Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • 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
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
  • C12Q1/701—Specific hybridization probes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
  • C12N2770/00011—Details
  • C12N2770/32011—Picornaviridae
  • C12N2770/32611—Poliovirus
  • C12N2770/32622—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
  • C12N2770/00011—Details
  • C12N2770/32011—Picornaviridae
  • C12N2770/32611—Poliovirus
  • C12N2770/32634—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• DNA fragments carrying parts of the genetic information of polioviruses, in particular of the PV1 type, and method for obtaining them are obtained.
• the invention relates to DNA fragments having, in terms of the genetic information which they contain, parts in common with the polioviruses, preferably but not exclusively of the PV-1 type.
• the invention relates to DNA coding for the capsid proteins and for any proteins necessary for cutting them, however essentially to the exclusion of a fragment hybridizable with the terminal part of the poliovirus coding for proteins involved in the replication of poliovirus.
• the invention relates to such DNAs, which also comprise the nucleotides of the 5 ′ end of the PV-1 (or PV1) type poliovirus.
• RNA has, however, been the subject of extensive studies. The information that has been drawn from it certainly contributes to a better understanding of the poliovirus genome itself. It was possible thanks to the digestion of this RNA by a RNase T1 (or nuclease T1) to isolate a certain number of oligonucleotides resistant to P-Nase T1, to characterize them (reference 6) and even to determine the locations relative nucleotide sequences of the genome to which said oligonucleotides correspond (21). These oligonucleotides will be designated in the following by the same numbers as those used by YF LEE (in publication 6).
• the object of the invention is to remedy the abovementioned difficulties, in particular as regards the production of DNA fragments of determined length, more particularly those carrying the information necessary to direct the synthesis of antigenic properties, preferably also.
• immunogenic specific to the poliovirus, however in the absence of the parts of the genome of the latter which carry the genes conferring them / an infectious character.
• a more particular object of the invention is also the production of such fragments, these however comprising all of the nucleotides specific for the 5 ′ end region of the poliovirus.
• the invention results from the discovery that the cloning into a bacterium of a cDNA-RNA hybrid had in fact resulted in the production of a multitude of clones containing fragments of different lengths and corresponding respectively to different regions of the whole cDNA capable of being synthesized from the RNA of the PV-1 poliovirus.
• the invention takes advantage of the combination which has been carried out of the techniques of analysis of these various fragments by restriction enzymes, on the one hand, and of the in situ hybridization technique which can be observed between said fragments.
• the DNA according to the invention is characterized in that it consists of or is formed of a segment hybridizable with part of the PV-1 type poliovirus, this segment coding for the capsid proteins and for any proteases necessary for cutting. of these, however essentially excluding a fragment hybridizable with the terminal part of the poliovirus, comprising at least 1.7 to 1.8 kilobases from its 3 'terminal end and coding for the intervening proteins in replication of poliovirus.
• the method according to the invention for obtaining such DNA is then characterized in that a genetic recombination is carried out between the cDNA fragments, as they were obtained above, after their analysis under the conditions already mentioned, in particular by the use of appropriate restriction enzymes to which correspond specific sites in the regions that the various fragments have in common, in particular according to the methodology which will be given below with regard to the production of an example.
• the above-defined DNA comprises, near its 5 'end nucleotides of the 3 poliovirus PV-1 type, in particular the sequence
• the DNA according to the invention can also be defined by reference to the series of restriction sites it contains, the positions of which can then be determined, for example with respect to a BamHI site, itself located at a distance of the order of 0.7 kilopairs of bases (kb or kbp according to abbreviations used below) from the 5 'end of the poliovirus genome. These positions are then as follows (distances expressed in kb and assigned a "-" sign, for those of the sites located between said 5 'end of the genome of the poliovirus and of the BamHI site considered):
• the translation initiation region of the PV-1 poliovirus genome is located at a distance of the order of 741 base pairs from the 5 ′ end of the genome, it is possible to envisage renounce in the fragments according to the invention the corresponding region, case in which the DNA as it was defined above could be modified by deletion of the corresponding 5 'end region.
