DE3701301A1 - Preparation of expression plasmids for the viral coat protein regions of HRV2 and HRV89 - Google Patents

Abstract

The present invention relates to the preparation of expression plasmids for viral proteins of HRV2 and HRV89, to the proteins which can be prepared in this way, and to the use thereof. Also described are the preparation of monoclonal antibodies against these proteins and their use.

Description

The present invention relates to Production of expression plasmids for viral Proteins of HRV2 and HRV89 that can be produced with this Proteins and their use. Continue to be described the preparation of monoclonal Antibodies to these proteins and their use.

Much of the upper infections respiratory tract is caused by rhinoviruses (Gwaltney, J.M., Jr., 1975, Yale J. Biol. Med. 48, 17-45). The existence of well over 100 serologically distinguishable rhinovirus types possibly one of the main reasons that obtaining immunity to rhinovirus infection so far is practically impossible (Fox, J.P., 1976, Amer. J. Epidemol. 103, 345-354; Stott, E.J. and Killington, R. A., 1972, Ann. Rev. Microbiol. 26, 503-525). Rhinoviruses represent the largest group within the Picornaviridae and exhibit all characteristic physical and structural properties of this Virus family (Matthews, R.E.F., 1982, Intervirol., 17, 1-201; Melnick, J.L., 1980, Proc. Virol. 26, 214-232; Cooper, P.D., Agaol, V.I., 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., Rückert, R. R., Schaffer, F. L. and Tyrell, D.A. J., 1978, Intervirol. 10, 165-180; MacNaughton, M.R. 1982, Current Top. Microbiol. Immuno. 97, 1-26). Using hyperimmune sera it is possible, 50 out of a total of 90 rhinovirus Divide serotypes into 16 groups (Cooney, M.K., Fox, J.P. and Kenny, G.E., 1982, Infect. immune. 37, 642-647). Another form of classification results  due to the binding to cellular receptors. In spite of the pronounced heterogeneity in the immunological The rhinoviruses behave in particular with respect to binding to cell surface receptors similar to each other. With the help of Competition experiments could be determined that it was found for the 88 strains of rhinovirus examined HeLa cells (clone R-19) only two different ones Receptors exist (Colonno, R.J., Callahan, P.L. and Long, W.L., 1986, J. Virol. 57, 7-12).

Rhinoviruses contain a typical Picornavirus single-stranded RNA surrounded by a capsid, which consists of 4 polypeptides called VP1 (P1D), VP2 (P1B), VP3 (P1C) and VP4 (P1A) (Medappa, K.C., McLean, C. and Rückert, R. R., 1971, Virology 44, 259-270; the terms in parentheses are the new names according to the Proposed nomenclature by Rückert, R. R. and Wimmer, E., 1984, J. Virol. 50, 957-959). A single one Virus particles contain 60 copies of each of these Polypeptides. The relative molecular masses are at different rhinoviruses for VP1 34-36kd, for VP2 27-30kd, for VP3 24-28kd and for VP4 7-8kd (McNaughton, M.R., 1982, loc.cit.). It is also for rhinoviruses characteristic that they rapidly at pH values below 5 be inactivated and sensitive to saline solutions are high concentration. Furthermore, most show Rhinoviruses optimal growth at 33 ° -34 ° C (Luria, S.E., Darnell, J.E. Jr., Baltimore, D. and Campbell, A., 1978 General Virology, Third Edition, John Wiley & Sons, New York, 308 ff).

The single-stranded, monocistronic RNA with one The genome of the Virus and can also serve as a template for serve the synthesis of viral proteins. Doing so  Much of the nucleotide sequence is initially in a long polyprotein translated from by proteolytic cleavage of the finished viral proteins arise (Butterworth, B.E., 1973, Virology 56, 439-453; McLean, C. and Rückert, R. R., 1973, J. Virol. 11, 341-344; McLean, C., Matthews, T.J. and Rückert, R.R., 1976, J. Virol. 19, 903-914). As products of this Processing is carried out in addition to the viral coat proteins VP1, VP2, VP3 and VP4 still have a number of proteins formed such as P2A, P2B, P2C, P3A, P3B, P3C and P3D. P3B (usually referred to as VPg) is covalent to that 5′-terminal nucleotide of the HRV2-RNA bound. In Analogy to poliovirus, it can be assumed that P2A and P3C are proteases necessary for the processing of the viral polyproteins are responsible (McNaughton, M.R., 1982, loc.cit., Dunn, J.J., Studier, F.W. and Wimmer, E., 1986, Cell 45, 761-770). Accordingly P3D is the polymerase for replication of the viral RNA. About the function of the P2B, P2C and P3A proteins little is known so far. When processing the viral polyproteins become part of the amino acid sequence split off and then degraded (McLean, C., Matthews, T.J. and Rückert R.R., 1976, loc.cit.). To Time no statement can be made as to whether not one of these peptides yet undiscovered function.

The sequences of the rhinovirus strains HRV2 and HRV89 are known. (Skern, T., Sommergruber, W., Blaas, D., Pieler, Ch., And Küchler, E., 1984, Virology 136, 125-132; Skern, T., Sommergruber, W., Blaas, D., Gründler, P., Frauendorfer, F., Pieler, Ch., Fogy, I. and Küchler, E., 1985, Nucleic Acids Res. 13, 2111-2126). In EPA 0 192 175 and DE 36 28 558.3 are described DNA molecules that are either whole or partially the RNA sequence of HRV2 or HRV89 correspond, or are derived from this.  

In addition to these sequences is also that of the human Type 14 rhinovirus known from the literature (Stanway, G., Hughes, P.J., Mountford, R.C., Minor, P.D. and Almond, J.W. 1984, Nucleic Acids Res. 12, 7859-7877; Callahan, P.L., Mizutani, S. and Collono, R., 1985, Proc. Natl. Acad. Sci. USA 82, 732-736, 01 69 146).

Numerous works have already been published in which the synthesis of foreign proteins in bacteria with the help genetic engineering methods is described (For review article, see Harris, T. J. R. in "Genetic Engineering ". Williamson, R., ed., 1983, vol. 4, Academic Press, London, 127 ff). For this purpose the foreign DNA close to appropriate bacterial Control regions (promoters, ribosome binding sites) introduced by plasmids that make this possible Information - mostly in the form of fusion proteins - in to express high yields and so the to win corresponding proteins. Also on the The Picornavirus area already has a number of Publications in which the expression of viral genes in Bacteria (Küppler, H., Keller, W., Kurz, C., Forss, S., Schaller, H., Franzel, R., Strohmaier, K., Marquardt, O., Zaslavsky, V. G. and Hofschneider, P.H., 1981, Nature 289, 555-559; Dress, 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 Giraurd, M., 1983, EMBO J. 11, 2019-2024; Klump, W. Marquardt, O. and Hofschneider P.H., 1984, Proc. Natl. Acad. Sci. United States 81, 3351-3355; Hanecak, R., Semler, B.L., Ariga, H., Anderson, C. W. and Wimmer, E., 1984, Cell 37, 1063-1073; Strebel, K., Beck, E., Strohmaier, K. and Schaller, H., 1985, J. Virol. 57, 983-991; Toyoda, H. et al. 1986, loc. cit.).

