FR2464269A1 - Nucleic acid coding for hepatitis B antigen - and corresp. peptides, vectors and hybrid proteins - Google Patents

Abstract

The following are claimed:\ (A)A nucleic acid (I) comprising 1000-1100 nucleotide units which codes for an immunogenic peptide sequence capable of inducing anti- HBV antibody prodn. in vivo (HBV is hepatitis B virus).\ (B)Polypeptides including a peptide sequence coded by (I).\ (C)Phage- or plasmid-type vectors which contain at least part of the lactose operon (esp. its promoter and Z gene) and which are modified by insertion of at least a fragment of (I) into a suitable site in the Z gene.\ (D)Hybrid proteins coded by (I) in the vectors.. The polypeptides and hybrid proteins can be used as vaccines or diagnostic reagents for hepatitis B.

Description

 The invention relates to a nucleic acid comprising a nucleotide sequence capable of encoding an immunogenic peptide sequence corresponding to the surface antigen of the viral hepatitis B virus, and to the polypeptides and peptides obtained.

It also relates to a process making it possible to obtain such a nucleic acid.

 Hepatitis B is a common viral disease, particularly in tropical Africa, Southeast Asia and the Far East.

The etiological agent is a virus (HBV) or Dane particle, comprising an Australia antigen envelope or HBs antigen), a capsid (HBc antigen), an endogenous polymerase and a partially single-stranded circular DNA molecule; 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 72, 45974601).

 Endogenous DNA polymerase can be used to repair the shortest strand in vitro (T.A.LANDERS, H.B.

GREENBERG and J.S.ROBINSON, J.VIROL., 23, 1977, p.368-376).

 The electrophoretic analysis of the proteins of the envelope showed the presence of 2 to 7 polypeptides, the main ones of which are called: polypeptide I and polypeptide II.

(PETERSON, D.L., ROBERTS, I.M. And VYAS, G.N. (1977) Proc.Nat.

Acad. Sci., USA, 74, 1530-1534, and PETERSON, DL, DOG,
DY, VYAS, GN, NITECKI, D. and BOND, H. (1978) In Viral
Hepatitis, ed.G.VYAS, S.COHEN and R.SCHMID, The Franklin
Institute Press, Philadelphia, 569-573).

Polypeptide I has a molecular weight of -22,000 to
26,000 daltons. Polypeptide II is glycosilated and has a molecular weight of 28,000 to 30,000 daltons. The composition
the amino acid content of these two polypeptides is very sem
blable, so the assumption has been made that the poy-
peptide II may differ from polypeptide I only by
glycosilation.

 The sequences formed by their first 15 amino acids (from the N-terminus) and their last 3 amino acids, respectively, are identical.

The study of these polypeptides is extremely difficult since there is no cell culture system allowing the propagation of the virus. This difficulty has already been partially circumvented, more particularly with regard to the avw serotype of whole DNA.
(genome) of the virus, which has been identified and cloned, in particular in E. Coli, after its prior insertion into the uni site Eco RI of an Agt.WES vector. XB, according to the technique described by FRITSCH, A., POURCEL, C., CHARNAY, P. and TIOLLAIS, (P (1978) CR Acad. Of Paris, 287, 1453-1456).

 To date, the identification of the polypeptides I and II themselves, the localization in the viral DNA of the sequence encoding these peptides have not been carried out.

The object of the invention is to obtain a DNA sequence much smaller than the viral DNA itself, containing the sequence capable of encoding the peptide sequence endowed with immunogenic properties allowing, when it is introduced into the organism of a living host, to induce the manufacture by the latter of antibodies capable of subsequently protecting this same host with respect to the viral hepatitis B virus, in particular when the latter is in the state viruilent
The invention derives not only from the complete nucleotide analysis of the genome of the Dane particle carried out by the inventors, but from the idea that they had for identifying the coding gene (hereinafter referred to as "gene
S ") of the above polypeptides, to search in the complete nucleotide structure thus predetermined of the genome of the Dane particle, those of the nucleotide sequences capable of coding the known proximal and terminal peptide sequences of these polypeptides.

It will be recalled that PETERSON and collaborators reported, in particular in the articles whose references were mentioned above, that the proximal sequence (first N-terminal amino acid) of the first 15 amino acids was analyzed in principle 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 amino acid C terminal) was as follows
val tyr ile
Figure 1 is a schematic map of the genome of the Dane particle. This comprises two strands b1 and b2; the shortest of them (b2) being normally devoid of the part represented by a dashed line in the drawing.