• the DNA according to the invention can then be characterized by a 5 ′ end formed either by the reference Bam HI site, when the entire anterior part has been deleted, or by the Bal I end (-0.05 kb ), or Kpn I (-0.63 kb) or Bgl I (-0.66 kb), in particular in the event of partial deletions of said anterior part.
• the DNA fragment according to the invention is limited on the side of its 3 ′ end, either by a Bgl II site (4.9 kb), or by a Bgl I end (4.56 kb), or Bal I (4.25 kb), or Bam HI (3.9 kb).
• oligonucleotide probes derived from the RNA of the PV-1 poliovirus and resistant to the Tl nuclease and comprising 5 'nucleotide ends, the positions of which respectively in the total nucleotide sequence of the RNA of the poliovirus (identified in numbers (N) of base pairs counted from its own 5 'end) are the following: N806 (oligonucleotide No. 2) N3000 (oligonucleotide No. 1 ) N4434 (oligonucleotide No. 9).
• DNAs according to the invention can be obtained by variants of the process defined above consisting in cloning a cDNA resulting either from the total hydrolysis of the whole cDNA corresponding to the genome of the PV-1 poliovirus by the restriction enzyme Bgl I or Bgl II, or partial hydrolysis by the restriction enzyme Bal I or Bam I, to isolate the fragments having the desired lengths.
• the final cDNA obtained can then in turn be incorporated into a vector, for example pBP-322, under the conditions which are described below or similar conditions, the whole vector then being used to transform suitable host cells into which the fragment in question is likely to be expressed.
• the invention therefore also relates to the application of these DNAs to the production of anti-gene principles comprising the transformation with this DNA of a host cell in which it is capable of being expressed, where appropriate after prior incorporation of this DNA into an appropriate vector, containing a promoter capable of initiating the translation of this DNA in said host cell, and the recovery from the transformed cells or from their culture medium, as the case may be, of the antigenic principles produced.
• techniques that are now conventional can be used.
• FIG. 1 provides a representation of the restriction maps of several of the cDNA fragments which have been obtained after transformation of E. coli by a hybrid cDNA-RNA of the genus in question, said DNA fragments being placed in the relative positions making it possible to bring in alignment (or coincidence) the regions which are common to them, consequently the common restriction sites and the locations in which they exhibit in situ hybridizations with some of the numbered oligonucleotides resistant to RNase Tl above referred to (the corresponding numbers appearing under an arrowhead and being preceded by the sign "#").
• the signs used to identify the site-specific regions of various restriction enzymes are also defined in FIG. 1.
• the small arrows correspond to the orientation of the corresponding insertion fragments with respect to the carrier vector pBR322.
• FIG. 2 and Figure 3 are representations of heteroduplex molecules formed between frag elements delimited by EcoRI ends, obtained by opening the plasmids containing insertion fragments which themselves have common nucleotide sequences.
• FIG. 4 represents the restriction map obtained for the entire genome of the poliovirus. The positions at the different restriction sites are presented there. There are also shown schematically the relative positions vis-à-vis the whole genome of the insertion fragments of those of the clones which were selected to establish the restriction map of the genus in question.
• the lengths of the different fragments which separate either two sites which are identical to each other or a single site, when this is the case for the respectively 5 'and 3' ends of the entire genome of the poliovirus, are expressed therein in number of kilobases.
• Figures 5a to 5h are a schematic representation of the different operations which. were carried out to produce a cDNA according to the invention.
• a Mahoney poliovirus strain belonging to the PV-1 serotype is developed in cultures of HEla cells in suspension and the viral RNA is extracted according to the technique described by LEE and Coll. (reference 6).
• a cDNA is prepared from this RNA using a primer consisting of an oligo (dT) 10 and a reverse transcriptase according to the method described by KITAMURA et al. (reference 7).
• the hybrid cDNA-RNA molecules obtained are purified by centrifugation in a 15-30% neutral sucrose gradient - the ends of these hybrid cDNA-RNA are then trimmed with RNase A (25 micrograms / milliliter) and RNase T1 ( 0.05 U / milliliter) in the presence of 0.3 M NaCl.
• the 3 'ends of the molecules obtained by poly (dC) fragments are lengthened under the direction of a terminal transferase.
• the 5 ′ ends of a cloning vector pBR322 previously opened at its PstI site are lengthened by means of poly (dG), in particular according to the technique described by NELSON et al. (reference 8).