The present invention includes both the installation of the total DNA of human rhinovirus types 2 and 89, the Incorporation of parts of this DNA as well as varying Combinations of these DNA fragments into suitable ones Plasmid vectors, the plasmid vectors themselves, the so transformed host organisms, the synthesis continues of the proteins in question, the proteins themselves and their application.

As already mentioned, are becoming genetic engineering Production of proteins in host organisms Usually so-called expression vectors are used. These contain so-called regulatory sequences that for optimal transcription and translation of the heterologous DNA are responsible. To this regulatory sequences include strong promoters preferably adjustable promoters and in Direction of transcription of the promoter one or more unique interfaces for restriction enzymes, which in the must be at a suitable distance from the promoter. Some expression vectors are constructed so that before the first singular interface "downstream" from Promoter inserted a start codon (initiation codon) is. When using vectors without a start codon provide the DNA to be expressed with a start codon and inserted. Furthermore, the DNA to be expressed a so-called stop or termination codon in the vector consequences. Important when designing a Structural gene-bearing expression vector is that this Structural gene is inserted in phase to the start codon. The insertion of the heterologous DNA into the Expression vector can be done in a known manner be made. Since in the rarest of cases DNA has the same recognition site as the singular interface of the expression vector, need the interfaces in most cases be made compatible.  

This is possible, for example, by filling up or Digestion of overhanging single strand ends or through Add appropriate linkers. Has the selected one Expression vector no start codon, can at the same time with the addition of a linker also the start codon be added.

All of these operations must ensure that the heterologous DNA is in phase with the start codon.

Particularly suitable for the solution of the invention The task is to use the vector pRH100 and the expression plasmids derived therefrom for the DNA or DNA fragments of HRV2 or HRV89.

This vector - a pBR322 derivative - contains the tryptophan promoter from Serratia marcescens as an essential structural feature, which is an effective signal sequence for triggering protein synthesis and can be activated with 3- β- indole acrylic acid (= IAA; inducer of the trp operon). Behind the trp promoter is a synthetic ribosome binding site and the start codon ATG. A SacI interface is located immediately behind the start codon (see FIG. 1). For the purposes of the invention, the single strand ends of this interface were removed with a single strand specific nuclease. If the plasmid modified in this way is ligated with a DNA or a DNA fragment ("blunt-end"), for example the "blunt-end" XhoII fragment 969/1 of the HRV2 cDNA (see FIG. 2), expression plasmids are formed, which lead to the production of proteins without a fusion component in host cells transformed with this, for example prokaryotic or eukaryotic cell systems. The expression of such proteins has the advantage over a fusion protein that the expressed protein is available in its native structure and can be subjected to immunological or enzymatic tests.

By knowing the complete DNA sequence of the HRV2 and HRV89, it is possible according to the invention to insert any desired fragment of the DNA in the manner shown in suitable expression plasmids, preferably into the expression plasmid pRH100 and to bring it to expression, provided that the fragments are correct Reading frames for the initiation codon ATG are available. The desired fragments can be produced either by complete chemical oligonucleotide synthesis or by fragmentation of the plasmids obtainable according to EPA 0 192 175 or DE 36 28 658.3. The combination of both methods is also possible. For example, the DNA molecule shown in Fig. 2, e.g. B. from the plasmid pHRV2-969 by double digestion with the restriction enzymes BalI and BssHI at positions 930 and 1580, respectively. In order, for example, to express the mature VP2, two pieces of oligonucleotides are required which represent nucleotides 817 to 929 and 1581 to 1599 + tag from FIG. 2. The oligonucleotides can be produced chemically in a manner known per se, for example using an Applied Biosystems Model 381A DNA Synthesizer. After these fragments have been linked ( FIG. 5) and inserted into the "blunt" SacI site of the plasmid pRH100, an expression plasmid pRH969 / 2 suitable for expression of the mature VP2 is obtained ( FIG. 12).

This strategy is as above mentioned, also for the other viral proteins applicable.  

Another object of the present invention are monoclonal antibodies and their production, Antibodies against structural and not structural viral proteins (in native and denatured form) are directed. The connection monoclonal antibody to cellular receptors for HRV14 and its blocking is already through Collono et al. (European patent application 0 169 146) have been described.

The invention furthermore relates to expression plasmids, for example pRH100, which contain, as an insert, the DNA of polypeptides which have the biological activity of at least one of the viral proteins of the rhinovirus strains HRV2 or HRV89, in particular of polypeptides with the amino acid sequence for VP1, VP3, VP4, P2A, P2B, P2C, P3A or P3C preferably with the amino acid sequence according to FIGS . 7-11 or parts thereof, of polypeptides which correspond in part or in total to at least one of the viral proteins of the rhinovirus strain HRV2 or HRV89, of polypeptides which comprise at least two of the viral proteins contain linked in any combination and order and of polypeptides which are derived from the amino acid sequence according to FIG. 7 or 11 or from parts thereof and / or bind to and / or block the cellular receptors for the rhinoviruses strain HRV2 or HRV89. Based on the knowledge of the amino acid sequence, synthetic oligopeptides can be produced which correspond to parts of one or more viral proteins. The synthesis can be carried out by expressing the DNA derived therefrom in pro- or eukaryotes, as well as in "in vitro" translation systems or by using automatic peptide synthesizers (eg Beckman, Model 990).

The viral proteins produced according to the invention can according to known, for insulation and Purification of proteins commonly used procedures be isolated and cleaned.

The proteins or protein fragments obtained can then to stimulate one against intact viruses or against non-structural, virally encoded proteins Immune response can be used. 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 30, 699-703); are similar studies also carried out with 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 298, 30-33; Pfaff, E., Mussgay, M., Böhm, H. O., Schulz, G. E. and Schaller, H., 1982, EMBO J. 1, 869-874). object of the present invention are also the oligo- and Polypeptide portions of HRV2 proteins that are a result of Synthesis or for the purpose of application with others Oligo or polypeptides are linked.

The invention can be used Polypeptides also for binding and / or blocking the cellular receptors for the rhinovirus strain HRV2 or HRV89.

The inventive can be used Polypeptides in the form of drugs, in addition to pharmaceutically inert auxiliaries or carriers effective amount of at least one of the invention Contain polypeptides.  

Legend for the illustrations Fig. 1

Tryptophan promoter / operator region, synthetic Ribosome binding site (RBS) and start codon of the Expression vector pRH100. The is also listed Primer (17mer), which is used for sequencing the Expression plasmids pRH969 / 1 was used. Lower case letters and numbering characterize the Sequence of pBR322; Capital letters put the between pBR322 nucleotide numbers 1 and 29 inserted sequence.

Fig. 2

Sequence section of the HRV2 cDNA between nucleotide number 700 and 2000. The short, thick arrows indicate the Sequence of VP2, the slender arrows show that the most important restriction points in this section.

Fig. 3

Construction scheme for the expression plasmid pRH969 / 1 (NCR = 2 "non coding region", untranslated region; trp ^p / o = tryptophan promoter operator region).