It is known that this DNA has only one site
EcoRI.

The arrow f1 gives the direction of the numbering of the nucleotides of which the longest strand b1 is composed, and the arrow f2 gives the direction of the transcription of the DNA of the virus, in particular by the cellular machinery of the cells invaded by the hepatitis B virus, with regard to the expression of the S gene.

 The EcoRI site can therefore be numbered O or, as has now been determined more precisely for that of the hepatitis B viruses belonging to the serotype aym, 3182.

 The circle in solid inner line e gives the scale in% of DNA length and makes it possible to specify the nositiors Se certain of its parts.

 The numbers 3 ', 5' and 5 ', 3' at the bottom of the card target the terminal ends carrying the same numbers in the conventional representations of the ends of the nucleic acid chains.

According to the invention, it has been shown that the "S gene" essentially constitutes the fragment of the longest strand bl located between positions 73.6 and 95.1 of the schematic map of FIG. 1. The abbreviations "Start" and "Stop" represent the points of initiation and stop of the transcription
of the "S gene".

Figure 2 is representative of the terminal part of the above genome, in particular between positions 6O, and 100 (in% of DNA length). Each of the letters appearing in FIG. 2 conventionally corresponds to one of the 4 basic nucleotides of DNA
A: Adenine
G Guanine
T: Thymine
C: Cytosine
The lower lines, in each pair of lines of which FIG. 2 is made up, correspond to the nucleic acid corresponding to the nucleotide chain b2.

 The analysis technique used to establish the more detailed map, as shown in Figure 2, will be briefly recalled below.

 Characterization of the nucleotide sequence of the "S gene" as it is. proposed in the context of the present invention, and whose proximal E "S" and terminal t ends are indicated in FIG. 2, results from the observation that - the first 14 triplets (in the direction of reading f2) starting from the nucleotide numbered 3030 opposite the EcoRI terminal end, are respectively capable of coding the first 14 amino acids of the proximal sequence of the first 15 amino acids of the above polypeptides, - as the last 4 GTA triplets TAC ATT rAA read on hate complementary b2 to the transcribed chain bl correspond respectively to the 3 terminal aino acids of the above-mentioned polypeptides and to a stop codon - that this nucleotide sequence (678 nucleotides) does not include any stop codon, at least when we adopt the reading framework implying that the first triplet "read" by the cellular machinery is AITG, (corresponding on the other strand to qW 'the complete translation of the genetic information beginning with the codon in itial ATG leads to a theoretical polypeptide of 226 amino acids, having a molecular weight 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 FIG. 3.

These values are entirely consistent with the ahalytic data which result from the electrophoretic mobility of the polypeptide I on polyacrylamide gels which have already been described by the previous authors (references 9-12 according to the biblography appearing 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 the leucine polypeptide I according to the maps of FIGS. 2 and 3 mentioned above, and not serine according to the observations of the above-mentioned authors, can perhaps be attributed to the fact that these authors worked with a genetic variant different from that which was the subject of the present study. It will be noted that the difference can moreover be related to the substitution of a single nucleotide in the triplet concerned "TTA" in the particular "S gene" represented in the maps of FIGS. 2 and 3, instead of "TCA", one of the triples likely to be translated into serine.

 The invention therefore relates more particularly to the nucleic acid fragments which can be excised from the DNA of 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 that is responsible for the immunological properties of the hepatitis B virus.

 As such, the invention therefore relates to a nucleic acid comprising at most of the order of 1000-1100 nucleotides, more particularly characterized in that it is capable of coding an immunogenic peptide sequence, itself capable of inducing in vivo the production of antibodies active against the hepatitis B virus, this peptide sequence essentially containing the structure shown in FIG. 3, or any peptide sequence having equivalent immunogenic properties.

 It will be observed that the first amino acid of the above-mentioned sequence: methionine, is N-terminal and that the amino acid of the opposite end: isoleucine, is Cterminal.

 The invention also relates, more particularly, to the nucleotide sequence represented in FIG. 4 encoding the peptide sequence as it results from FIG. 5 or any analogous peptide sequence endowed with equivalent immunogenic properties.

 It goes without saying that by "equivalent peptide sequence" is meant any peptide sequence in which certain parts may not be strictly identical to the corresponding parts of the peptide sequence shown in FIGS. 3 and 5, these variations possibly being due to local mutations which do not affect the general immunogenic character of the protein or to structural modifications relating to the various serotypes under which the proteins of the genus in question are liable to appear (in particular serotypes adw, adr and air).