• the pBR322 DNA thus modified (450 ng) and cDNA-RNA molecules comprising the terminal extensions of poly (dC) (300 ng) are combined by hybridization, in particular by the techniques described by WOOD and Coll. (reference 9) and ZAIN et al. (reference 10), and used to transform E. co li 1106 803r k -m k - (MURRAY et al., reference 11) (CNCM May 19, 1981 under No. I-157).
• the bacterial clones containing recombinant DNAs are selected on the basis of their resistance to tetracycline and their sensitivity to ampicillin. 800 colonies were thus isolated.
• the plasmids contained in the above-mentioned clones are isolated and the cDNAs contained in these plasmids are extracted by the clear lysate technique according to CLEWEL et al. (reference 16).
• the cDNAs are separated by centrifugation until equilibrium in gra proliferatives of CsCl / ethidium bromide or according to the method described by BIRNBOIM et al. (reference 17).
• the cDNAs can be further purified by centrifugation in a 5-40% sucrose gradient, in particular for the analytical study by electron microscopy.
• the analyzes using restriction endonucleases use electrophoresis on agarose or polyacrylamide gel in TAE buffer (40 mM Tris, 20 mM acetate, pH 7.8, 2 mM EDTA). The fragments are visualized by UV, after staining with ethidium bromide (2 ⁇ g / ml). If necessary, an additional purification of the restriction fragments is carried out by electroelution from the gel in TAE buffer diluted to 1/10 and chromatography on hydroxyapatite columns, according to the technique described by MAY et al. (reference 18). The fragments are classified according to a linear map by appropriate double digestions.
• the clones were screened or sorted by in situ hybridization, using short single stranded PV-1 cDNA molecules radioactively labeled with 32 P, used as a 3 'end-specific probe of the PY1 poliovirus genome.
• Hybridization is carried out for 60 hours at a temperature of 42 ° C., in a solution whose composition is as follows: 1 x Denhart solution (15) 2 x SSC (0.30 M NaCl; 0.030 M sodium citrate), 2 mM EDTA, KH 2 PO 4 25 mil (adjusted to PH 7.2 with NaOH) , 0.5% SDS and yeast transfer RNA (Sigma) 5 ⁇ g / ml. Filters were washed 4 times at 42 ° C in a solution of 1 X
• 32 P-labeled cDNA hybridization probes were also used, which were prepared by limited incubation of PV-1 RNA, with reverse transcriptase and oligo (dT) 10 in the presence of ( ⁇ 32 P) or restriction fragments whose ends were marked with 32 P.
• the hybridization conditions were those described in
• the hybridizations observed are allowed, as explained below, to classify the clones, as follows from Table I below and to determine the relative positions (shown in FIG. 1) of the cDNA clones with respect to the initial genome.
• oligonucleobides-T1 Hybridization on a colony filter with oligonucleobides-T1 whose ends are labeled with phosphorus 32 or with a cDNA probe labeled with phosphorus 32.
• the oligonucleotides are numbered according to the indications provided in the publication under reference 21; the numbers in parentheses (for example N 806) 3e refer to the position of the 5'-terminal nucleotide of the corresponding oligonucleotide with respect to the total nucleotide sequence, as calculated in the direction 5'-3 '"The sizes cDNA insertion fragments were estimated thanks to their mobility in agarose.
• restriction maps of the clones hybridizing with the oligonucleotides T1 known to hybridize with the PV-1 genome in its central part were placed so as to bring the Bgl sites into alignment I and Bgl II, separated by about 0.3 kb, which both also had.
• a fragment 2.8 kb contained in pPYl-120 also met these conditions. It was also found to contain cleavage sites by additional restriction enzymes, most of which were also in the clone pPVl-341 (Fig. 1).
• pPV1-120 must have common parts with pPV1-142, pPV1-378 and pPV1-344; this has been confirmed by the study of the presumed homologous sequences, by analysis with an electron microscope of heteroduplex molecules formed between the digestion products by EcoRI containing these DNAs.
• the heteroduplex molecules formed between the DNAs pPV1-120 and pPV1-378 were then found to present two single-stranded D loops (B and D) having lengths of 2.3 + 0.35 and 1.16 + 0.21kb respectively . (fig.2), separated by a central region of homology (C) of 715 + 75 base pairs as the map of Figure 1 allowed to predict.
• the 5 'end of pPVl-366 also had a terminal end of 20 dG residues, followed by 50 nucleotides whose sequence is in perfect agreement with that determined for the 5' end of the corresponding RNA (23,24) to PV1.
• the insertion fragment of the clone pPVl-366 started exactly at the first nucleotide of the genome of the poliovirus.
• the difference, or even the impossibility for nucleotide No. 21 to form a stable hybrid with pPVl-366 or pPVl-846 in the hybridization test, can; likely to be attributed to the small size of the probe.