Fig. 4

Result of the expression of 969/1 in the maxicell system CSR603. Lanes A1 to A4 show a "Western Blot "experiment after staining with NBT and BCIP. In Lane A1 was denatured as a control HRV2 particles (approx. 50 ng HRV2) also separated. On track A2 and A4 were the cell lysates with the plasmid pRH969 / 1 (from clones 307 and 319) transformed E. coli strain CSR 603 cells separated. As a negative control in lane A3 that was Cell lysate separated from E. coli strain CSR 603 cells, which were transformed with the plamide from clone 224 were (inverted orientation of insert 969/1).  

Fig. 5

Insert the plasmid pRH969 / 2.

Fig. 6

Binding site of 8F5 on the viral coat protein VP2. Get the numbers 1-261 on the top line refer to the amino acid sequence of VP2, which is under the bottom line on the DNA sequences of HRV2. The DNA and amino acid sequences in the region of the binding site are shown with the exact details of each deletion.

Fig. 7

Sequence of the cloned HRV2 genome and that thereof deduced amino acid sequence. Split points in the Polyprotein are represented by arrows. Filled out Arrows indicate experimentally determined cleavage points, open arrows show due to the homology other Picornavirus predicted cleavage sites in Polyprotein.

Fig. 8

A: Nucleotide sequence of the polypeptide VP1 B. amino acid sequence of polypeptide VP1 C: Translated nucleotide sequence of the polypeptide VP1

Fig. 9

A: Nucleotide sequence of the polypeptide VP3 B. Amino acid sequence of polypeptide VP3 C: Translated nucleotide sequence of the polypeptide VP3  

Fig. 10

A: Nucleotide sequence of the polypeptide VP4 B. amino acid sequence of polypeptide VP4 C: Translated nucleotide sequence of the polypeptide VP4

Fig. 11

Sequence of the cloned HRV89 genome and those thereof deduced amino acid sequence. Split points in the Polyprotein are represented by arrows. Filled out Arrows show experimentally determined cleavage points, open arrows show due to the homology other Pivornavirus predicted cleavage sites in the Polyprotein.

Fig. 12

Construction scheme for the expression plasmid pRH 969/2.  

Explanations of abbreviations

ABTS: 2,2′-azino-di- (3-ethylbenzthiazoline sulfonate) AMP ^r : ampicillin resistant BCIP: 5-bromo-4-chloro-3-indolyl-phosphate BME: bromophenol blue BSA: bovine serum albumin cccPlasmid: "circular-covalently- closed "plasmid DE-81 paper: diethylaminoethyl cellulose paper DMEM: Dulbecco's Modified Eagle's Medium dNTPs: equimolar solution of all 4 deoxytriphosphates DTT: dithioticness EDTA: ethylenediaminotetraacetic acid FKS: fetal calf serum g : gravitational acceleration HAT medium: medium acceleration HIFKS: heat-inactivated, fetal calf serum HRV2 or 14: human rhinovirus type 2 or 14 IAA: β- indolacrylic acid kb: kilobases kd: kilodalton Klenow (enzyme): Klenow fragment of the DNA polymerase I LB medium: the Luria-Bertani Medium consists of 1% of a pancreatic digestion of milk casein (bactotrypton), 0.5% yeast extract and 1% NaCl in distilled water and was heated to 120 ° C in an autoclave for 20 min. M: aqueous solution containing one mole of the substance in question per liter. MEM: Minimum Essential Medium mM: aqueous solution containing one millimole of the substance in question per liter. ml: milliliter NBT: nitro-blue-tetrazolium nm: nanometer OD₆₀₀: optical density at 600 nm PBS: aqueous buffer mixture containing 137 nMol NaCl, 2.7 nMol KCl, 8 mmol Na₂HPO₄, 1.5 mmol KH₂PO₄, Contains 5 mmol MgCL₂ · 6 H₂O and 1 mmol CaCl₂ and has a pH of 7.4. PBS: like PBS only without Ca and Mg salts PEG: polyethylene glycol PFU: "plaques forming units" RPMI medium: Roswell Park Memorial Institute medium SDS: sodium dodecyl sulfate TE: aqueous buffer mixture containing 10 mmol TrisHCl pH per liter = 7.4 and mmol EDTA contains. Tris: trishydroxymethylaminomethane TWEEN 20: polyoxyethylene (20) sorbitan monolaurate U: unit µCi: microcurie µg: microgram µl: microliter µM: aqueous solution containing one micromole of the substance in question per liter. VP1, 2, 3, 4: viral coat protein VP1, 2, 3, 4. VPO: viral precursor protein of the coat proteins VP2 and VP4 XC: xylene cyanole

The invention is illustrated in the following examples explained without restricting it:

Example 0 Construction of the expression plasmid pRH 100

All enzyme reactions were among those of the Manufacturers specified conditions carried out.

7 µg plasmid pER103 (Eva Dworkin-Rastl et al., Gene 21 (1983) 237-248, EP-A-0 115 613) were in 50 ul Reaction medium with the restriction endonuclease HindIII linearized. After an hour Incubation at 37 ° C was 50 ul 2 × CIP buffer added (2 × CIP buffer = 20 mM Tris, pH = 9.2, 0.2 mM EDTA). By adding 2 units more alkaline Calf intestine phosphates (CIP) became the 5'terminal Phosphate residues removed; was incubated at 45 ° C 30 For minutes. The reaction was started by adding 4 ul 0.5 mM EDTA solution and addition of 10 µl 1M Tris, pH = 8.0 solution stopped. The proteins were made by two phenols and one Removed phenol / chloroform extraction. The DNA was from the aqueous phase after adding 0.1 vol 3M Sodium acetate solution pH = 5.5 and 250 µl ethanol precipitated, the DNA precipitate after centrifugation washed once with 70% ethanol solution. The DNA was dried, and then the pellet in 20 ul TE buffer (10 mM Tris pH = 8.0, 1 mM EDTA) dissolved.  

1 µg each of the synthetically produced Oligonucleotides d (AGCTTAAAGATGAGCT) and d (CATCTTTA) were in 10 ul reaction solution with the addition of 10 units of T4-PNK (polynucleotide kinase) and 1 mM rATP phosphorylated. The reaction took place at 37 ° C and takes 45 minutes. The reaction was through Heating stopped at 70 ° C for 10 minutes.

5 µl each of the plasmid solution and the phosphorylated oligonucleotides were mixed and 5 Heated to 70 ° C for minutes. After that, the solution was on Cooled to 0 ° C, 2 µl 10 × ligase buffer (500 mM Tris, pH = 7.5), 100 mM MgCl₂ 200 mM DTT (dithiothreitol), 1 mM rATP, 500 µg / ml BSA (bovine serum albumin), and 2 µl Water and 10 units of T4 DNA ligase were added. The reaction lasted 40 hours and was at 4 ° C carried out. It was opened by heating for 10 minutes 70 ° C canceled.

2 ul of this ligase reaction were in a total of 30 ul Solution with 10 units of Restriction endonuclease Sacl (New England Biolabs) Digested for 3 hours at 37 ° C. The reaction was through Heating stopped at 70 ° C for 10 minutes. 5 µl this reaction mixture was in a total of 30 ul by adding 10 units of T4-PNK at 14 ° C 16 Ligated for hours.