The invention relates more particularly to the nucleotide sequence containing the peptide sequence as shown in FIG. 6 or any analogous peptide sequence endowed with equivalent immunogenic properties.

The invention relates more particularly still to the following peptide sequences
Alanine - Glutamine - Glycine - Threonine - Serine
Threonine - Alanine Glutamine - Glycine - Threonine - Serine
Threonine - Threonine - Alanine - Glutamine - Glycine - Threonine - Serine
In the first peptide mentioned above, the alanine end is N-terminal and the serine end is C-terminal.

 In the second and third peptide mentioned above, the threonine end is N-terminal and the serine end is C-terminal.

 By way of example, the pentapeptide can in particular be prepared starting from the C-terminal serine to which threonine is hooked by the Castro method described in Tetrahedron Letters, 1975, no 14, pages 1219-1222.

Then the amino acids glycine, glutamine, alanine are added by the so-called repeated mixed anhydride method (rema method) described by Beierman in Chemistry and Biology of Peptides, Ed. J. Meienhofer, Ann. Arbor Science
Publ., Ann. Arb. Mich. 351 [1972).

The invention also relates to the products resulting from the attachment of the pentapeptide to a larger carrier molecule, in particular of the polypeptide or protein type, the compositions containing this pentapeptide as binding products, in particular in association with a pharmaceutically acceptable vehicle, and more particularly to hepatitis B vaccines. These pharmaceutical vehicles are suitable, in a conventional manner, for the chosen mode of administration, in particular by oral, parenteral, rectal route or by nebulization on mucous membranes, in particular nasal.

 The hexapeptide and the 7 amino acid polypeptide can be synthesized by standard peptide synthesis techniques.

 These peptides are, according to the present invention, held to be the antigenic site of the larger polypeptides of which question above and responsible for the vaccinating power of the viral envelope journal of Biol.

Stand. 1976, 4, 295-304, Rao -and Vyas "Biocheminal Characterization of Hepatitis
B Surface Antigen in Relation to Serologic Activity ").

The invention also also relates to the DNA fragments capable of encoding the production of such pentapeptide, hexapeptide and polypeptide with 7 amino acids. It's about
for pentapetide, in particular the polynucleotide of
formula
5 'GCT CAA GGA ACC TCT 3'
3 'GGA GTT OCT TGG AGA 5'
for the hexapeptide, in particular of the polynucleotide of
formula
5 'ACT GCT CAA GGA ACC TCT 3'
3 'TGA CGA GTT OCT TGG AGA 5'
for the 7 amino acid polypeptide of the polynucleotide
of formula
5 'ACT ACT GOT CAA GGA ACC TCT 3'
3 'TGA TGA CGA GTT OCT TGG AGA 5' or in each of the three cases, complementary plynucleotide relating to the three preceding respective polynucleotides or any polynucleotide in which each of the triplets can be replaced by any analogous triplet capable of coding the production of the same amino acid.

 The nucleic acid according to the invention can also be characterized in that it comprises at least one of the two mutually complementary strands of a DNA sequence as represented in FIG. 4 (in which also appear the corresponding numbers at the positions of the first nucleotides of each of the successive fragments of 10 nucleotides shown with respect to the EcoRI position not shown in the figure: it goes without saying that these numbers do not occur at the level of the characterization of the nucleotide sequence of the genre in question). This DNA fragment is delimited by two HincII sites.

 It will be appreciated that this nucleotide sequence corresponds to the genetic information, the translation of which leads to the peptide sequence represented in FIG. 5.

 The invention naturally relates to the equivalent single-stranded or double-stranded nucleotide sequences, in particular the strand having the structure which results from the succession of the lower lines of FIG. Lt, the corresponding double-stranded DNA, or the corresponding messenger RNAs. , in particular that represented by the complementary sequences of nucleotides formed by the infrared lines of the pairs of lines in FIG. Lt (direction of the arrow f2).

 Likewise, the nucleotide chains which differ from the preceding ones by certain triplets or small triplet sequences fall within the scope of the invention, insofar as these nucleotide sequences remain capable of encoding a polypeptide conserving the immunogenic activities characteristic of the viruses. viral hepatitis B. In general, these are nucleotide chains which, if necessary, after denaturing the double-stranded DNA to produce the corresponding single-stranded nucleic acids, remain susceptible to hybridization over at least about 90% of their length with one of the strands of the AON in Figure 4.

