DE3628658A1 - Polypeptides of the rhinovirus strain HRV89 and the DNA molecules coding therefor - Google Patents

Abstract

The present invention relates to peptides which correspond in whole or in part to those of the viral proteins of human rhinovirus strain 89 (HRV89), to the deoxyribonucleic acid molecules coding for these peptides, and to the preparation and use of these substances.

Description

The present invention relates to peptides which are entirely or partly those of the viral proteins of human Rhinovirus strain 89 (HRV 89) correspond to this Deoxyribonucleic acid molecules encoding peptides, as well the production and use of these substances.

Rhinoviruses are RNA viruses and make after the conventional division of viruses into a genus within the Picornavirus family (Cooper, P. D., Agol, H.L., Bachrach, H.L., Brown, F., Ghendon, Y., Gibbs, A.J. Gillespie, J.H., Lonberg-Holm, K. Mandel, B. Melnick, J.L., Mohanty, S.B. Povey, R.C., Rueckert, R.R., Schaffer, F.L. and Tyrrell, D.A.J., 1978, Intervirology, 10, 165-180; MacNaughton, 1982, Current Top. Microbiol. Immunol. 97, 1-26).

They are common, affecting the upper one respiratory tract of humans and cause acute Infections causing runny nose, cough, hoarseness etc. lead and are commonly referred to as colds (Stott, E.J. and Killington, R.A., 1972, Ann. Rev. Microbiol. 26, 503-524). Rhinovirus infections are among the most common human diseases. Even though the course of these diseases is mostly harmless, colds lead to a temporary one Weakening of the organism. This can cause it to Secondary infections from other viruses or bacteria come, which may lead to serious illnesses Have consequence. It is also caused by rhinoviruses economic damage considerable. It is has been calculated that in the U.S.A. by Rhinovirus infections annually more than 200 million Working days or school days are lost (Davis, B.D., Dulbecco, R., Eisen, H.N. and Ginsberg H.S., 1980, Microbiology, Third Edition, Harper Row, Publ., New York, p. 1114).  

In addition, in recent years there has been a considerable one Increase in rhinovirus infections in metropolitan areas been found. While it is with most others Infectious diseases to a long-term or permanent immunity to the concerned Pathogens can come through infections Rhinoviruses occur again and again. The reason for that The lack of permanent immunity is the great variety on strains of rhinovirus. So far, over 100 Strains of rhinovirus isolated, none or between each other show little immunological cross-reaction (Fox, J.P. 1976, American J. Epidemiol. 103, 345-354; Melnick, J.L., 1980, Proc. Virol. 26, 214-232). To If the infection is successful, antibodies against the detect the virus strain in question, but grant it no protection against other strains of rhinovirus. Due to the large number of tribes in the population circulate are repeated infections caused by rhinoviruses possible.

The object of the present invention was therefore to provide funds to provide protection against infection by Enable rhinoviruses.

It is possible using hyperimmune sera 50 of a total of 90 rhinovirus serotypes in 16 To divide groups (Conney, M.K., Fox, J.P. and Kenny, G.E., 1982, Infect. Immune. 37, 642-647). Another The form of the division results from the binding to cellular receptors. Despite the pronounced heterogeneity behave in immunological properties namely the rhinoviruses in terms of binding to cells surface receptors similar to each other.  

With the help of competition experiments it was possible to determine that there are 88 rhinovirus strains studied HeLa cells (clone R-19) only have two different receptors (Abraham, G. and Colonno, R.J., 1984, J. Virol. 51, 340-344; Colonno, R.J., Callahan, P.L. and Wong, W.J., 1986, J. Virol. 57, 7-12). This must be restrictive however, it can be said that these results come with HeLa cells have been obtained and not necessarily also for the receptors on the natural host cells in the upper respiratory tract of man are valid.

Another object of the invention was To provide oligopeptides, all or part of the viral proteins correspond to either excitation an immune response against intact viruses used or for the formation and blocking of cellular Receptors can be used.

Rhinoviruses contain a typical Picornavirus single-stranded RNA surrounded by a capsid that consists of 4 polypeptides called VP1 (P1D), VP2 (P1B), VP3 (P1C) and VP4 (P1A) are referred to (Medappa, K.C., McLean, C. and Rueckert, R. R., 1971, Virology 44, 259- 270; the terms in parentheses are the new names according to the nomenclature proposed by Rueckert, R. R. and Wimmer, E., 1984, J. Virol. 50, 957-959). A a single virus particle contains 60 copies of each of these Polypeptides. The relative molecular masses are at different rhinoviruses for VP1 34-36 000, for VP2  27-30,000, for VP3 24-28,000 and for VP4 7-8000 (MacNaughton, M.R., 1982, loc. Cit). It is also for Rhinoviruses characteristic that they are at pH values below 5 are quickly inactivated and sensitive to Salt solutions are high concentration. The show further most rhinoviruses have optimal growth at 33-34 ° C (Luria, S.E., Darnell, Jr., J.E., Baltimore, D. and Campbell, A., 1978, General Virology, Third Edition, John Wiley & Sons, New York, 308 ff.).

The single-stranded RNA with a length of approx. 7100 Nucleotides represent the genome of the virus and can at the same time as a template for the synthesis of viral Serve proteins. Most of the Nucleotide sequence first into a long polyprotein translated, from which by proteolytic cleavage finished viral proteins are formed (Butterworth, B.E., 1973, Virology 56, 439-453; McLean, C. and Rueckert, R. R., 1973, J. Virol. 11, 341-344, Mc Lean, C., Matthews, T.J. and Rueckert, R.R., 1976, J. Virol. 19, 903- 914). In addition to the viral envelope proteins VP1, VP2, VP3 and VP4 another one Series of proteins formed, such as P2A, P2C, P3B, and P3C P3D. P3B (mostly referred to as VPg) is covalent to that 5′-terminal nucleotide of the viral RNA bound. In Analogy to poliovirus, it can be assumed that P2A and P3C proteases are partially used for processing of the viral polyprotein are responsible (MacNaughton, M.R., 1982, loc. cit; Toyoda, H., Nicklin, M.J.H. Murray, M.G., Anderson, C.W., Dunn, J.J., Studier, F.W. and Wimmer, E. 1986, Cell 45, 761-770).  

Accordingly, P3D is the polymerase for replication the viral RNA. About the function of the P2C protein is little known so far.

When processing the viral polyprotein, parts become cleaved the amino acid sequence and then degraded (McLean, Matthew, T.J. and Rueckert, R.R., 1976, loc. cit.) Currently no statement can be made about it whether these peptides are not one yet undiscovered function.

Knowledge of rhinoviruses have been around recently experienced a strong expansion that succeeded is the nucleotide sequence of two human rhinovirus To determine serotypes, namely HRV 2 (German Patent application P 35 05 148.5; Skern, T., Sommergruber, W. Blaas, D. Gruendler, P. Fraundorfer, F., Pieler, C., Fogy, I. and Kuechler, E., 1985, Nucleic Acids Res. 13, 2111- 2126) and HRV 14 (Stanway, G., Hughes, P.J., Mountford, R, C., Minor, P.D. and Almond, J.W., 1984, Nucleic Acids Res. 12, 7859-7875; Callahan, P.L., Mizutani, S. and Colonno, R.J., 1985, Proc. Natl. Acad. Sci. USA 82, 732- 736; European patent application 8 54 01 465.1). Continue was the structure of HRV14 with the help of X-ray structure analysis with a resolution of 3 Å enlightened (Rossmann, M.G., Arnold, E., Erickson, J.W., Frankenberger, E.A., Griffith, J.P., Hecht, H.J. Johnson, J.E., Kramer, G., Luo, M., Mosser, A.G., Rueckert, R.R., Sherry, B. and Vriend, G., 1985, Nature (London) 317, 145- 153) and the position of 4 epitopes in HRV 14 required for who are responsible for neutralization, with the help of HRV 14 mutants resistant to monoclonal antibodies are identified (Sherry, B. Mosser, A.G., Colonno, R. J. and Rueckert, R. R., 1985, J. Virol. 57, 246-257). All of these findings point to great similarity Poliovirus.  

This affects both the sequences of the nucleic acids and of the proteins as well as the virus structure as from the Comparison with the X-ray structure analysis of poliovirus (Hogle, J.M., Chow, M. and Filman, D.J., 1985, Science 229, 1358-1365). This leaves a near Close relationship of rhinoviruses to enteroviruses. Still, one would have expected the two to be humane Rhinoviruses are more similar to each other than to Poliovirus. The fact is that the homology between HRV 2 and HRV 14 is not larger than that in some genes to poliovirus. A possible explanation for the comparatively low degree of kinship between the two rhinoviruses would be the facts that HRV 2 and HRV 14 bind to various cellular receptors and thus represent prototypes for the respective group. Accordingly, other rhinoviruses that target the same should Like HRV 14, receptors also bind with this in sequence are strongly homologous. For this reason, the Sequencing work of the human rhinovirus strain 89 (HRV 89) that belongs to the same receptor group as HRV 14 (Abraham, G. and Colonno, R.J., 1984, loc. Cit.).  