200 µl of competent E. coli Hb101 were mixed with 10 µl this ligase reaction. The bacteria turned 45 Held on ice for minutes and then on for 2 minutes Heated at 42 ° C to allow DNA uptake. The bacterial suspension was then added again Incubated 0 ° C for 10 minutes. In conclusion, the transformed bacteria on an LB agar containing 50 µg / ml Ampicillin contained, streaked.  

Of the bacterial colonies that emerged, 12 became arbitrary selected and the plasmids isolated on a microscale (Birnboim and Doly, Nucl. Acids Res. 7 (1979) 1513-1523). The resulting DNA was cut with the restriction endonuclease SacI cut and the DNA on an agarose gel (1%, 1 × TBE buffer) separated.

Migration of the DNA as a linear molecule approximately 4400 bp in size confirmed the introduction of a SacI recognition site into the plasmid. One of these plasmids was chosen arbitrarily. E. coli HB101 was transformed again with the DNA from the associated mini preparation. A colony was selected from the resulting transformed bacteria and grown on a larger scale. The plasmid isolated therefrom was cut with the restriction endonucleases EcoRI and BamHI, the DNA was separated on a 1% agarose gel, and the smaller fragment was isolated from the gel by electroelution. This approximately 460 bp long EcoRI-BamHI DNA fragment was sequenced according to Sanger (F. Sanger et al., Proc. Natl. Acad. Sci. (1977) 5463-5467). The plasmid analyzed in this way was called pRH100 ( FIG. 0).

Example 1 Isolation of the Xho fragment (969/1) and preparation of the Expression plasmids pRH969 / 1

Starting, for example, from the DNA shown in FIG. 2, a fragment of the HRV2 genome obtainable according to EPA 192 175, which is located as an insert in a vector, for example pUC9, was digested with the restriction enzymes HindIII (Biolabs) and EcoRI (Boehringer Mannheim) in 100 ul Eco-Hind buffer (7.5 mM MgCl₂ 75 mM TrisHCl pH = 7.5, 50 mM NaCl) with 15 U each of EcoRI and HindIII cut out the DNA insert at 37 ° C for 15 hours.

The insert DNA was separated from the linearized vector by separation on a 1% agarose gel. For this purpose, the 100 ul with 11 ul 10 × agarose gel "loading buffer" (0.1% bromophenol blue, 0.1% xylene cyanole, 35% Ficoll, 0.5% SDS) was added. The separated EcoRI / HindIII fragment from pHRV2-969 (approx. 1.1 kb) was isolated from the agarose gel using DE81 paper (Whatman) (Dretzen, GM, Bellard, P., Sassone-Corsi and Chambon, P ., 1981, Anal. Biochem. 112, 295-298). For this purpose, after the DNA fragments had been separated on the agarose gel, a slit was cut in front of and behind the DNA band to be isolated, into which a strip of DE81 paper was inserted. Electrophoresis was continued (gel should not be covered with buffer) until the desired DNA fragment was completely bound to the front DE81 strip. The rear DE81 strip prevented contamination by larger DNA fragments. The DE81 paper with the bound DNA fragment was transferred into a 1.5 ml Eppendorf tube (with a discharge hole in the bottom of the vessel and polyalomer wool superimposed thereon) and twice for 5 min with 500 .mu.l wash buffer (0.2 M NaCl, 25 mM TrisHCl pH = 8.0, 1 mM EDTA), the washing solution being collected in the second Eppendorf vessel underneath by brief centrifugation (approx. 1 sec). The bound DNA was then washed twice from the DE81 paper by incubation for 15 minutes in 200 μl elution buffer (1 mM NaCl, 25 mM TrisHCl pH = 7.5, 1 mM EDTA) (the ethidium bromide remains absorbed). The 400 µl eluate was centrifuged in the Eppendorf centrifuge (15,000 g) for 10 min to remove pieces of paper. The supernatant was carefully transferred to a new Eppendorf tube, mixed with 800 μl 96% ethanol, precipitated at -20 ° C. (approx. 2 hours), washed twice with 70% ethanol, dried and in 50 μl (10 mM TrisHCl pH = 8 , 10 mM MgCl₂ with 10 U Xho II (Boehringer) cut for 2 hours at 37 ° C. In order to inactivate the restriction enzyme, the mixture was incubated for 2 min at 70 ° C. and then the reaction mixture was slowly cooled to room temperature × Klenow buffer (660 mM TrisHCl pH = 7.5, 66 mM MgCl₂ 3 mM dNTPs, 1 mM DTT, 0.1 mg / ml BSA), 2 ul Klenowenzym (4 U / ul; Amersham) and 2 ul H₂O added and incubated for 30 min at 22 ° C. The reaction was stopped by adding 2 μl 0.5 M EDTA pH = 8, the aqueous phase once with the same volume of phenol / CHCl₃ / isoamyl alcohol (25: 24: 1), once with the same Volume CHCl₃ extracted and mixed with 7 ul 10 × agarose gel "loading buffer. To separate the 1092 bp long Xho II fragment from the resulting oligonucleotides the fragment (969/1) provided with a "blunt-end" structure was isolated from a 1% agarose gel using DE81 paper. The expression vector pRH100 used essentially represents a pBR322 derivative, the modification of which consists in inserting a 123 bp fragment between bases 4362/0 (EcoRI site in pBR322) and 29 (HindIII site in pBR322), which has the tryptophan promoter region of Serratia marcescens, a synthetic ribosome binding site and the start codon ATG (see Fig. 1).

The construction of the expression plasmid pRH969 / 1 is shown schematically in FIG. 3. About 10 μg of the expression vector pRH100 were digested overnight in 200 μl (6 mM TrisHCl pH = 7.5, 6 mM β- mercaptoethanol (BME), 6 mM MgCl₂ with 100 U Sac I (Biolabs) at 37 ° C. The plasmid DNA was precipitated in a conventional manner with ethanol, washed, dried and taken up in 200 .mu.l (200 mM NaCl, 1 mM ZnCl₂ 30 mM sodium acetate pH = 4.75, 5% glycerol) and treated with 150 U Mung bean nuclease.