Preferred nucleic acids according to the invention are also those which can be excised from the DNA of viral hepatitis and which, when they are double stranded, are characterized by the existence at one of their ends. from a HincII, HhaI, AvaI or EcoRI end, and at their other end by an Avalîl, HincII or HhaI end.

The positions of these various ends vis-à-vis
of the EooRI site are shown diagrammatically in Figure 2.

The nucleic acid according to the invention is intended for incorporation into a vector allowing its expression in a bacterium and in eukaryotic cells, notarmellt for the production of a protein or a peptide capable of inducing in the organism of a living host producing antibodies against the viral hepatitis B virus. The protein or peptide resulting from the translation of the nucleotide sequence according to the invention can be used as a vaccinating agent or diagnostic agent
The nucleic acid according to the invention can also be used as a probe to detect the presence or not in samples of blood or of test serum, of Dane particles, of HBs antigen or of fragments thereof, etc. . (by conventional DNA-DNA hybridization technique).

 Other characteristics of the invention will also result from the brief description which follows of the techniques (analysis, identification and obtaining of DNA fragments in accordance with the invention. Reference will naturally be made to the drawings, the figures of which have already been taken into consideration in the foregoing The figures or numbers in brackets correspond to the references in the bibliography annexed to this description.

The products and methods used.

- The enzymes and chemicals used ~
The restriction enzymes used: BamHI, HhaI, Hincîl, HaeIII, XbaI, MboI, Hindi, HpaII, XhoI, are those manufactured by BIOLABS. The DN-EcLymerase I of
BOEHRINGER. Bacterial alkaline phosphatase and polynucleotide kinase were supplied by PLBIOCHEMICALS. The chemical agents were:
Dimethyl sulfate (ALDRICH)
Hydrazine (Eastman Kodak)
Acrylamide and bis-acrylamide (twice crystallized, (Serva)
Dideoxy nucleotide triphosphates and
deoxynucleotide triphosphates (PLBiochemicals)
Piperidine (MERCK) redistilled under vacuum.

- HBV DNA preparation
The entire HBV genome (subtype avw) has been cloned
in E.Coli by bringing into play the unique EooRI restriction site
of the Agt.WES vector. AB (14). The cloned DNA is hereinafter called "Eco HBV DNA".

 The recombinant bacteriophage was amplified in a Petri dish on Agar and the DNA sought was extracted in a manner known per se. After digestion of the DNA with the restriction enzyme EcoRI, the Eco HBV DNA sequence was purified by ultra centrifugation, in a sucrose gradient, according to the technique described in the bibliographical references (16,17).

- Prep ~ ration ~ e ~ f ~ aqments ~ of A ~ N ~ marked ~ 5'3 ~ P ~
10 to 20 picomoles of ECO HBV DNA were completely hydrolyzed by the various restriction enzymes, under the conditions recommended by the manufacturer. The DNA fragments were dephosphorylated by alkaline phosphatase, which was then inactivated by alkaline treatment. The DNA was then precipitated with ethanol, according to the technique described in article (18). After redissolution in a spermidine-based buffer, the DNAs were labeled at their 5 ′ ends with ATP {γ p} (3000 Ci / mM manufactured by New England Nuclear) and with poly-nucleotide kinase (according to the technique referred to in article (19)).

The restriction DNA fragments were separated by polyacrylamide gel electrophoresis, then eluted.

The labeled ends were subjected to segregation by polyacrylamide gel electrophoresis in a manner known per se, after restriction with another enzyme or by denaturation of the DNA fragments of the genus in question.

- Determination of ~ the structure of DNA nucleotide sequences.

The primary structure of the double-stranded or single-stranded DNA fragments was determined essentially by the technique described by Maxam and Gilbert (19). The terminal chain inhibitor method described by SANGER et al was also used. (20) and adapted by MAAT and
SMITH (21), with regard to the double-stranded fragments marked at one of their 5 'ends.

The chemical and enzymatic reaction products were analyzed by electrophoresis in gels of acrylamide sequence at 8.16 or 25 ide 1 mm thick.

 Techniques and results of the analysis.

In order to determine whether the HBV genome is capable of encoding polypeptides I and II, all of the HaeIII fragments (HaeIII restriction sites of the HBV genome represented in FIG. 1 by small arrows) have been marked at their 5 'ends. Substantial parts of their primary structures have been determined by the method of Maxam and Gilbert. The nucleotide sequences capable of encoding the proximal and terminal amino acid sequences of polypeptides I and II have been located in the HaeIII E and HaeIII F fragments, previously located on the restriction map of the HBV genome (according to the technique described in reference (17)). It is these nucleotide sequences which have been considered to consist of the ends of the "gene
Occupying themselves positions 73.6 and 95.1 vis-à-vis the EcoRI restriction site (FIG. 1) for the reasons already indicated.