To obtain the viral starting material HeLa cells in a suitable infection medium with HRV 89 infected and several hours with optimal growth incubated conditions.

Virus could come from both the cells and the medium be won.

Various purification steps resulted in a virus preparation, the protein pattern of which is typically shown in FIG. 1.

The viral RNA was made from the virus preparation obtained for example by phenol extraction and cleaned; it was subsequently reversed Transcriptase and oligo dT as primers in cDNA transcribed. The RNA / cDNA hybrids obtained were extended homopolymer and into a suitable plasmid for example the pBR 322 installed.

The use of reverse transcription methods from RNA, for the integration of RNA-cDNA hybrids in plasmids and to transform bacteria are in the literature have been adequately described (Kitamura, N. and Wimmer, E., 1980, Proc. Natl. Acad. Sci. USA 77, 3196-3200; Nelson, T. and Brutlag, D., 1979, Methods in Enzymology 68, 41-50, Zain, S., Sambrook, J., Roberts, J.J. Keller, W., Fried, M. and Dunn, A., 1979, Cell 16, 851- 861; Roychoudhury, R. and Wu, R., 1980, Methods in Enzymology 65, 42-62; Kitamura, N., Semler, B.L., Rothberg P.G., Larsen, G.R., Adler, C.J., Dorner, A.J., Emini, E.A., Hanecak, R., Lee, J.L., van der Werf, S., Anderson, C. W. and Wimmer, E., 1981, Nature (London) 291, 547-553;  van der Werf, S., Bregegere, F., Kopecka, H., Kitamura, N. Rothberg, P.G., Kourilsky, P., Wimmer, E. and Girad., M., 1981, Proc. Natl. Acad. Sci. USA 78, 5983-5987; Namoto, A., Omata, T., Toyoda, H., Kuge, S., Hosie, H., Kataoka, Y., Genba, A., Nakano, Y. and Imura, N., 1982, Proc. Natl. Acad. Sci USA 79, 5793-5797; Stanway, G., Cann, A.J. Hauptmann, R., Hughes, P., Clarke, L.D., Mountford, R.C., Minor, P.D., Schild, G.C. and Almond, J.W., 1983, Nucleic Acids Res. 11, 5629-5643; Skern, T., Sommergruber, W., Blaas, D., Gruendler, P., Fraundorfer, F., Pieler, C., Fogy, I. and Kuechler, E., 1985, loc. cit.)

After the transformation of a suitable one Host organism, for example E. coli. HB 101 became the Plasmid DNA using the "mini plasmid preparation technique" isolated and the size of the recombinant DNA determined. For this purpose, the recombinant plasmids were analyzed using the Restriction endonuclease PstI cut the fragments electrophoretically separated and with known Lambda-HindIII marker DNA compared.

To subclone the DNA, the PstI digestion of the recombinant pBR 322 clones obtained and purified DNA fragments in a suitable vector, for example incorporated into the plasmid pUC 9 and the recombinant Vectors into a suitable host, for example E. coli. JM 101 transformed. The subclones with recombinant Vectors had been successfully transformed grown up and their plasmid DNA isolated and purified.

Three different lengths of primer fragments [54 nucleotides (fragment A), 122 nucleotides (fragment B) and 139 nucleotides (fragment C)] were isolated and purified from three subclones. In addition, a 20 nucleotide synthetic primer (D) was used. Using this complementary single-stranded DNA, a reverse transcript of HRV 89-RNA labeled with ³² P was produced.

For the detection of HRV 89 sequences in recombinant DNA the plasmid DNA was digested with PstI, electrophoterically analyzed and the DNA fragments from the gel Nitrocellulose filter transferred and fixed. These DNA-carrying filters were labeled with the radioactive HRV 89 cDNA hybridizes, the filters subsequently exposed. Of the HRV 89-RNA determined in this way corresponding, recombinant DNA fragments were the Restriction sites were mapped and the DNA sequenced. Man received clones that represent the genome of HRV 89.

The sequence of the HRV 89 genome is shown in Fig. 4. It is shown in the form of the DNA as obtained from the sequencing of the recombinant DNA. To determine the sequence, 21 clones shown in FIG. 3 and an additional 5 clones were used. The sequence comprises 7152 nucleotides, whereby the 3'-terminal poly-A is not counted. It contains an open reading frame which codes for a polyprotein with a length of 2164 amino acids. The reading frame begins with an AUG in position 619 and ends with a UAA stop codon 42 nucleotides before the start of Poly-A.

The 5'-terminal sequence was obtained from clones Nos. 5 and 10, fragment D being used as a primer (see FIG. 3). Cleavage with PstI gave a fragment of constant length. It was determined by sequencing that both clones contained the sequence TTAAAAC immediately subsequent to oligo-G, which comes from the integration site in the plasmid. From this it was concluded that these clones represent the 5'-terminus of the HRV 89 genome. This sequence corresponds to the first 7 nucleotides of the three poliovirus types, Coxsackie virus B1 (Hewlett, MJ and Florkiewicz, RZ, 1980, Proc. Natl. Acad. Sci, USA 77, 303-307; Stanway, G., Cann, AJ, Hauptmann, R., Hughes, P., Clarke, LD, Mountford, RC, Minor, PD, Schild, GC and Almont, JW, 1983, loc.cit.) HRV 2 (Skern, T., Sommergruber , W., Blaas, D., Gruendler, P., Fraundorfer, F., Pieler, C., Fogy, I. and Kuechler, E., 1985, loc. Cit.) And HRV-14 (Stanway, G. , Hughes, PJ, Mountford, RC, Minor, PD and Almond, JW, 1984, loc. Cit.).

Analysis of the 618 nucleotides between the 5'-terminus and the beginning of the long open reading frame shows that The presence of several short reading frames. A comparison the nucleotide sequence of the 5'-terminal region of HRV 89 with that of the HRV 2 shows - taking into account Insertions - a strikingly high degree of homology of 79%, while homology to HRV 14 67% and to poliovirus Type 1 is 62%. The homology is in some Areas particularly large. There are a total of 9 different ones Blocks of 17 or more in a row identical nucleotides in the 5'-terminal region of the HRV 89 and HRV 2 genome present.

The comparison of the amino acid sequences showed that the regions of homology also continue in the region coding for the polyprotein ( FIG. 5). The pronounced homology between HRV 89 and HRV 2 is particularly striking, while surprisingly - contrary to expectations - the homology to HRV 14 is much lower. The highly conserved sections encompass both the envelope proteins and the area of non-structural proteins (P2A-P3D). This obviously means that despite the fact that HRV 89 belongs to the same receptor group as HRV 14, the relationship to HRV 2 is greater. Due to the sequence, HRV 2 and HRV 89 form a common group that clearly distinguishes itself from HRV 14 and poliovirus. This could possibly mean that human rhinoviruses are more similar to each other than was previously assumed based on the homology comparison of HRV 2 and HRV 14. Thus, the proposal to group rhinoviruses and enteroviruses together as one genus within the family of picornaviruses (Stanway, G., Hughes, PJ, Mountford, RC, Minor, PD and Almond, JW, 1984, loc. Cit.) Seems to be in question posed.

The viral proteins are obtained from the polyprotein by proteolytic cleavage. To determine the cleavage sites, the viral coat proteins were isolated and the N-terminal amino acid sequence was determined (see FIG. 6). This not only unambiguously identified the cleavage sites in the viral envelope proteins, but also the reading frame which had been derived from the nucleotide sequence was confirmed. A comparison with the amino acid sequence derived from the nucleotide sequence shows that the VP4 / VP2 cleavage between glutamine and serine, the VP2 / VP3 cleavage between glutamine and glycine and the VP3 / VP1 cleavage between glutamine and asparagine. These cleavage sites are identical to those of HRV 2, but differ from those in HRV 14 (Stanway, G., Hughes, PJ, Mountford, RC, Minor, PD and Almond, JW, 1984 loc. Cit.)

The present invention enables DNA to be obtained generate the information for the viral RNA of HRV 89 contains.

However, the subject of the invention is not only the genetic Sequences specific to the viral proteins code, but also the modifications that for example by mutation, degradation, transposition or Addition can be obtained. Each sequence that is in the Comparison with those shown is degenerate locked in. Even the sequences that are under stringent  Conditions, for example, under conditions that are for more than 85%, preferably more than 90% homology select, with the sequences shown or parts thereof hybridize and for the proteins with the viral Encode activity spectrum are included. The Hybridizations are carried out in 6 × SSC / 5 × Denhardt's solution / 0.1% SDS performed at 65 ° C. The degree of stringency is determined in the washing step. So are the conditions for selection on DNA sequence with approx. 85% or more homology are suitable, as follows: 0.2 × SSC / 0.1% SDS / 65 ° C, for selection on DNA sequences with approximately 90% or more homology: 0.1 × SSC / 0.01% SDS / 65 ° C.