The mixture was previously divided into two 100 μl portions and incubated on the one hand for 2 hours at 14 ° C. and on the other for 30 min at 37 ° C. The reaction was stopped by adding 5 ul 0.5 M EDTA and extraction with phenol / CHCl₃ / isoamyl alcohol (25: 24: 1) or with CHCl₃ and the two batches were combined. The linearized plasmid was precipitated in the usual way with sodium acetate and ethanol, washed and dried. The DNA was then incubated in 1000 μl (100 mM TrisHCl pH = 8) with 40 U alkaline phosphatase (Boehringer Mannheim) at 37 ° C. for 1 hour. After the addition of EDTA to 20 mM, the mixture was extracted twice with phenol / CHDl₃ / isoamyl alcohol (25: 24: 1) and twice with CHCl₃ in a conventional manner and the DNA was precipitated by ethanol. Approx. 50-175 ng pRH100 (linearized with Sac I; treated with mung bean nuclease and alkaline phosphatase) and approx. 1-2 µg of the Xho II fragment from the subclone pHRV2-969 (treated with Klenow) were used for the ligation. precipitated together with ethanol, dried and taken up in 10 μl of freshly prepared 1 × ligase buffer (50 mM TrisHCl pH = 7.5, 10 mM MgCl₂ 20 mM DTT, 0.1 mM rATP and 50 μg / ml BSA). The ligation was carried out for 70 hours at 14 ° C. with 1 U T4 ligase (Boehringer Mannheim). In order to obtain cells competent for the transformation, a modified regulation by Mandel and Higa (Mandel, M. and Higa, A., 1979, J. Mol. Biol. 53, 159-162) was used. For this purpose, 0.5 ml of an "overnight culture" of E. coli strain HB 101 in 50 ml LB medium (10 g / l tryptone, 5 g / l yeast extract, 10 g / l NaCl) was inoculated up to an OD₆₀₀ of grown approximately 0.4 and then pelleted for 5 min at 5 k and 4 ° C. The bacteria were then carefully resuspended in 25 ml 0.1 M MgCl₂ (ice cold), placed on ice for 5 min and centrifuged again at 5 k and 4 ° C. for 5 min. The pellet was resuspended in 25 ml 0.1 M CaCl₂ (ice cold), placed on ice for 4 hours and centrifuged for 5 min at 5 k and 4 ° C. The cells were taken up in 2.5 ml of 1 × storage buffer (0.1 M CaCl₂ / glycerol = 4/1% v / v), placed on ice for 20 min, aliquoted in 100 μl portions and snap frozen in liquid nitrogen, and Stored at 80 ° C. 5 μl of the ligation mixture described above were added to 100 μl of competent cell suspension thawed on ice, the cells were incubated on ice for 1 hour and at 42 ° C. for 2 minutes and finally placed on ice for 5 minutes. Before the cells were plated out, 900 μl of LB medium were added, incubated for 10 min at 37 ° C. and 200 μl of cell suspension each was applied to LB agar plates (1.5% agar in LB medium with 100 mg / l ampicillin) and incubated overnight. A total of 351 Amp ^r clones were isolated, from which the plasmid DNA was isolated using the "mini-plasmid preparation technique" (Birnboim, HC and Doly, J. 1979, Nucleic Acids Res. 7, 1195-1204). By HindIII digestion of the plasmid DNA, 8 clones with an insert corresponding to the size of the Xho II fragment could be determined. A restriction analysis was carried out since this construction required a blunt end ligation on both sides and there were therefore basically two possible orientations of the insert. By digestion with 3.5 U Bal I / 100 ng plasmid DNA in 10 mM TrisHCl pH = 7.5, 10 mM MgCl₂ BME and by double digestion with 8 U BssHII / 100 ng plamide DNA in 25 mM NaCl, 6 mM TrisHCl pH = 7.5, 6 mM MgCl₂ 20 mM BME (2 hours at 50 ° C) or with 20 U BamHI / 100 ng plasmid DNA in 150 mM NaCl, 6 mM TrisHCl ph = 7.5, 6 mM MgCl₂ 20 mM BME (2 hours at 37 ° C) 6 clones (no .: 156, 165, 289, 301, 307 and 319) with correct and 2 clones (no .: 1 and 224) with inverted insert orientation were identified (see restriction map in Fig. 2). In order to check whether the Xho II fragment of 969 is in phase with the start codon ATG, the base sequence of the transition region between the non-coding and coding section was determined using a 17mer sequence primer, directly on the cccPlasmid. The sequence primer is complementary to the first 17 bases of the non-codogenic strand of the Trp promoter region (see Fig. 1).

For this purpose, the clones listed above became the Plasmid DNA according to the "mini-preparation technique" obtained, mixed in 100 µl of 5 M LiCl and 5 min Incubated at 0 ° C. After centrifugation in the Eppendorf centrifuge (15,000 g, 5 min, 4 ° C) was the 400 µl of 96% ethanol were added to the supernatant, again for 2 min Centrifuged at room temperature, the pellet once washed with 70% ethanol and dried. The Plasmid DNA was then in 11 ul H₂O dissolved, of which 5 ul aqueous plasmid solution with 5 ul 1 × denaturing solution (267 mM NaOH, 0.267 mM EDTA) were transferred. After an incubation of 15 min Room temperature was 1 µl 17mer sequence primer (50 ng / µl) and 2 µl 2 M CH₃COONH₄ (pH = 4.5) added and incubated for 5 min at room temperature. To Add 75 µl 96% ethanol and precipitate -70 ° C (15 min) was pelleted at 15,000 g for 5 min, with Washed 70% ethanol, dried and with the Primer hybridized plasmid DNA in 8.5 ul H₂O added. 1 µl was added 10 × to each of these 8.5 µl Binding buffer (100 mM TrisHCl pH = 8, 50 mM MgCl₂, 3 µl ³⁵S-ATP (1000 Ci / mmol; Amersham) and 1 µl Klenow enzyme (4 u / µl; Biolabs) added. The other Sequencing followed the Chain termination method by Sanger et al. (Sanger, F., Nicklen, S. and Coulsen, A.R., 1977, Proc. Natl. Acad. Sci. USA 74, 5463-5467).  

In the expression plasmid pRH969 / 1 from clones 307 and 319 became an insert in phase with the ATG detected.

Example 2 Expression of the viral coat protein region 969/1 of HRV2 in E. coli strain CSR603 cells

In order to be able to detect the expression of plasmid-encoded proteins in E. coli, a maxicell system developed by Sancar (Sancar, A., Hack, AM and Rupp, WD 1979, J. Bacteriol., 137, 692-693) was used. The E. coli maxicell strain CSR 603 (CGSC 5830; F ^- , thr-1, leuB6, proA2, phr-1, recA1, argE3, thi-1, uvrA6, ara-14, lacY1, galK2, xyl-5, mtl- 1, gyrA98 (nalA98), rps31, tsx-33, lambda ^- , supE44) has no mechanisms for repairing DNA damage caused by UV radiation. For the experiment, the radiation dose is optimized so that the bacterial chromosome is often hit, the comparatively very small plasmid DNA, which is also present in several copies per cell, mostly remains undamaged. The damaged chromosomal DNA is broken down by the cell, whereas plasmids that have not been hit by the UV radiation continue to replicate and represent the main part of the remaining DNA. After all the undamaged, multiplying cells have been killed by the antibiotic D-clycoserin and the endogenous mRNA has been broken up, only genes coded on the plasmid are transcribed and translated in the cells which are to be replaced. After transferring the bacteria into a sulfate-free medium, the proteins formed can be radioactively labeled and detected by incorporating ³⁵S-methionine.