The nucleotide sequence between these two positions
was analyzed using known chemical techniques, in particular by the chemical degradation method with hydrazine dimethyl sulfate and the chain termination method. Among the various chemical reactions proposed by Maxam and Gilbert, partial depurination with formic acid and cleavage with piperidine have been used, methods which give bands of scant intensity slXr ics alltoradiograns, the for the terminated fragpents with guanine and adenine. Reactions to hydrazine followed by cleavage with piperidine were also used to obtain bands of equal intensity for the nucleosides cytosine and
: thymidine: the electrophoretic fractionation of the products of these two reactions gives a stain for all bases
in either. gel columns used. This procedure facilitates the reading of the gel autoradiogram.

The reaction to hydrazine in the presence of sodium chloride specific for cytosine makes it possible to distinguish this nucleotide from thymidine utla reaction to dimethylsulfate followed by cleavage by piperidine specific for guanine makes it possible to distinguish this latter nucleotide from adenine .

In order to ensure the greatest possible degree of precision, distinct nucleotide sequences forming different overlapping fragments were produced by hydrolysis of Eco HBV DNA by various restriction enzymes
BamHI, HinfI, HpaII, HaeIII and HincII.

 In this way the analysis of each of the restriction sites used as starting points for the first fragments studied was confirmed by the analysis of the distinct fragments in which the restriction sites of the first fragments were between the new ends of these fragments. separate.

 LeXgène S "represented in FIG. 3, which begins because the initiation codon ATG, comprises 227 triplets, including a stop codon TAA. The 3 codons corresponding to the 3 amino acids of the carboxy terminus of the corresponding polypeptide are located in the same reading frame, immediately before the stop codon TAA. One of the other two reading frames (respectively offset from the previous one by 1 and 2 nucleotides) is also devoid of stop codon, but encodes a protein quite different from the above-mentioned polypeptides I and 11. The third reading frame comprises 10 stop codons (5 TAG, 4 TGA, 1 TAA).

On the other DNA strand, the three reading frames are respectively closed by 11, 11, and 6 stop codons distributed along the DNA sequence.

 As already mentioned above, the complete translation of the genetic information starting with the initiation codon ATG leads to a theoretical polypeptide of 226 amino acids corresponding to a molecular weight of 25,422 daltons.

 It is interesting to emphasize that the nucleotide sequence corresponding to the "S gene" should normally be read entirely during translation.

Also part of the invention are nucleotide chains of the type of the "S gene" described above, which oom
carries small additional sequences which may contain up to a hundred nucleotides or which, on the contrary,
may be lacking without altering the corresponding genetic information (22, 23).

 The different fragments of the invention which have been defined above can be obtained from the DNA sequence known as Eco HBV DNA, by using the corresponding restriction enzymes and known fractionation techniques for DNA fragments, in particular on polyacrylamide gel and taking advantage of their migrations over distances which are a function of their molecular weights.

Thus we can for example obtain the fragment one end of which is delimited by an EcoRI site and the other by an Avalîl site by operating an Eco HBV DNA restriction by the enzyme Avalîl, the desired fragment consisting in the smallest fragment obtained (a single AvalII 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 with the restriction enzyme HhaI. One then recovers from the 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 treatment orders with restriction enzymes to isolate, in particular from Eco HBV DNA, fragments having useful restriction ends.

For all practical purposes, it will be recalled that these restriction operations can be carried out within a 10 mM Tris buffer; py 7.8; ydgC12 6 mM, 6-mercapto-ethanol 6 mM, the same medium additionally and preferably containing 50 mM NaCl
when EcoRI is used.

 As has already been said, the invention relates to the use of the DNA fragments described as a probe allowing the diagnosis of the presence in a serum of Dane particles or particles derived from the previous one, carrying DNA capable of encoding an immunogenic protein characteristic of hepatitis B.

 The DNA according to the invention can also be incorporated into a vector allowing, provided that the incorporation has been carried out in phase, the expression of this DNA in a bacterium or other microorganism, or in eukaryotic cells.

 The invention can therefore allow the production of a protein of lower molecular mass than the above-mentioned polypeptides I or II, endowed with the same immunogenic properties.

 As it goes without saying, and as it already follows from the foregoing, the invention is in no way limited to those of its modes of application and embodiments which have been more especially envisaged; on the contrary, it embraces all its variants.