As shown, this DNA can be both complete and incorporated into suitable plasmid vectors in fragments to be suitable after the transformation Host organisms either reproduce the DNA or to achieve the expression of the proteins themselves. Suitable Hosts, vectors and the conditions for these operations are well known to the expert. So are many Published works in which the synthesis of strangers Proteins in bacteria with the help of genetic engineering Methods (review article, see Harris, T. J. R. in "Genetic Engineering", Williamson, R., ed., 1983, Vol. 4. Academic Press, London, 127 ff.). To this For this purpose, the foreign DNA becomes more suitable in the vicinity bacterial control regions (promoters, Ribosome binding sites) of plasmids introduced it make this information possible - mostly in the form of Fusion proteins - to express in high yields and such to win the corresponding proteins. Also on the The Picornavirus area already has a number of Publications in which the expression of viral genes in Bacteria (Küpper, H., Keller, W., Kurz, C., Forss, S., Schaller, H., Franzel, R., Strohmaier, K.,  Marquardt, O., Zaslavsky, V. G. and Hofschneider, P. H., 1981, Nature (London) 289, 555-559; Kleid, D.G., Yansura, D., Small, B., Darbenko, D., Moore, D.M., Grubman, M.J., McKercher, P.D., Morgan, D.O., Robertson, B. H. and Bachrach, H. L., 1981, Science 214, 1125-1129; Wychowski, C., van der Werf, S., Siffert, O., Crainic, R., Bruneau, P. and Girard, M., 1983, EMBO J. 11, 2019-2024; Klump, W., Marquardt, O. and Hofschneider, P.H., 1984, Proc. Nat. Acad. Sci. USA 81, 3351-3355; Hanecak, R., Semler, B.L., Ariga, H. Anderson, C.W. and Wimmer, E., 1984, Cell 37, 1063-1073; (Skern, T., Sommergruber, W., Blaas, D., Gruendler, P., Fraundorfer, F., Pieler, C., Fogy, I. and Kuechler, E., 1985, loc. cit.).

Prokaryotes, for example E. coli, are preferred for expression. K 12, strain 294 (ATCC No. 31 446). Other strains that are suitable include E. coli. X 1 1776 (ATCC No. 31 537). Like the strains mentioned above, E. coli. W 3110 F ^- , Lambda ^- , Prototroph, ATCC No. 27 325), bacilli such as Bacillus subtilis, and other Enterobacteriaceae such as Salmonella typhimurium or Serratia marcescens and various Pseudomonads can be used.

In general, plasmid vectors, the replicon and Control sequences derived from species that are compatible with the host cells are included, in conjunction with these hosts are used. The vector bears usually next to a replication site Recognition sequences that allow in to selectively transform transformed cells. To the Example is E. coli. usually with pBR 322 transformed, a plasmid derived from E. coli. Species originates (Bolivar, et al., Gene 2, 95 (1977)).  

pBR 322 contains genes for ampicillin and tetracycline resistance and thus provides simple means to identify transformed cells. The pBR 322 plasmid or other plasmids must also contain promoters on their own or must be modified in such a way that they contain promoters which can be used by the microbial organism to express their own proteins. The promoters most commonly used in the production of recombinant DNA include beta-lactamase (penicillinase) and lactose promoter systems (Chang et al., Nature 275, 615 (1978); Itakura et al., Science 198, 1056 (1977); Goeddel et al., Nature 281, 544 (1979) and tryptophan (trp) promoter systems (Goeddel et al., Nucleic Acids Res. 8, 4057 (1980); European application, disclosure 00 36 776 While the mentioned promoters are the most common, other microbial promoters have also been developed and used, for example, the gene sequence according to the invention can be used under the control of the leftward promoter of the bacteriophage lambda (P _L ) the promoter which is known to be particularly strong and which can be controlled. The control is made possible by the lambda repressor, from which neighboring restriction interfaces are known.

A temperature sensitive allele of this gene can be inserted into a vector containing a viral DNA sequence. If the temperature is increased to 42 ° C, the repressor is deactivated and the promoter is expressed up to its maximum concentration. The sum of the mRNA produced under these conditions should be sufficient to obtain a cell that contains approximately 10% of its new synthetic ribonucleic acids derived from the P _L promoter.

In this way it is possible to establish a clone bank in which a functional viral DNA sequence is placed in the vicinity of a ribosome binding site at varying distances from the lambda P _L promoter. These clones can then be checked and selected with the highest yield.

Expression and translation of a viral DNA sequence can also be under the control of other regulatory systems as "homologous" to the organism in its untransformed form can expire. So contains e.g. B. chromosomal DNA from a lactose-dependent E. coli. a lactose or lac operon by Release of the enzyme beta-galactosidase Lactose breakdown enables.

The Lac control elements can be from the bacteriophage Lambda pLac 5, the infectious for E. coli. is received will. The phage's lac operon can be by transduction come from the same bacterial species. Regulation systems in the inventive Processes can be used from plasmid DNA originate from the organism. The Lac promoter operator system can be induced by IPTG will.

Other promoter-operator systems or parts thereof can be used just as well: for example arabinose operator, colicine E ₁ operator, galactose operator, alkaline phosphatase operator, trp operator, xylose A operator, tac promoter and the like. Ä.

The genes can preferably be in the expression plasmid pER 103 (Rastl-Dworkin et al., Gene 21, 237-248 (1983); see also European patent application 8 31 12 812.9, deposit DSM 2773, December 20 1983) can be expressed.  

These vectors contain all regulatory elements related to that lead to a high expression rate of the cloned genes.

In addition to prokaryotes, eukaryotic can also be used Microorganisms such as yeast cultures are used. Saccharomyces cerevisiae is the most widely used among the eukaryotic microorganisms, although one Number of other species is commonly available. To Expression in Saccharomyces will be, for example Plasmid YRp 7 (Stinchcomb, et al. Nature 282, 39 (1979); Kingsman et al., Gene 7, 141 (1979); Tschumper, et al., Gene 10, 157 (1980) and the plasmid YEp 13 (Bwach et al., Gene 8, 121-133 (1979)) commonly used. The Plasmid YRp 7 contains the TRP 1 gene, the one Selection marker for a mutant yeast, the is unable to grow in medium containing tryptophan, provides; for example ATCC No. 44076.

The existence of TRP 1 damage as a characteristic of the yeast host genome then represents an effective one Tools to demonstrate the transformation by is cultivated without tryptophan. It is very similar the plasmid YEp 13 that the yeast gene LEU 2, the can be used to supplement a LEU-2 mutant, receives. Suitable promoter sequences for yeast vectors include the 5′-flanking region of ADH I (Ammerer, G., Methods of Enzymology 101, 192-201 (1983), 3-phosphogylcerate kinase (Hitzeman, et al., J. Biol. Chem. 255: 2073 (1980) or other glycolytic enzymes (Kawasaki and Fraenkel, BBRC (Biochem. Biophys. Res. Comm.) 108, 1107-1112 (1982), such as enolase, Glyceraldehyde-3-phosphate dehydrogenase, hexokinase, Pyruvate decarboxylase, phosphofructokinase, glucose-6 Phosphate isomerase and glucokinase.  

In the construction of suitable expression plasmids the termination sequences associated with these genes also in the expression vector at the 3'-end of the expressing sequence can be used to To provide polyadenylation and termination of the mRNA.

Other promoters that also have the advantage of transcription controlled by growth conditions are the promoter regions of alcohol dehydrogenase-2, isocytochrome C, acid phosphatase, degrading enzymes that are coupled with nitrogen metabolism, the glyceraldehyde mentioned above. 3-phosphate dehydrogenase and enzymes that are responsible for the processing of maltose and galactose. Promoters that are regulated by the yeast mating type locus, for example promoters of the BARI, ME α 1, STE 2, STE 3, STE 5 genes, can be used in temperature-regulated systems by using temperature-dependent sir mutations. (Rhine, Ph. D. Thesis, University of Oregon, Eugene, Oregon (1979), Herskowitz and Oshima, The Molecular Biology of the Yeast Saccharomyces, part I, 181-209 (1981), Cold Spring Harbor Laboratory). These mutations influence the expression of the resting mating type cassettes of yeasts and thereby indirectly the mating type dependent promoters. In general, however, any plasmid vector which contains a yeast-compatible promoter, original replication and termination sequences is suitable.