E. coli strain CSR603 cells were mixed with the appropriate Expression plasmid pRH969 / 1 from clone 307 and 319 or 224 (with inverted orientation of 969/1) as above described transformed. The transformed cells were up to 15 ml tryptophan-free expression medium to an optical density at 600 mnm of 0.5-0.7 Grown at 37 ° C. The expression medium used was manufactured as follows (data per liter of medium):

• a) 10 g (NH₄) ₂HPO₄ 3.5 g KH₂PO₄ and 0.5 g NaCl were dissolved in 500 ml H₂O, the solution with NaOH brought to pH = 7.3 and to approx. 800 ml replenished.
• b) 21 g casamino acids (acid hydrolyzed, vitamin free; Merck) were dissolved in 100 ml of H₂ =.
• c) 50 ml of 20% glucose
• d) 1 ml of 1 M MgSO₄
• e) 1 ml 0.1 M CaCl₂
• f) 1 mg thiamine HCl and 20 mg L-cysteine were mixed in 1 ml H₂O solved.

These autoclaved or sterile filtered solutions were mixed shortly before use, made up to 1000 ml and mixed with 100 mg / l ampicillin. 10 ml of the harvested bacterial cultures were transferred to an open 13.5 cm petri dish, irradiated with a UV-Germicid lamp (15 watts) from 50 cm away for 5 seconds with gentle swirling and incubated further at 37 ° C. The cultures were mixed with 100 μg / ml D-cycloserine (fresh, 100 × concentrated solution in 50 mM phosphate buffer pH = 8) and incubated at 37 ° C. on a shaker for 14-16 hours. The bacteria were harvested by centrifugation (5 min, 5 k at room temperature), washed twice with 5 ml of Hershey's saline solution, suspended in 5 ml of Hershey's medium with 20 μg / ml of 3- β- indole acrylic acid (= IAA; inducer of tryptophan operon) and shaken at 37 ° C for 1 hour.

Hershey salt solution (per liter):

5.4 N NaCl 15 mg 3.0 g KCl 0.2 g 1.1 g NH₄Cl 0.2 mg CaCl₂ · 2 H₂O87 mg KH₂PO₄ MgCl₂ · 6 H₂O12.1 g Tris + HCL pH = 7.4 FeCl₃ · 6 H₂O

Hershey medium (per 100 ml Hershey salt solution):

2.0 ml 20% glucose 0.5 ml 2% threonine 1.0 ml 1% leucine 1.0 ml 2% proline 1.0 ml 2% arginine 0.1 ml 0.1% thiamine HCl

About 15 µCi / ml ³⁵S-methionine were added to each culture (1000 Ci / mmol; Amersham) and this one more incubated for half an hour at 37 ° C. Then the Cells by centrifugation (5 k, 5 min at room temperature) harvested and in 200 µl SDS sample buffer (10 mM TrisHCl pH = 7.4, 125 mM BME, 2% SDS, 10% sucrose, 0.05% Bromophenol blue) lysed. The samples were 5 minutes at 95 ° C incubated and centrifuged at 15,000 g for 2 min. The Supernatant was expressed on the presence of Proteins checked.

20-60 μl of cell lysate supernatant in SDS sample buffer (see above) were separated according to their size on a 10% SDS-containing polyacrylamide gel (Laemmli, UK, 1970, Nature 277, 680-686). The gels with a thickness of 1.5 mm consisted of an approximately 10 cm long 10% separating gel and a 3 cm long 3% collective gel. The electrophoresis was carried out at 3 watts (constant) in the collecting gel, at 12 watts (constant) in the separating gel (total duration 4.5 hours). A protein mixture consisting of BSA (66 kd), ovalbumin (45 kd), pepsin (34.7 kd), β- lactoglobulin (18.4 kd) and lysozyme (14.3 kd) was also separated as a size marker. The gel trace of the marker proteins was separated from the rest of the gel after electrophoresis, incubated for 30 min in the staining solution (50% methanol, 10% acetic acid, 0.1% Coomassie brilliant blue 2250), overnight in the decolorizing solution (5% methanol, 10% acetic acid) shaken, placed in a glycerol bath (7% acetic acid, 2% glycerol) for 30 min and dried on Whatman 3 M paper at 60 ° C on a gel dryer (see Fig. 4, lane M) .

The proteins of the cell lysates were after the "Western blot" method by electrotransfer from the gel Nitrocellulose filter (Schleicher and Schüll, BA 85, 0.45 µm) transferred (Burnette, W.N., 1981, Analyt. Biochem. 112, 195-203). The transfer was carried out in the transfer buffer (20 mM Tris, 150 mM glycine, 20% methanol p. a .; pH = 8.8) at 60 Volts, 5 hours in a biorad protein blot apartment in Presence of 0.1% Empigen (Marchon Italiana S.P.A.) carried out (Mandrell, R.E. and Zollinger, W.D., 1984 J. Immunol. Meth. 67, 1-11).

Expression of the proteins encoded exclusively by plasmid was demonstrated by incorporation of ³⁵S-methionine. In FIG. 4 the autoradiogram (lane B2 to B3) of the "Western blot" after exposure to Kodak X-Omat X-ray film S shown. Lane B2 (corresponds to lane A2 of the Western blot) and lane B4 (corresponds to lane A4) have both signals of the expressed insert 969/1 and of the β- lactamase encoded by the plasmid gene for ampicillin resistance, while lane B3 (corresponds to lane A3) with the inverted insert 969/1 no signal at 40 kd, but it does for β- lactamase. Because there are several termination codons at the beginning of the inverted coding region, no protein is expressed.

Example 3 Production and isolation of monoclonal antibodies

Explanation of the solutions and media used:

100 × aminopterin

1.8 mg aminopterin in 50 ml of H₂O by adding 1 M NaOH dissolved and made up to 100 ml (storage at -20 ° C in the dark).

100 × OPI mix

7.5 g of oxalic acid and 2.5 g of pyruvate are in 450 ml of H₂O dissolved and with 1000 U insulin (in a few ml 0.1 M HCL solved). Then with H₂O to 500 ml replenished.

100 × HT mix

0.6805 g hypoxanthine dissolved in 200 ml H₂O and with NaOH be adjusted to pH = 10 with 200 ml of an aqueous Thymidine solution (0.1937 g in 200 ml) combined and made up to 500 ml replenished.  

HT medium

500 ml Dulbecco's Modified Eagle's Medium (DMEM; Flow laboratories; high glucose) 6 ml 200 mM L-glutamine 6 ml 100 × HT mix 6 ml 100 × OPI mix 50 mlHIFKS (heat inactivated fetal Calf serum) 0.6 ml gentamycin (50 mg / ml)

HAT medium

100 ml HT medium 1 ml 100 x aminopterin

ABTS solution

10 mg / ml aqueous solution of 2,2'-azino-di (3-ethyl benzothiazoline sulfonate).

PBS

137 mM NaCl 2.7 mMKCl 8 mM Na₂HPO₄ 1.5 mMKH₂PO₄ 0.5 mMMgCl₂ · 6 H₂O 1 mMCaCl₂ · 2 H₂O  

PBS def.

Like PBS only without Ca and Mg salts.

PBS wash solution

PBS def. with 0.05% Tween 20.

29% FKS-DMEM

445 ml Dulbecco's Modified Eagle's Medium (Flow Laboratories) 50 mlHIFKS (heat inactivated fetal Calf serum) 5 ml penicillin / streptomycin (10,000 Z / ml)

RPMI compl.