• the insertion fragment 846 having a length of 3.2 kb was found to also contain a second Kpn.J site located 3 kb from the first, and close (approximately 0.1 kb) to a Hae II site. Since the insertion fragment 120 also presented, near its 5 ′ end, Kpn I and Hae II sites separated by approximately 0.1 kb, it could be hypothesized that the 3 ′ end region of the fragment Insert 846 and the 5 'end region of insert 120 showed the common nucleotide sequence shown schematically in Figure 1.
• the configuration of the overlap or overlap between pPVl-846 and pPVl-120 was obtained by examination in the electron microsphere of the heteroduplex molecules formed between the fragments obtained by dirrestion of these plasmids in the presence of EcoRI.
• the heterocuplex molecules presented those double-stranded D loops (G and I), having respective lengths of 3.28 + 0.52 and 2.82 + 0.4, separated by a central homologous region (H) of 195 + 50 base pairs, as expected from the map of Figure 1.
• the restriction map of the PV-1 genome could therefore be reconstituted from that of successive clones giving rise to mutual overlap, in particular insertion fragments 846, 120, 142 and 751.
• the restriction map shown in FIG. 4 of the total genome of the PV1 poliovirus consequently results from analyzes by the restriction enzymes of the above-mentioned insertion fragments.
• fragment 366 coincides with the actual 5' terminal nucleotide of PV1.
• fragment 751 coincides with the 3 'end region of PV1.
• the entire genome comprises from 7,400 to 7,500 nucleotides, which is in agreement with previous determinations.
• Example of production of a characteristic cDNA in accordance with the invention. Obtaining a plasmid carrying the 5 'end of cDN A corresponding to the genome of the poliovirus. _P V1 _ et_ co u vran t les 2 / 3_du_génome_viral_
• the two plasmids of clones pPVI-846 and pPVI-120 are recombined.
• the clone pPVI-846 covers the region of the genome of the poliovirus which extends from nucleotide 1 (+ 35) to nucleotide 3100 approximately
• the clone pPVI-120 covers the region of the genome of the poliovirus which extends from nucleotide 2950 to nucleotide 5650 approximately. It carries 2 Kpn I sites (positions 3050 and 3548. It has no EcoRI or Downstream site ( Figure 5b).
• the plasmid pBR322 from which the two aforementioned plasmids are derived has in its genome an EcoRI site (position 4359.) a downstream site (1424), no Kpn I site.
• FIGS. 5a to 5h The plasmids and DNA fragments produced during the recombination operations are shown diagrammatically in FIGS. 5a to 5h.
• the DNA of the clone pPVI-120 is hydrolyzed in a complete digestion with Downstream and EcoRI thus forming 2 fragments of different sizes (FIG. 5e).
• the DNA is then partially hydrolyzed with Kpn I.
• the fragments thus obtained (FIG. 5f) are separated by electrophoresis in 0.7% agarose gel.
• the fragment of size 3-55 kbp is selected. It indeed represents the cDNA sequence of the allarfc poliovirus from nucleotide 3050 (2nd site Kpn I) to nucleotide 5650 approximately, extended by that of the 752 base pairs of the Pst I-EcoRI segment of the plasmid pBR322. 2. Extraction of DNA fragments frozen
• the fragments are visualized in the gels by staining with ethidium bromide; those of the desired size are extracted from the gels by electroelution in a dialysis bag.
• the genome of the plasmid pBR322 is thus reconstituted without modification or deletion in the recombinant plasmid.
• the regions necessary for its replication and for the expression of resistance to tetracycline are not affected.
• the plasmid DNA of the resistant tetracycline bacteria is purified. Its mass is determined by agarose gel electrophoresis. It is equal to that of the plasmid pBR322-increased by the 5650 base pairs of the viral cDNA formed by recombination.
• the cDNA carried by the recombinant plasmid (pPV1-X) carries the genetic information necessary for the synthesis of the pi protein, precursor of the capsid proteins VP4 (nucleotides 741 to 975), VP2 (nucleotides 976 to 1818), VP3 ( 1819 to 2562) and VP1 (2563 to 3501), followed by those which correspond to the p2 protein (precursor in particular of the NCVPX protein) and at the beginning of the p3 protein.
• the set covers approximately 5,650 of the 7,432 Su viral genome bases.
• the plasmid pPVI-846 was deposited at the C.N.C.M. under No. 1-155 and plasmid 120 under No. 1-156 on May 19, 1981.
• the invention naturally extends to all equivalent DNA fragments, in particular those derived from polioviruses belonging to other serotypes, such as those designated by the abbreviations PV2 and PV3, and devoid of the parts of gene coding for the enzymes involved in the replication of these viruses.
• the methods for manufacturing gene fragments derived from PV1 apply in the same way to obtaining corresponding DNAs from PV2 and PV3.
• the invention also relates to the smaller fragments, in particular such as those coding for the p1 protein. (see part P1 in FIG. 4), in particular those extending up to 3 to 4 kilobases from the 5 'end of the genome of the poliovirus. They are useful inter alia as intermediate products for the manufacture of the larger fragments described above. They are also of interest for the expression products whose production they direct in competent bacteria.