In addition to microorganisms are cultures multicellular organisms are also suitable Host organisms. In principle, each of these cultures usable, whether from vertebrate or invertebrate Animal cultures. However, there is greatest interest Vertebrate cells, so that the multiplication of Vertebrate cells in culture (tissue culture) in recent Years has become a routine method.  

(Tissue Culture, Academic Press, Kruse and Patterson, Editors (1973)). Examples of such useful ones Host cell lines are VERO and HeLa cells, hamster Ovary (CHO) cells and WI 38, BHK, COS-7 and MDCK cell lines. Expression vectors for these cells usually contain (if necessary) a replication place a promoter in front of the gene to be expressed is localized, together with any necessary Ribosome binding site, RNA splice site, Polyadenylation site and transcriptional Termination sequences.

When used in mammalian cells, the Control functions on the expression vectors often made of viral material. For example, come from the polyoma promoters commonly used, Adenovirus 2, and particularly often from Simian Virus 40 (SV 40). The start and end promoters of the SV 40 are particularly useful since both are easily identified as virus Preserve fragment that is also viral Replication site of SV 40 contains (Fiers et al., Nature 273, 113 (1978). Smaller or larger ones can also be used Fragments of SV 40 are used, provided they contain the approximately 250 bp long sequence derived from the HindIII interface to the BglI interface in the viral replication site is sufficient. Besides, it is also possible and recommended, promoter or Use control sequences normally associated with the desired gene sequences are linked, provided these control sequences are compatible with the Host cell systems.

A replication site can either by appropriate Vector construction can be provided to an exogenous Place to install, for example from SV 40 or others viral sources (e.g. Polyoma, Adeno, VSV, etc.)  or may be due to chromosomal replication mechanisms of the host cell are provided. Will the Vector integrated into the host cell chromosome is enough the latter measure mostly.

Transformation of the cells with the vehicles can be done by a Variety of procedures can be achieved. For example they are done by calcium, with either the cells in Magnesium can be washed and the DNA in calcium suspended cells is added or the cells one Coprecipitate of DNA and calcium phosphate exposed will. With subsequent gene expression, the cells transferred to media used for transformed cells select.

After transformation of the host, expression of the Gene and fermentation or cell cultivation under Conditions under which the protein is expressed can the product usually by known chromatographic separation methods are extracted so to obtain a material that contains the viral protein or contains no leader and tailing sequences. The protein can be expressed with a leader sequence at the N-terminus are (pre-protein) removed by some host cells can be. If not, it is a split off of the Leader polypeptide (if any) required to to get mature protein. Alternatively, the mature one Protein can be produced directly in the microorganism. To can the precursor sequence of the yeast mating pheromone MF-alpha-1 used to correct "maturation" of the fused protein and the excretion of the Products in the growth medium or the periplasmic To ensure space. The DNA sequence for functional or mature protein can be suspected with MF-alpha-1 Interface.  

In addition to using the DNA according to the invention for Production of the proteins in question in bacteria or eukaryotic cells can also serve them out the amino acid sequences derived from the nucleotide sequence synthetically, in whole or in part.

These oligopeptides can then, like the genetically engineered proteins either for Suggestion of one directed against intact viruses Immune response used or for binding and blocking of cellular receptors. Studies on the use of oligopeptides to stimulate an against Poliovirus-directed immune responses have been published (Emini, E.A., Jameson, B.A. and Wimmer, E., 1983, Nature (London) 30, 669-703); similar studies are also in foot-and-mouth disease viruses (Bittle, J.L., Houghton, R.A., Alexander, H., Shinnick, T.M., Sutcliffe, J.G., Lerner, R.A., Rowlands, D.J. and Brown, F., 1982, Nature (London), 298, 30-33; Pfaff, E., Mussgay, M., Böhm, H. O., Schulz, G. E. and Schaller, H., 1982, EMBO J. 1, 869-874). This invention extends also on the oligo- and polypeptide parts of HRV 89 proteins used as a result of synthesis or Application purposes, e.g. B. as a vaccine with others Oligo or polypeptides are linked.

Other properties and characteristics of the present Invention are described in the following examples, the embodiments of the present invention concern and not restrict them.

Used in the context of this notification of biological activity spoken in connection with proteins (polypeptides), this means that the protein (polypeptide) in question stimulate an immune response in a biological test and / or any reaction with the cellular receptors for the rhinoviruses.  

Legend for the illustrations

Fig. 1 Electrophoretic separation of the envelope proteins of HRV 89 on a 12.5% polyacrylamide gel in the presence of sodium dodecyl sulfate. VP4 is not visible because the front of it has migrated out of the gel.

Fig. 2 electrophoresis of HRV 89-RNA on a 1.2% agarose gel in the presence of sodium dodecyl sulfate. The positions of the ribosomal RNA markers are shown on the right.

Fig. 3 restriction map of the HRV 89 genome. 21 overlapping clones used for sequencing are shown. Characteristic interfaces of some restriction enzymes are listed. The arrows (AD) represent fragments that were used as primers for reverse transcription.

Fig. 4 Sequence of the cloned HRV 89 genome and the amino acid sequence derived therefrom. Cleavage points in the polyprotein are represented by arrows. Filled arrows (↓) indicate experimentally determined cleavage sites, open arrows (↓) indicate predicted cleavage sites in the polyprotein due to the homology with other Picornaviruses.

Fig. 5 Comparison of homology in the amino acid sequence (in%) of individual genes between HRV 89, HRV 2, HRV 14 and poliovirus type 1.

Fig. 6 N-terminal amino acid sequences of the envelope proteins of HRV 89th

example 1 Preparation of HRV 89

HeLa cells (HeLa-Ohio strain, 03-147, Flow Laboratories, England) were grown in suspension at 37 ° C. The suspension medium (Thomas, DC, Conant, RM and Hamparian, VU, 1970, Proc. Soc. Exp. Biol. Med. 133, 62-65; Stott, EJ and Heath, GF, 1970, J. Gen. Virol, 6 , 15-24) consisted of a Joklik modification of MEM for suspension (Gibco 072-1300) and 7% horse serum (Seromed 0135). The inoculation density was 5-10 × 10 ⁴ cells / ml, the volume 500 ml. The suspension was centrifuged at a cell density of 1 × 10 ⁶ cells / ml under sterile conditions at 300 g for 10 min. The supernatant was aspirated and the cells were resuspended in 100 ml of infection medium (Joklik modification of MEM for suspension culture with 2% horse serum and 2 mM MgCl ₂ ). The cells were distributed homogeneously in the infection medium by repeated careful suction in a 20 ml pipette. It was then made up to 500 ml. The cell suspension was then brought to 34 ° C. and infected with HRV 89 (twice plaque-cleaned) at a multiplicity of 0.1 viruses / cell. The HRV 89 strain was obtained from the American Type Culture Collection (ATCC VR-1199). The strain used was neutralized by antiserum against HRV 89 (American Type Culture Collection, Cat. No. ATCC VR-1199 AS / GP). An antiserum against HRV 2 (Cat. No. ATCC VR-1112 AS / GP), which showed no neutralization, served as control serum. The virus was harvested after 60 hours at 34 ° C. Virus was obtained from the cells or cell fragments as well as from the medium.

For this purpose, the medium from infected cells and cell fragments by a 10 min centrifugation 1500 g separated and suction filtered. The precipitation was frozen at -70 ° C.

The cell precipitates from 12 l suspension culture were combined, resuspended in 40 ml TM buffer (20 mM Tris / HCl, pH 7.5, 2 mM MgCl ₂ ), placed on ice for 15 min, then broken up in a Dounce homogenizer and the mixture 30 centrifuged at 6000 g for min. The precipitate was then washed again in 10 ml of TM buffer. The two supernatants were combined and centrifuged at 110,000 g for 3 hours to pellet the virus. The virus pellet was then taken up in 10 ml of KTMP buffer (50 mM KCl, 50 mM Tris / HCl, pH 7.5, 5 mM MgCl ₂ , 2 mM mercaptoethanol, 1 mM puromycin, 0.5 mM GTP) and after the addition of 150 µg DNase I (Sigma, ribonuclease free) incubated on ice for 1 hour.

The virus was stirred in from the infection medium 4 ° C with polyethylene glycol 6000 (PEG 6000; Merck) at one Concentration of 7% and 450 mM NaCl precipitated (Korant, B. D., Lonberg-Holm, K., Noble, J. and Stasny, J. T., 1972, Virology 48, 71-86). After 4 hours in the cold centrifuged the virus at 1500 g for 30 min, the Precipitation in 10 ml KTMP buffer containing 75 µg DNase I contained, resuspended, the mixture on ice for 1 hour incubated and then frozen at -70 ° C.