500 ml RPMI 1640 Medium (Flow Laboratories; No .: 12-604-54) 50 ml FCS 5 ml 200 mM L-glutamine

PEG-4000 solution

20 g PEG are def in 28 ml PBS. solved and in one Incubate in a water bath (60 ° C) until the PEG is dissolved (approx. 30 min).

RPMI wash solution

500 ml RPMI 1640 medium 5 ml penicillin / streptomycin (10,000 U / ml) 5 ml 200 mM L-glutamine  

HAT thymocyte medium

The sterile removed thymus organ of a 3-4 month old Balb-c mouse (if possible without connective tissue) is transferred to a sieve and passed through the sieve with a sterile syringe stamp (slow, circular movements; stir in the sieve for approx. 1 min). The cell suspension in 25 ml of RPMI wash solution is centrifuged off at room temperature and 300 g for 10 min. The supernatant is aspirated with a Pasteur pipette and completely in 20 ml RPMI. resuspended and further diluted to approx. 5.10⁶ cells / ml. This medium containing thymocytes is stored in small culture bottles at 37 ° C in the CO₂ incubator (shelf life approx. 14 days). To produce the HAT thymocyte medium, the cells are centrifuged off, washed in RPMI washing solution as described above, centrifuged again and the cell pellet is taken up in HAT medium.

In 2-3 month old Balb-c mice became intraperitoneal 5-10µg cleaned and in complete Freund's Adjuvant injected with emulsified HRV2. Subsequently became the same after 27, 49, 100, 117 and 118 days Amount of HRV2 (in incomplete Freund's adjuvant emulsified) administered. One day after the last one Inoculation, the spleen was removed and the cells with Myeloma cells, e.g. B. NS-1 (ATCC TIB 18), where essentially according to the protocol of Galfre, G. and Milstein, C. 1981, Methods Enzymol. 73, 3-46) has been.

The spleen removed under sterile conditions was washed in a petri dish (chilled on ice) in about 5 ml of RPMI washing solution. The spleen was crushed in 5 ml of RPMI washing solution and passed through a sieve with a syringe plunger (slow, round movements). The cell suspension was then allowed to rest on ice in order to sediment cell clumps and parts of the connective tissue. The finely suspended cells were aspirated with a pipette and transferred into a 50 ml centrifuge tube with a round tube bottom, made up to 50 ml with freshly prepared RPMI washing solution and centrifuged for 5 min at 300 g and room temperature. The supernatant was removed and the pellet was suspended in 5 ml of lysis buffer (155 mM NH₄Cl, 10 mM KHCO₃ and 1 mm EDTA; pH = 7.2) and incubated at 0 ° C. for 2 min. The suspension was brought to 50 ml with RPMI wash solution and carefully stirred with a Pasteur pipette. Lipid-containing compounds and fats stuck to the pipette and could be easily removed. The suspension was centrifuged again for 5 min at 300 g and room temperature. The pellet was resuspended in 10 ml RPMI wash solution and approx. 5.10⁷ mouse myeloma cells, eg. B. NS-1 (from four 75 cm² tissue culture bottles to 50% confluent) mixed. The cells were previously washed three times with 50 ml RPMI wash solution and centrifuged at 300 g (5 min and room temperature) to remove all serum. The mixture on spleen and myeloma cells was resuspended in 50 ml RPMI wash solution and centrifuged as described above. The supernatant was carefully and completely removed, the pellet was immediately mixed with 1.5 ml of thermostatted PEG-4000 solution at 37 ° C. and the tube was gently tapped on the edge of the table for 2 minutes until the pellet and the PEG-4000 solution were mixed (" gritty consistency "). The mixture was then incubated for 2 min at 37 ° C. with gentle shaking, and 20 ml of RPMI wash solution were then added dropwise, the drops being allowed to run down individually on the wall of the vessel.

Another 30 ml of RPMI wash solution was carefully added. After the cells had been centrifuged (300 g , 5 min at room temperature), the supernatant was suctioned off and the pellet was carefully resuspended in 10 ml HAT thymocyte medium with a pasteupipette. This suspension was divided into aliquots of 0.1 ml in microtiter plates (96 wells / plates) and incubated at 37 ° C and 5% CO₂ (do not move plates in the incubator). After 3 days, about 0.1 ml of HAT medium was added. After a further 3 days, approximately 0.05 ml of HAT medium were added. 10 days after the fusion, 70% of the medium was removed and 0.2 ml of HT medium were replenished. 12 days after the fusion, the plates were examined microscopically, the medium was removed for an ELISA and neutralization test and about 0.3 ml of 20% FCS-DMEM were added per well. Positive hybrids were selected by the two tests mentioned above and the hybrids split 1: 3 to 1:10; ie the content of a well was divided into 3 to 10 wells of the microtiter plates. After growing up, the hybrids were tested again and those with the highest titer were selected. These hybrids were cloned twice by "endpoint dilution", ensuring under the microscope that the hybrid cells were derived from a single cell. The cloning was carried out in the presence of thymus cells and 20% FCS. The individual clones were grown up to 50% confluence in 75 cm² cell culture flasks. The cells of such a tissue culture bottle were injected into a mouse which had been pretreated by intraperitoneal pristane injection (0.5 ml) 10 days beforehand. Ascites fluid was withdrawn through the abdominal wall after 2 to 6 weeks.

ELISA

50 µl of one were used per well of a microtiter plate Virus dilution (1-2 µg HRV2 / ml) in carbonate buffer incubated. By adding 100 µl PBS def., Which is 1% Contained BSA and by incubation at 37 ° C for 1 hour was saturated. The microtiter plate was opened Unclip cellulose and the wells with 50 µl Hybrid protrusion offset. As a negative control pure HAT medium used. Then one Incubated for one hour at 37 ° C, tapped again and three times washed with PBS wash solution (PBS def. + 0.05% Tween 20). Then 50 ul of a 1/500 dilution of a Peroxidase conjugated antiserum (RAM = "rabbit anti mouse ") in PBS def. (+ 1% BSA and 2% Tween 20) per well upset. After incubation (1 hour at 37 ° C) the wells five times with PBS wash solution as above described, washed. On the development of the Elisatest 50 μ / l developer solution (1 ml 50 mM Citrate buffer pH = 4, 10 µl ABTS solution and 5 µl 30% Hydrogen peroxide) added. It was a positive result recognizable by the green color.

Neutralization test

The cell culture supernatants from hybridoma colonies were found on Presence of virus neutralizing antibodies tested using microneutralization as follows: 50 ul hybridoma supernatant were used per well applied and each with 50 ul MEM (Minimal-Essential-Medium; Flow Laboratories), which Contains 1000 PFU HRV2, 30 mM MgCl₂ and 2% HIFKS, Incubated for 1 hour at 34 ° C in 5% CO₂. Every bowl was  then with 3.10⁴ HeLa cells (HeLa-Ohio strain, 03-147, Flow Laboratories), plates 48 Incubated for hours at 34 ° C in 5% CO₂ and with 0.1% Crystal violet stained in 20% ethanol. Bowls that one intact cell lawn were considered positive viewed. Under the conditions mentioned, a 1/1000 dilution of the ascites fluid a 50% Neutralization can be determined.