In appendix of this description appears the bibliography, and in particular the references which were quoted within the framework of the present description.

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 6, 63-67.

Claims (19)

1. Nucleic acid characterized in that it comprises at most of the order of 1000-1100 nucleotides and that it is capable of coding an immunogenic peptide sequence, itself capable of inducing in vivo the production of active antibodies to with regard to the hepatitis B virus. 2. Nucleic acid according to claim 1, characterized in that it comprises a nucleotide sequence capable of encoding the peptide sequence represented in FIG. 3 or an analogous peptide sequence endowed with equivalent immunogenic properties. 3. Nucleic acid according to claim 2, characterized in that it comprises a nucleotide sequence represented in FIG. 4, capable of encoding the peptide sequence represented in FIG. 5 or an analogous peptide sequence endowed with equivalent immunogenic properties. 4. Nucleic acid according to claims 1 to 3, characterized in that it comprises a nucleotide sequence capable of encoding the peptide sequence represented in FIG. 6 or an analogous peptide sequence endowed with equivalent immunogenic properties represented in FIG. 6. 5. Nucleic acid according to any one of claims 1 to 4; characterized in that it contains a sequence capable of encoding the peptide sequence: Alanine tN-terminal) - Glutamine - Glycine - Threonine - Serine (C-terminal).  6. Nucleic acid according to any one of claims 1 to 4, characterized in that it contains a sequence capable of encoding the peptide sequence: Threonine (N-terminal) - Alanine - Glutamine - Glycine - Threonine - Serine ( C-terminal)  7. Nucleic acid according to any one of claims 1 to 4, characterized in that it contains a sequence capable of coding the peptide sequence: Threonine (N-terminal) - Threonine - Alanine - Glutamine - Glycine - Threonine - Serine ( C-terminal). 8. Nucleic acid according to any one of claims 1 to 7, characterized in that it comprises at least one of the two strands mutually complementary to a DNA sequence as shown in Figure 4 or a strand capable to hybridize with the previous one.  9. Nucleic acid according to claim 8, characterized in that it comprises at least one of the two mutually complementary strands of a DNA sequence as shown in FIG. 3 or a strand capable of hybridizing with the previous one. 10. Nucleic acid according to any one of claims i è 7, characterized in that it is double-stranded and in that it is delimited at one of its ends by a Hindi, HhaI, AvaI end, or EccRI, and at its other end by an AVILI, HincII, or HhaI end.  11. Nucleic acid according to claim 5, characterized in that it contains the sequence 5 ′ GCT CAA GGA ACC TCt 3 ′ or the sequence complementary to the preceding one, or else a sequence in which at least one of the above-mentioned triplets is replaced by another triplet, however capable of coding the same amino acid.  12. Nucleic acid according to claim 6, characterized in that it contains the sequence 5 ACT GCT CAA GGA ACC TCT 3 ', or the sequence complementary to the previous one, or also a sequence in which at least one of the above triplets is replaced by another triplet, however capable of coding the same amino acid.  13. Nucleic acid according to claim 7, characterized in that it contains the sequence 5 'ACT ACT GCT CAA CGA ACE TACT 3', or the sequence complementary to the previous one, or a sequence in which at least one of the above triplets is replaced by another triplet, however capable of coding the same amino acid.  14. Application of the DNA according to any one of claims 1 to 13, to the constitution of a vector capable of being expressed in a microorganism or in a eukaryotic cell for the production of a protein comprising a peptide sequence encoded by said nucleic acid.  15. Peptide, characterized in that it comprises a peptide sequence encoded by the nucleic acid according to any one of claims 1 to 13. 16. Peptide according to claim 15, characterized by the following sequence: Alanine - Glutamine - Glycine Threonine - Serine, in which the alanine end is N-terminal, and the serine end is C-terminal. 17. Peptide according to claim 15, characterized by the following sequence: Threonine - Alanine - Glutamine Glycine - Threonine - Serine, in which the threonine end is N-terminal and the serine end is C-terminal.  18. Peptide according to claim 15, characterized by the following sequence: Threonine - Threonine - Alanine Glutamine - Glycine - Threonine - Serine, in which the threonine end is N-terminal and the serine end is C-terminal.  19. Pharmaceutical composition, in particular of a vaccine with a hepatitis B diagnostic reagent, containing in association with a pharmaceutically acceptable vehicle the polypeptide according to any one of claims ie, 17 and 18, either alone or previously fixed on a carrier molecule, in particular of the polypeptide or protein type.