The virus suspension coming from the cells and from the medium were pooled for 5 min at 37 ° C incubated by adding 60 ml of cold TE buffer (10 mM Tris / HCl, pH 7.4, 1mM EDTA) cooled and then sonicated in an ice bath for 5 minutes.  

The mixture was then centrifuged at 6000 g for 30 min. 920 ml of TE buffer containing 7% PEG 6000 and 450 mM NaCl were added to the supernatant, the mixture was carefully stirred at 4 ° C. for 4 hours and the precipitate formed was pelleted at 6000 g for 30 minutes. The precipitate was again taken up in 100 ml of TM buffer, the virus was precipitated as above by adding PEG 6000 and NaCl and pelleted. The precipitate was resuspended in 40 ml of TM buffer, the suspension was centrifuged at 6000 g for 30 min, and the virus was pelleted at 110,000 g for 3 hours. The precipitate was dissolved in 1 ml of TM buffer, after the addition of 50 μg DNase I incubated at 4 ° C. for 1 hour and then 1 ml of TE buffer was added. For further purification, the virus suspension was centrifuged on sucrose gradients (10-30% w / w in TE buffer) for 4 hours at 4 ° C. at 110,000 g. The fractions containing the virus were determined from the absorbance at 260 nm and diluted with TM buffer so that the final concentration of sucrose was 10%. The mixture was then centrifuged at 85,000 g for 8 hours. The virus pellet was taken up in 1 ml of TM buffer and stored at -70 ° C. To check the purity of the virus preparation, electrophoresis was carried out on a 12.5% polyacrylamide gel in the presence of 0.1% sodium dodecyl sulfate (Laemmli, UK, 1970 Nature (London) 277, 680-685) and the protein bands with Coomassie Brilliant blue colored. A typical image of the protein pattern of a preparation of HRV 89 is shown in FIG. 1.

Example 2 Cloning of the cDNA-RNA hybrid RNA extraction from the HRV 89 preparation

The virus was dissolved in 1 ml NTES buffer (100 mM NaCl, 10mM Tris / HCl, pH 9, 1mM EDTA, 0.1% Sodium dodecyl sulfate) suspended and with phenol extracted. Chloroform was used for better phase separation added. 20 μl of 5 M NaCl and 20 ul 3 M sodium acetate (pH 5.6) added and the viral RNA precipitated with twice the volume of ethanol.

In order to remove the VPg covalently bound to the 5'-end of the viral RNA, the RNA was then digested in NTES buffer with 1 mg / ml Proteinase K (Merck) for 15 min at 37 ° C. In order to remove contaminations from ribonuclease, the proteinase K stock solution (50 mg / ml) had been preincubated at 37 ° C. for 15 minutes before use. After proteinase K digestion was complete, the solution was extracted with phenol / chloroform as described above and the RNA was precipitated by adding ethanol. A small part of the RNA was then electrophoresed on a 2% agarose gel in TAE buffer (10 mM Na acetate, 40 mM Tris / acetate, pH 8.2, 2 mM EDTA) with 0.1% sodium dodecyl sulfate. After staining with ethidium bromide, the band of intact HRV 89 RNA was visible ( Fig. 2). A weak, diffuse band underneath indicated a slight degradation of HRV 89.

Reverse transcription of HRV 89 RNA with oligo-dT as a primer

4 µg HRV 89-RNA were dissolved in 10 µl H ₂ O, 5 µl 10 × RT buffer (1 × RT buffer = 100 mM KCl, 110 mM MgCl ₂ , 50 mM Tris / HCl, pH 8.3), 5 µg oligo-dT (12-18) (Pharmacia PL Biochemicals), 10 µCi [ α ³² P] -dCTP (3000 Ci / mmol Amersham International, England), 100 U reverse transcriptase (Anglian Biotechnology Co, Cambridge) and 20 each nmol of dATP, dGTP, dTTP, dCTP added, and incubated in a total volume of 50 μl for 2 hours at 42 ° C.
After adding 2 .mu.l 250 mM EDTA (pH 8) was extracted with phenol / chloroform and the aqueous phase applied to a Biogel P 30 (or Sephadex G-25) column in a Pasteur pipette. A TE buffer was used for elution. The formed cDNA-RNA hybrid was so excess [α ³² P] dCTP separated and precipitated (pH 5.6) and 2 parts by volume of ethanol after addition of ¹ / _10th volume 3 M sodium acetate units.

Homopolymeric extension of the HRV 89-RNA-cDNA hybrids ("Tailing")

The extension of the HRV 89-RNA-cDNA hybrids was carried out according to the method of Roychoudhury and Wu (1980, loc. Cit.). For this, the HRV 89-RNA-cDNA hybrid was dissolved in 50 µl TT buffer (200 mM potassium cocodylate, 25 mM Tris / HCl, pH 6.9, 2 mM MnCl ₂ , 2 mM dithiothreitol, in the presence of 2 nmol [ α - ³² P] -dCTP (5 Ci / nmol) with 25 U terminal transferase (Pharmacia PL Biochemicals) incubated for 5 min at 37 ° C (Deng, GR and Wu, R., 1983, method Enzymol. Vol. 100, Part B, 96-116) After addition of 2 μl of 0.25 M EDTA (pH 8), the mixture was extracted with phenol / chloroform, the reaction mixture was then chromatographed on a Biogel P 30 column, as described above, and the oligo-dC-carrying RNA cDNA hybrid precipitated with ethanol.

Incorporation of the oligo-dC-bearing HRV 89-RNA-cDNA hybrid into the Plasmid pBR 322 ("annealing") and transformation of Escherichia coli HB 101

The oligo-dC-bearing RNA-cDNA hybrid was in 100 ul NTE buffer (100mM NaCl, 10mM Tris / HCl, pH 7.6, 1mM EDTA) with 0.3 pmol pBR 322 plasmid (that with PstI was cut and polymerized onto the oligo-dG residues were; Bethesda Research Laboratories), only 5 min at 65 ° C, then heated at 42 ° C for 2 hours, overnight slowly cooled to room temperature and at 4 ° C kept.

For cell transformation, strain HB 101 (DSM 1607) was grown in 50 ml LB medium (10 g tryptone, 5 g yeast extract, 10 g NaCl in 1 l) (Mandel, M. and Higa, A., 1979, J. Mol. Biol. 53, 159-162). In order to obtain cells ("competent cells") suitable for the transformation, the bacteria were pelleted and in 25 ml of TR buffer (150 mM KCl, 50 mM CaCl ₂ , 1 mM Tris / HCl, pH 7, 3 mM MgCl ₂ ) taken up, placed on ice for 30 min, centrifuged again, resuspended in 2 ml of TR buffer and placed on ice for 1 hour. 100 .mu.l of the mixture containing pBR 322 with the inserted HRV 89-RNA-cDNA hybrid and 5 .mu.l of 1 M CaCl _{2 were} added to 200 .mu.l of the cell suspension, at 0.degree. C. for 1 hour and then at 42.degree. C. for 90 seconds incubated. Then 2 ml LB medium was added and incubated for 1 hour at 37 ° C. The cell suspension was applied to LB agar plates (1.5% agar in LB medium) containing 10 μg / ml tetracycline (Sigma) and incubated overnight.
Tetracycline-resistant clones were then tested on ampicillin agar plates (100 µg ampicillin / ml; Sigma) for ampicillin sensitivity.

Characterization and isolation of the recombinant DNA molecules

Clones of tetracycline-resistant, ampicillin-sensitive bacteria were grown up in 6 ml LB medium (10 μg tetracycline / ml) overnight, plasmid DNA was isolated using the "mini-plasmid preparation technique" (Birnboim, HC and Doly, J., 1979, Nucleic Acids Res. 7, 1195-1204) and the size of the recombinant DNA was determined by digestion with the restriction enzyme PstI. The plasmid DNA was in 25 ul RE buffer (6 mM MgCl ₂ , 10 mM Tris / HCl, pH 7.5, 6 mM mercaptoethanol) with 50 mM NaCl in the presence of 2 U of the restriction enzyme PstI (Bethesda Research Laboratories) and 5 µg ribonuclease A incubated at 37 ° C for 2 hours. The samples were then electrophoresed on a 1.4% agarose gel. The sizes of the inserts could be determined by staining with ethidium bromide and comparison with Lambda HindIII marker DNA. These were between 300 and 3300 base pairs.

In order to isolate larger amounts of DNA inserts, Plasmids as described above from 200 ml cultures of Tetracycline-resistant, ampicillin-sensitive Bacterial clones obtained and digested with PstI. The recombinant DNA fragments were as described above, The bands separated on a preparative agarose gel cut out the DNA in 0.5 × TBE buffer (1 × TBE buffer = 100 mM Tris / borate, pH 8.3, 2 mM EDTA) electroeluted and precipitated with ethanol.  