From the group of monoclonal antibodies which are directed against the native HRV2, a monoclonal antibody was selected which, in addition to neutralizing properties, also binds to the denatured form of its antigen - the viral capsid protein VP2 - which is carried out using the "Western blot" could be shown ( Fig. 4). One such antibody was named 8F5. With this monoclonal antibody 8F5 you have the possibility not only to neutralize HRV2 or to detect expression products which contain VP2 in "Western blots", but with this antibody z. B. characterize the immunogenic site of HRV2 on VP2 in more detail, as this could be shown in poliovirus system for VP1 (Wychowski, C. et al., 1983, loc. Cit.).

A monoclonal antibody with the above Properties, for example 8F5 was used to control the antigenic properties of the expressed protein of Check pRH969 / 1 from clones 307 and 319.

To check the antigenic properties of the expressed protein of pRH969 / 1 from clones 307 and 319 the monoclonal antibody 8F5 was used. This As already mentioned, Antibody has the for Expression studies favorable property, VP2 (and thus also 969/1) not only in the native, but also in to recognize denatured condition.  

This type of monoclonal antibody is rare and has so far only been found in the poliovirus system (Wychowski, C. et al., 1983, loc. Cit.). The filters with the proteins bound to them were overnight at room temperature in 50 ml "blocking solution", that is PBS (137 mM NaCl, 2.7 mM KCl, 8 mM Na₂HPO₄, 1.5 mM KH₂PO₄, 0.5 mM MgCl₂ · 6 H₂O, 1 mM CaCl₂ · 2 H₂O with 1% BSA, 1% Tween 20 and 10% heat inactivated fetal calf serum (HIFKS), followed by a 3-hour incubation in 4 ml "blocking solution" with the antiserum 8F5 (mouse ascits Supernatant 1/200 diluted in "blocking-solution"), the filter was sealed in between household foil (fixed on a seesaw) and then the filter was rinsed well under running tap water (approx. 20 min), three times for 15 min at 50 ml PBS containing 1% Tween 20. In the last step, the filter in 50 ml "blocking solution" was diluted with the alkaline phosphatase-conjugated anti-mouse IgG (approx. 1/3000 diluted in "blocking solution") for 3 hours The filter was again rinsed well under running tap water d washed three times as described above with 50 ml of PBS containing 1% Tween 20. The staining was carried out in 10 ml of phosphatase buffer (100 mM TrisHCl pH = 9.5, 100 mM NaCl, 5 mM MgCl₂) in the presence of the dyes nitro-blue-tetrazolium (NBT; 165 µg / ml) and 5-bromo-4-chloro -3-indolyl phosphate (BCIP; 82.5 µg / ml). The alkaline phosphatase-conjugated anti-mouse IgG (H + L), as well as the dyes NBT and BCIP were taken from the Protoblot ™ system from Promega Biotec. The dyeing reaction was also stopped after about 5 minutes by rinsing under running tap water. The result of the expression of 969/1 in the maxi-cell system CSR603 is summarized in FIG. 4. Lanes A1 to A4 show a "Western blot" experiment after staining with NBT and BCIP. In trace A1, the entire denatured HRV2 particle (approx. 50 ng HRV2) was also separated as a control. As can be seen from FIG. 4, the 8F5 monoclonal antibody reacts exclusively with VP2 (28.7 kd); the very weakly recognizable higher or lower molecular band represents VPO (precursor of the envelope proteins) or a proteolytic degradation product of VP2. In lane A2 and lane A4, the cell lysates of the E. coli strain CSR603 cells transformed with plasmid pRH969 / 1 (originating from clones 307 and 319) were separated. After staining of the "Western blot", the desired specific signal of the expression product 969/1 can be recognized in both lanes as a band at approximately 40 kd (40.2 kd). As a negative control in lane A3, the cell lysate of E. coli strain CSR603 cells was separated, which had been transformed with the plasmid from clone 224. As described above, this plasmid has an inverted orientation of insert 969/1. Due to the associated loss of specific antigenicity and the fact that there are several termination codons at the beginning of the inverting coding region, no signal can be obtained on the "Western blot" in lane A3. Lane M shows the Coomassie blue staining of the marker proteins (see above).

Essentially the strategy of Wychowsky et al. following, an expression plasmid containing the XhoII fragment from FIG. 2, for example the plasmid pRH969 / 1, was linearized at the single BssHII site and treated with the exonuclease Bal-31 for 2, 4, 6, 8 and 10 minutes. The linearized plasmids shortened in this way were recircularized and transformed into competent E. coli cells. The expressed proteins were then checked for their ability to bind the 8F5 monoclonal antibody. By comparing the sequences of the plasmids that expressed bindable proteins with those of the plasmids that expressed non-bindable proteins, it was found that the immunogenic site of HRV2 on VP2 for the monoclonal antibody 8F5 is between the amino acids that of the nucleotides 1274-1309 encoding the HRV2 (s. Fig. 2 and Fig. 5). Lane M shows the Coomassie blue staining of the marker proteins. Lanes B2 to B4 represent the radio program from lanes A2 to A4 (for detailed information see example 2 loc. Cit.).

Claims (14)

1. Expression vector, characterized in that it contains, in addition to the regulatory sequences required for the expression of heterologous polypeptides / proteins, a DNA in the direction of transcription which corresponds to the entire viral RNA of HRV2 or HRV89 or parts of the viral RNA of HRV2 or HRV89. 2. Expression vector according to claim 1, characterized characterized in that in a singular interface Direction of transcription of the promoter if necessary with an appropriate linker provided DNA according to any one of the preceding claims was inserted. 3. Expression vector according to claim 2, characterized characterized that the singular interface at a suitable distance from the transcription start point located. 4. Expression vector according to claim 2 or 3, characterized characterized in that the linker is in phase for contains the DNA encoding the intitation codon.   5. expression vector according to one of claims 1 to 4, characterized in that the DNA Contains termination codon. 6. Expression vector, according to one of the preceding Claims, characterized in that the vector portion are derived from the plasmid pRH100. 7. Expression vector, according to one of the preceding Claims, characterized in that in the SacI Interface of the plasmid pRH100, the overhanging Single-strand ends had been digested, one corresponding "blunt" DNA according to one of the previous claims was inserted in phase. 8. Expression vector, according to claim 7, characterized in that the XhoII fragment from FIG. 2, the overhanging single-strand ends had been filled in, was inserted. 9. Expression vector according to claim 7, characterized in that the DNA according to the formula was inserted. 10. Host organism, preferably a prokaryote, eukaryote or a mammalian cell line, in particular E. coli, a Expression vector according to one of the preceding Containing claims. 11. A method for producing a polypeptide having the amino acid sequence as shown in Fig. 7 or Fig. 11, or parts thereof, characterized in that a host organism is cultured according to claim 10 under suitable conditions and the Polypeptin formed is isolated and purified by conventional methods. 12. Polypeptides producible according to claim 11. 13. Use of one of the polypeptides according to claim 12 for therapeutic treatment. 14. Medicament suitable for use according to claim 13, characterized in that it is pharmaceutical inert auxiliaries and / or carriers an effective Amount of at least one of the polypeptides after Claim 12 contains.