Landscapes

• Chemical & Material Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Health & Medical Sciences (AREA)
• Organic Chemistry (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Wood Science & Technology (AREA)
• Zoology (AREA)
• Biophysics (AREA)
• Genetics & Genomics (AREA)
• Engineering & Computer Science (AREA)
• General Health & Medical Sciences (AREA)
• Biochemistry (AREA)
• Molecular Biology (AREA)
• Virology (AREA)
• Immunology (AREA)
• Microbiology (AREA)
• Analytical Chemistry (AREA)
• Bioinformatics & Cheminformatics (AREA)
• General Engineering & Computer Science (AREA)
• Biotechnology (AREA)
• Physics & Mathematics (AREA)
• Gastroenterology & Hepatology (AREA)
• Medicinal Chemistry (AREA)
• Saccharide Compounds (AREA)
• Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Applications Claiming Priority (2)

Publications (1)

Family

ID=9258647

Family Applications (2)

Family Applications After (1)

Country Status (5)

Families Citing this family (10)

• 1981
  • 1981-05-19 FR FR8109968A patent/FR2506326A1/fr active Granted
• 1982
  • 1982-05-18 CA CA000403182A patent/CA1195938A/fr not_active Expired
  • 1982-05-19 WO PCT/FR1982/000084 patent/WO1982004067A1/fr not_active Ceased
  • 1982-05-19 EP EP82901515A patent/EP0079353A1/fr not_active Withdrawn
  • 1982-05-19 JP JP57501549A patent/JPS58500759A/ja active Pending
  • 1982-05-19 EP EP82400941A patent/EP0065924A1/fr not_active Withdrawn

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events