Subcloning in Escherichia coli strain JM 101- Cells using the pUC 9 vector

The plasmid pUC 9 (Vieisa, J. and Messing, JG 1982, Gene 19, 259-268) contains a gene for ampicillin resistance, a region for the start of replication, which originate from the plasmid pBR 322, and part of the E. coli Lac-Z gene. A small section of DNA containing a number of restriction sites is in this Lac-Z region, so cloning DNA in one of these sites disrupts the Lac-Z gene region. Colonies containing DNA inserts therefore appear on X-Gal (X-Gal = 5-bromo-4-chloro-3-indolyl- β- D-galactoside, Bethesda Research Laboratories) - indicator plates white, those without inserts appear blue (Rüther, U., 1980, Mol. Gen. Genet. 178, 475-478). In order to subclone DNA inserts into pUC 9, recombinant pBR 322 clones (approx. 7 μg) were digested with PstI and, after separation on the agarose gel (1.2% -1.4%), the DNA inserts from the vector -DNA separated. The DNA was recovered from the gel by electroelution and ethanol precipitation as described above. The isolated DNA insert was in 20 ul RE buffer with 0.4 ug pUC 9 vector (cut with PstI and pretreated with bacterial alkaline phosphatase), in the presence of 1 mM ATP and 3 UT ₄ ligase (Bethesda Research Laboratories) Incubated for 1 hour at 15 ° C and stored at 4 ° C (Vieisa, J. and Messing, JG, 1982, loc. Cit). At the same time, E. coli. Strain JM 101 cells that were competent for transformation were prepared in the manner described above. 200 µl of the competent cell suspension were mixed with 20 µl mixture of the pUC 9 ligase reaction and incubated for 1 hour at 0 ° C.

After a heat shock (90 sec, 42 ° C) the cells with 10 µl 200 mM isopropylethia galactoside (Sigma), 50 µl of a solution of 20 mg X-Gal in 1 ml Dimethylformamide and 1 ml LB medium and 1 hour incubated at 37 ° C. 200 µl each of this cell suspension were then placed on an ampicillin LB agar plate (100 µg / ml) transferred and overnight at 37 ° C in Incubator incubated. Positive transformants were considered white column identified using the "Mini-plasmid preparation technique" for DNA inserts were examined.

Preparation of pUC 9 plasmids with inserts from recombinant DNA

The E. coli subclones obtained. JM 101 with pUC 9 were transformed and contained DNA inserts were in 200 ml LB medium (with 100 µg ampicillin / ml) grown up and the plasmid DNA isolated (Birnboim, H.C. and Doly, J., 1979 loc. cit.) The plasmid DNA was then dissolved in 100 µl TE buffer and the solution over a Sephacryl 1000 column (1 × 20 cm) with TE buffer chromatographed. The fractions that cleaned up Plasmid contained were based on the absorbance localized, pooled and lyophilized. The plasmid was taken up in 500 ul TE buffer, 5 min at 65 ° C. incubated and extracted again with phenol / chloroform and precipitated with ethanol.

Extraction of primer fragments from the plasmids pHRV 89-100, pHRV 89-136 and pHRV 89-193 on synthetic ways

Clones 100, 136 and 193, which contained corresponding DNA inserts, were subcloned into pUC 9, grown up in 200 ml LB medium (with 100 μg ampicillin / ml) and the plasmids isolated as described above. The primer fragment (54 nucleotides) from clone 100 was obtained by digestion with the restriction enzymes NcoI and PvuII (the primer fragment is designated as fragment A in FIG. 3). For this purpose, 200 μg of purified plasmid from clone 100 in 200 μl of RE buffer in the presence of 50 mM NaCl with 30 U NcoI (New England Biolabs.) Were incubated at 37 ° C. for 15 hours. After the reaction had ended, ethanol was precipitated and the cut plasmid in 100 .mu.l RE buffer in the presence of 50 mM NaCl with 20 U PvuII (New England Biolabs) incubated at 37 ° C for 15 hours. The course of the digestion with restriction enzymes was checked by electrophoresis on 1.4% agarose gels. The NcoI / PvuII fragment was taken up in 100 ul OG solution (1% Ficoll, 1mM EDTA, 0.01% Orange G) and the DNA on a preparative 15% polyacrylamide gel (acrylamide / bisacrylamide = 19: 1 , Gel thickness 1.2 mm) in 1 × TBE buffer. The band corresponding to the 54 base NcoI / PvuII fragment was cut out after ethidium bromide staining, the fragment was electroeluted in 0.5 × TBE buffer and the DNA was precipitated with ethanol. The DNA fragment was then incubated in 50 μl of 100 mM Tris / HCl, pH 8, with 200 U of bacterial alkaline phosphatase (Bethesda Research Laboratories) at 65 ° C. for 1 hour. After the reaction, 2.5 μl of 0.5 mM EDTA, pH 8 were added, the aqueous phase was extracted twice with phenol / chloroform and the DNA was precipitated by ethanol precipitation. The DNA was dissolved in water and in 50 µl K buffer (10 mM MgCl ₂ , 50 mM Tris / HCl, pH 8.5, 5 mM dithioerythritol) with 20 µCi γ - [ ³² P] -ATP (specific activity 5000 Ci / mmol; Amersham International) and 5 U polynucleotide kinase (Pharmacia-PL Laboratories) at 37 ° C for 30 min.

The fragment labeled with ³² P was then precipitated and taken up in 30 μl of 30% dimethyl sulfoxide which contained 1 mM EDTA and 0.01% xylene cyanogen bromophenol blue. This solution was incubated at 90 ° C. for 2 min, cooled in ice, applied to a 15% polyacrylamide gel (acrylamide / bisacrylamide = 59: 1, Gelicke 1.2 mm) in TBE buffer and the two DNA strands were electrophoresed separated from each other (15 hours at 200 volts). The two strands were located, cut out and electroeluted using the autoradiogram. The strand hybridizing with HRV 89-RNA was determined by a "dot blot" experiment. Two 2 × 2 cm strips of nitrocellulose (Schleicher & Schüll, BA 85, 0.45 µm) were moistened with H ₂ O, once with 20 × SSC (1 × SSC = 150 mM NaCl, 15 mM sodium citrate, pH 7.4) ) washed and air dried. Approximately 1 µg of HRV 89-RNA was applied in spots to each strip, dried and incubated at 80 ° C. for 2 hours. The strips were then moistened with 2 × SSC and in a plastic film together in 1 ml of H buffer (400 mM NaCl, 40 mM PIPES, pH 6.4, 1 mM EDTA, 80% formamide) with 4 μg denatured salmon sperm DNA (Sigma; incubated at 100 ° C for 2 min and cooled to 0 ° C) incubated at 42 ° C for 1 hour. The two nitrocellulose strips were then sealed separately in plastic films with 0.5 ml H buffer, 4 µg denatured salmon sperm DNA and an aliquot of the isolated strands (20,000 cpm) and hybridized overnight at 42 ° C. After this incubation, the filters were washed twice with 2 × SSC for 10 minutes at 50 ° C. and twice with 0.1 × SSC, 0.1% sodium dodecyl sulfate for 30 minutes at 50 ° C. and the radioactivity was determined.

The primer fragments from HRV 89-136 and HRV 89-193 were isolated in the same way. The fragment from pHRV 89-136 (designated as fragment B in FIG. 3) lies between a DraI and a HindIII restriction site (122 nucleotides) and was obtained by digestion with these restriction enzymes. Fragment C was isolated from pHRV 89-193, is 139 nucleotides long and lies between two RsaI restriction sites ( Fig. 3). Strand separation and hybridization were performed as described above. Primer D was chemically synthesized using established methods on an Applied Biosystems device.

Reverse transcription of HRV 89 RNA using Primer fragments

5 pmol of the single-stranded DNA complementary to HRV 89-RNA, which, as described above, were isolated or chemically synthesized from clones 100 (fragment A), 136 (fragment B) and 193 (fragment C), were each together with 0, 25 pmol of the HRV 89-RNA precipitated from an aqueous solution with ethanol. The precipitate was taken up in 20 μl of H buffer, welded into a capillary, incubated at 72 ° C. for 10 min, transferred to 50 ° C., slowly cooled to 35 ° C. and then placed on ice. The solution was then in 100 ul RT buffer in the presence of 140 U reverse transcriptase (Anglian Biotechnology Co, Cambridge), 8 U ribonuclease inhibitor (RNasin, Bethesda Research Laboratories), each 0.2 mM dATP, dCTP, dGTP and dTTP , 30 µCi [ α ³² P] -dCTP and 5 mM dithioerythritol incubated at 42 ° C for 2 hours. The resulting reverse transcript was processed as described above.

