obtained as a result of the expression of the modified vector of Figure 2b.
The expression was obtained by transfecti ~ n of a 30 strain of E. cGli bacteria, including a strain Hfr ~ lacX74.
E. coli strains, including a strain of E. coli Hfr ~ lacX74 were transformed by plac 5-1, ~ lacHBs-1 and ~ lacHBs-2 respectively. After cultivation 35 cells are lysed and the lysates obtained analyzed by 3 ~

SDS polyacrylamide gel electrophoresis (24 ~ and proteins are detected by staining with coomas blue sie. We see the presence of a more intense band among the expression products of ~ plac 5 1 at the level of the 5 sition corresponding, for a witness, to that of the ~ -galactosidase (molecular weight 116 24B) and a ban-of distinct among the expression products of ~ lacHBs-1 ~ not present among the expression products of ~ lacHBs-2) corresponding to a new protein having a molecular weight 10 cular in the range of 135,000-141,000.
Proteins synthesized by bacteria transfected ~ ant by ~ lacHBs-1 than by ~ ac 5-1 are marked by the (355) methi ~ nine. The bringing together of these proteins with an anti-HBsAg serum and making a 15 autoradiogram of SDS polyacrylamide gel reveals the presence among expression products of only ~ lacHBs-1 a band to which there is no equivalent band slow among the expression products of other vectors.
This band disappears specifically when the immuno-20 precipitation is carried out in the presence of unlabelled H ~ sAg. The same band is still observed among the products of ex-pressure ~ lacHBs-1, when immunoprecipitation is re-edged with an antiserum with respect to ~ -galactosidase.
The presumed structure of the protein part 25 hybrid obtained, at the level of the fusion between the lacZ gene and the HBs-1 fragment results from FIG. 2c which appears raitre the fragment "~ -gal", corresponding to the ~ -galactosi-dase (1,005 amino acids), the HBsAg fragment (lg2 acids amino), these fragments being separated by an amino acid 30 prolyne, corresponding to a part of "EcoRI linker"
held in the ~ ector ~ lacHBs-l.
C - METHOD FOR MANUFACTURING AN IMMUNOGENEMETTING MOLECULE

~ L ~ ~ 6 ~

IN OPERATING THE VECTOR ACCORDING TO THE INVENTION
The inventiGn can therefore allow the low molecular weight protein production than the aforementioned polypeptides I or II, endowed with the same 5 immunogenic properties.
The results demonstrate that E. coli, or all other suitable microorganism, such as bacteria or a eukaryotic cell culture, can be infected with the ~ lacHBs-l and synthesize a protein having a weight 10 molecular of the order of 138,000 and having deter- - ~
antigenic minants of both HBsAg and ~ -galac-tosidase. This molecule is representative of polypep-hybrid tides which can be obtained by the process according to the invention, in which HBsAg is linked to a pro-15 support tine (resulting from partial substitution or of the ~ -galactosidase ~ fragment, these hybrids have however, the antigenic properties of HBsAg. These new molecules are useful for the production of vac-cins active against viral hepatitis B.
As it goes without saying, and as it results from their already from the above, the invention is not limited in no way to those of its modes of application and implementation which have been more specifically envisaged, it brews, on the contrary, all variants.
Annexed to this description is the library spelling, and in particular the references which have been tees in the context of this description.

~ 3763 REFER ~ NCES
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Claims (3)

The embodiments of the invention about which an exclusive right of property or privilege is claimed, are defined as follows: 1. The hybrid protein capable of being coded by the DNA fragment inserted into the vector. 2. Hybrid protein according to claim 1.
holding a polypeptide sequence having the properties immunologicals specific for HBsAg, adjoining a sequence polypeptide consisting of most of the .beta.-galac-tosidase. 3. Hybrid protein according to claim 2, characterized in that all or part of the .beta.-galacto- part sidase is replaced by any other non-carrier molecule immunogenic, or whose possible immunological properties, if these exist, do not interfere with those of the peptide part presenting the immunological properties of HBsAg.