Detection of HRV 89 sequences in recombinant DNA and Restriction mapping

Plasmid DNA from the recombinants was isolated from 3 ml cultures (Birnboim, HC and Doly, J. 1979, loc. Cit.). The DNA was then incubated with the restriction enzyme PstI and the samples were analyzed by electrophoresis on 1.4% agarose gel. The gels were stained with ethidium bromide. The DNA was then transferred from the gel to nitrocellulose filters. (Southern, EM, 1975, J. Mol. Biol. 98, 503-517) and fixed on the nitrocellulose by a 2 hour incubation at 80 ° C. The filters were washed in 50% formamide, 1x Denhardts solution (Denhardt, DT, 1966, Biochem. Biophys. Res. Comm. 23, 641-646), 900 mM NaCl, 50 mM sodium phosphate, pH 7.4, 5 mM EDTA pre-incubated with 80 µg / ml denatured salmon sperm DNA for 2 hours at 42 ° C in a plastic wrap. Radioactive HRV 89 cDNA was prepared as described above, only the reaction set contained 50 µCi [ α - ³² P] dCTP. The cDNA-HRV 89-RNA hybrid was denatured at 100 ° C for 90 seconds. For hybridization, the filters were incubated with radioactive HRV 89 cDNA, as described above, at 42 ° C. for 18 hours, then washed twice in 2 × SSC and twice in 0.1% sodium dodecyl sulfate at 50 ° C. for 30 minutes at which Air dried and exposed at -70 ° C (Kodak XAR-5, with intensifying screen 18-40 hours). The appearance of a radioactive band indicated the presence of recombinant DNA that was complementary to HRV 89 RNA.

For mapping, the DNA inserts were digested using restriction enzymes (New England Biolabs and Bethesda Research Laboratories) using the incubation conditions specified by the manufacturers. The result of the restriction mapping is shown in Fig. 3. The plasmids pHRV 89-80 and pHRV 89-82 contained immediately after the oligo-C, which is due to the chain extension by the terminal transferase reaction - a longer sequence of A residues, which is a part of the 3'-terminal poly -A represent the HRV 89 RNA. The other plasmids were arranged relative to pHRV 89-80 and pHRV 89-82 with the aid of characteristic cleavage sites of individual restriction enzymes. DNA inserts that were smaller than 500 base pairs were not classified by restriction enzyme mapping, but were immediately subcloned into pUC 9 and sequenced (see Example 3).

The identification of the remaining clones is obtained by colony hybridization using already mapped DNA inserts as "nick translation" samples according to the method of Grunstein and Hogness (Grunstein, M. and Hogness, DS, 1975, Proc. Natl. Acad. Sci USA 72, 3961-3965). ³² P-labeled DNA samples are obtained using a "nick translation kit" from Amersham International (England; Amersham Kit No. 5000) according to the manufacturer's instructions with [ α - ³² P] -dCTP (3000 Ci / mmol ). The labeled DNA was separated on a Biogel P-30 column in a Pasteur pipette in TE buffer. Fractions corresponding to the exclusion volume were pooled, heated at 100 ° C for 2 min and quickly placed in ice water. The hybridization was carried out as described above. Colonies which showed a positive hybridization signal were prepared from 50 ml cultures (overnight in LB medium with 10 μg tetracycline / ml) and the DNA inserts were isolated from the plasmid DNA using PstI. These were then characterized by digestion with various restriction enzymes and by sequencing. In this way, clones were obtained that represent the genome of HRV 89.

Example 3 DNA sequencing

HRV 89 cDNA clones were obtained after modification of the Maxam and Gilbert's method (Maxam, A. and Gilbert, W., 1980, Methods Enzymol. 65, 499-560) or after M 13 chain termination method according to Sanger et al. (Sanger, F., Nicklen, S. and Coulsen, A.R., 1977, Proc. Natl. Acad. Sci USA 74, 5463-5467).  

For Maxam and Gilbert sequencing, the DNA inserts were subcloned into pUC-9 as described above and thus competent E. coli. JM 101 cells transformed. Positive transformants were isolated as white colonies and grown up in LB medium (with 100 μg / ml ampicillin). 10-20 fig. Μg DNA were digested in 100 µl overnight under standard reaction conditions with restriction enzymes, which cleaved a site in the plasmid z. B. in the polyclinic region of pUC 9 (z. B. BamHI, EcoRI, AccI, HindIII). The restricted fragment was then dephosphorylated. For the restriction digestion, 5 μl of 2 M Tris / HCl, pH 8 and 100 U of bacterial, alkaline phosphatase (Bethesda Research Laboratories) were added and incubated at 65 ° C. for 3 hours. After adding EDTA to 20 mM, the mixture was extracted twice with phenol / chloroform and the DNA was precipitated by ethanol. The DNA was then in 50 µl 50 mM Tris / HCl, pH 8, 10 mM MgCl ₂ , 5 mM dithioerythritol, with 25 µCi [ α - ³² P] -ATP (5000 Ci / mmol, Amersham International) and 4 UT ₄ - Incubated polynucleotide kinase (Pharmacia-PL Biochemicals) at 37 ° C for 30 min and the labeled DNA was precipitated with ethanol. With the help of another restriction enzyme that cleaves in the polylinker region of pUC 9, recombinant DNA, which was labeled with ³² P in one strand, was electrophoresed in agarose gel (1.2-1.4%) with subsequent electroelution and ethanol precipitation as described above.

DNA sequencing after a modification of the method of Maxam and Gilbert

The sequencing reaction was carried out with the following Modifications according to Maxam and Gilbert (Maxam, A. and Gilbert, W., 1980, loc. cit.):  

• - No carrier DNA was added.
• - The DNA solution was divided into aliquots: guanine (G) -specific reaction 7.5 µl, guanine and adenine (G / A) -specific reaction 10 µl, cytosine and thymine (C / T) -specific reaction 10 µl, cytosine (C) - specific reaction 6 µl.
• - The (G / A) reaction mixture was 25 µl 96% Formic acid added and the mixture at 4.25 min Incubated at 19 ° C. To stop the reaction, were 200 µl hydrazine stop solution and 750 µl 96% ethanol added. The (G / A) reactions were then the same as treated all 3 other reactions.
• - Instead of hydrazine, hydrazine hydroxide (Merck) used in the (C / T) and C reactions. The Response times were 7.5 minutes.
• - The piperidine reactions were at 95 ° C for 30 min incubated. After lyophilization, the fragments were in 3-20 µl buffer (80% deionized formamide, 1 × TBE, 0.05% bromophenol blue and 0.05% xylene cyanol) 90 sec heated to 95 ° C and quickly cooled to 0 ° C.

6% polyacrylamide gels were used for sequencing (40 cm × 20 cm × 0.4 mm) with 8 M urea and 1 × TBE used at 1 hour before applying the samples 50 watts were subjected to electrophoresis. Between 1 µl and 3 µl of each reaction batch were applied to the Gel applied. Usually two were timed shifted gel loads carried out.  

The first run of a reaction took so long until the bromophenol blue marker, the second to the Xylene cyanol marker reached the end of the gel. The gels were then 20 min in 10% acetic acid and 10% methanol (approx. 2 liters) fixed, transferred to 3 MM filter paper and dried at 80 ° C on a gel dryer. Then were using the gels without intensifying screen at -70 ° C of an XAR-5 film (Kodak) exposed (approx. 18 to 36 Hours).

M 13 sequencing

DNA inserts with a size greater than 1000 bp were sequenced by the "shotgun" method (Deininger, P. L. 1983, Anal. Biochem. 129, 216-223). For that were recombinant clones with suitable restriction enzymes digested the DNA inserts as described above Separation on the preparative agarose gel (1.2-1.4%) with subsequent electroelution and ethanol precipitation isolated.

The DNA inserts were present in 20 μl RE buffer of 1 mM ATP and 3 U T 4 DNA ligase (New England Biolabs) 15 Incubated at 14 ° for hours. The circularized DNA was with TE buffer (10 mM Tris / HCl, pH 7.5, 1 mM ETDA) Final volume of 500 µl diluted, with ice cooling sonicated (4 times 5 seconds maximum energy; MSE ultrasonic power unit) and precipitated with ethanol.

The ends of the DNA fragments obtained were in 20 ul RE buffer, each of 50 µM dATP, dCTP, dGTP and dTTP and 2 U Klenow fragment (Boehringer Mannheim) contained Incubated for 2 hours at 14 ° C. After separation on Agarose gels (1.4%) were DNA fragments with a size of 400-900 bp by electroelution and ethanol precipitation recovered.  

These DNA fragments were inserted into the M 13 mp 9 vector linearized the restriction enzyme Sma I and with bacterial alkaline phosphatase had been treated (Insert: vector = 5: 1), in the presence of 1 mM ATP and 1 U T 4 DNA ligase inserted (15 h at 15 ° C).

E. coli competent for transformation. Strain JM 101 Cells were mixed with the ligase reaction incubated at 0 ° C for at least 2 hours. After one Heat shock (90 sec, 42 ° C) was the cells with 40 ul 100 mM IPTG, 40 µl of a solution of 2% X-Gal in DMF and 3 ml top agard (10 g tryptone, 8 g NaCl, 8 g agar in 1 l), which had been melted and heated to 42 ° C, mixed and on slightly preheated H-plates (10 g Trypton, 8 g NaCl, 12 g agar in 1 l) applied. To The plates solidified at 37 ° C above the agar Incubated overnight.

One was used to isolate the single-strand phage DNA colorless plaque in a tube with 1.5 ml one diluted E. coli. JM 101 culture (1 ml stationary culture in 100 ml 2 × TY medium diluted (16 g tryptone, 10 g Yeast extract, 5 g NaCl in 1 l)) transferred. After 6 The bacteria were incubated for hours at 37 ° C centrifuged and the phage from the supernatant Add 200 µl of a solution of 20% PEG 8000, 2.5 M NaCl precipitated for 15 min at room temperature. The Precipitate was pelleted and the supernatant was left completely away. The pellet was dissolved in 100 ul TE buffer, with phenol / chloroform - as already described - extracted and the single-stranded DNA from the aqueous Phase precipitated with Na acetate / ethanol. The size of everyone Single-stranded DNA was analyzed by 0.8% electrophoresis Agarose gel determined relative to the wild-type phage DNA. The Sequencing was carried out without modifications after the Chain termination method carried out (Sanger, F., Nicklen, S. and Coulsen, A. S., 1977, loc. cit.)  

Analysis of the sequencing data

The sequencing results were done using a cyber 170 computers analyzed. The programs of Staden (Staden, R. 1980, Nucleic Acids Res. 8, 3673-3694) and the modified forms of Isono (Isono, K. 1982, Nucleic Acids Res. 10, 85-89).

Example 4 Proteolytic cleavage sites in the P1 coat protein region of the polyprotein

The viral proteins are obtained from the polyprotein by proteolytic cleavage. In order to identify the cleavage sites, the viral coat proteins were isolated and the N-terminal amino acid sequence was determined. For this purpose, the proteins of 2 mg HRV 89 were separated electrophoretically on a 12.5% polyacrylamide gel (Laemmli, UK, 1970, loc. Cit.). The gels were stained with a saturated solution of Coomassie brilliant blue in 50 mM Tris / HCl, pH 7.4, and the protein bands were cut out. The individual proteins were electroeluted in an ISCO elution apparatus at 50 V for 16 hours and precipitated with trichloroacetic acid. The N-terminal amino acids were determined using an AB-470A protein sequencer (Applied Biosystems, Inc. Foster City, CA, USA). 2 nmol VP1 and VP2 and 1 nmol VP3 were used for sequencing. The derivatized amino acids were analyzed by HPLC. The N-terminal sequences of the individual proteins are given in FIG. 6.

Claims (50)

1. DNA molecule coding for at least one viral Protein of the rhinovirus strain HRV 89. 2. DNA molecule according to claim 1, characterized in that that it is the total viral RNA of the rhinovirus strain HRV 89 corresponds. 3. DNA molecule according to claim 1, characterized in that it shares the viral RNA of the rhinovirus strain HRV 89 corresponds. 4. DNA molecule according to claim 1, characterized in that it is a viral protein composed of the viral proteins VP1, VP2, VP3, VP4, P2A, P2B, P2C, P3A and P3C, encoded. 5. DNA molecule according to claim 4, characterized in that it is for at least two of the above, in any Combination of linked viral proteins coded. 6. DNA molecule according to claim 1, containing the sequence according to FIG. 4. 7. DNA molecule according to claim 1, characterized in that that it codes for the viral protein VP1. 8. DNA molecule according to claim 1, characterized in that it encodes the viral protein VP2.   9. DNA molecule according to claim 1, characterized in that that it codes for the viral protein VP3. 10. DNA molecule according to claim 1, characterized in that that it codes for the viral protein VP4. 11. DNA molecule according to claim 1, characterized in that that it codes for the viral protein P2A. 12. DNA molecule according to claim 1, characterized in that it codes for the viral protein P2B. 13. DNA molecule according to claim 1, characterized in that that it encodes the viral protein P2C. 14. DNA molecule according to claim 1, characterized in that that it codes for the viral protein P3A. 15. DNA molecule according to claim 1, characterized in that it codes for the viral protein P3C. 16. DNA molecule coding for part of the viral Proteins according to claim 1, characterized in that the biological activity of the protein at least one corresponds to the viral proteins. 17. DNA molecule according to claim 1, characterized in that that the coding sequence under stringent Conditions that allow homology recognize that is greater than 85% with a DNA molecule according to one of claims 4 to 16 or with degenerate variations of these molecules hybridized.   18. DNA molecule according to one of the preceding claims, characterized in that it is in a suitable Expression vehicle replicable in microorganisms or in mammalian cells. 19. DNA molecule according to claim 18, characterized in that the vehicle is a plasmid, replicable in Prokaryotes. 20. DNA molecule according to claim 18, replicable in Eukaryotes. 21. Transformed active organism for the viral Genetic coding protein according to claim 1 Containing information. 22. Host organism according to claim 21, characterized characterized as being a prokaryote. 23. Host organism according to claim 21, characterized characterized as being a eukaryote. 24. Host organism according to claim 21, characterized characterized as being a mammalian cell line. 25. Host organism according to claim 21, characterized characterized as being E.coli. 26. Host organism according to claim 21, characterized characterized in that the genetic information in a vehicle is contained in the Host organism is replicable.   27. Polypeptide, characterized in that it is the biological activity of at least one of the viral ones Contains proteins of the rhinovirus strain HRV 89. 28. Polypeptide according to claim 27, characterized in that that it is from a DNA molecule according to any one of the claims 1 to 17 is encoded. 29. A polypeptide according to claim 27, characterized in that that there are parts or at least one of the total viral proteins of the rhinovirus strain HRV 89 corresponds. 30. Polypeptide according to claim 27, characterized in that that at least two of the viral proteins or parts the viral proteins in any combination and Order are linked together. 31. Polypeptide according to claim 27, characterized in that it has the amino acid sequence acc. Fig. 4 contains. 32. Polypeptide according to claim 27, characterized in that that it contains the amino acid sequence for VP1. 33. Polypeptide according to claim 27, characterized in that it contains the amino acid sequence for VP2. 34. Polypeptide according to claim 27, characterized in that it contains the amino acid sequence for VP3. 35. Polypeptide according to claim 27, characterized in that it contains the amino acid sequence for VP4.   36. Polypeptide according to claim 27, characterized in that it contains the amino acid sequence for P2A. 37. Polypeptide according to claim 27, characterized in that it contains the amino acid sequence for P2B. 38. Polypeptide according to claim 27, characterized in that it contains the amino acid sequence for P2C. 39. Polypeptide according to claim 27, characterized in that that it contains the amino acid sequence for P3A. 40. Polypeptide according to claim 27, characterized in that that it contains the amino acid sequence for P3C. 41. Polypeptide derived from the amino acid sequence according to Claim 31 or parts thereof, thereby characterized that it is cellular receptors for the rhinoviruses strain HRV 89 and / or binds them blocked. 42. A method for producing a DNA molecule according to one of claims 1 to 17, characterized in that

• a. the viral RNA of the rhinovirus strain HRV 89 is isolated,
• b. produced a DNA complementary to the viral RNA,
• c. the viral cDNA / RNA hybrid is inserted into a suitable replicable vector and
• d. a suitable host organism is transformed with the vector produced under c.

43. Process for the preparation of the polypeptide according to one of claims 27 to 41, characterized in that a suitable host organism is transformed with genetic information coding for a viral polypeptide according to one of claims 27 to 41,
that this information is expressed to produce a viral polypeptide according to any one of claims 27 to 41 in the host organism and
that the viral polypeptide is isolated according to one of claims 27 to 41. 44. The method according to claim 43, characterized in that that the genetic information in a DNA molecule according to one of claims 1 to 17 is included. 45. The method according to claim 43, characterized in that that the host organism according to one of claims 21 to 26 is defined. 46. The method according to claim 43, characterized in that that the viral polypeptide according to any one of claims 27 to 41 is defined. 47. Use of one of the polypeptides according to the claims 27 to 41 for therapeutic treatment. 48. Use of one of the polypeptides according to one of the Claims 27 to 40 for stimulating the immune system.   49. Use of the polypeptide according to claim 41 for Binding and / or blocking of cellular receptors for the Rhinovirus strain HRV 89. 50. Medicinal product suitable for use after a of claims 47 to 49, characterized in that it in addition to pharmaceutically inert auxiliary or Carriers an effective amount of at least one of the Contains polypeptides according to claims 27 to 41.