JPS63219384A - Polypeptide of rhinovirus hrv89 and dna molecule for encoding the same - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

Detailed Description of the Invention (Field of Industrial Application) The present invention provides peptides corresponding to part or all of the viral protein of human rhinovirus strain 89 (HRV89), DNA molecules encoding these peptides, and the use of these substances. Concerning preparation and use.

According to the classical classification, rhinoviruses are RNA viruses, and they form a genus of the Picornavirus family [Cooper P.
, D, ; Agol H, L, ; Bachrach H, L, ; Brown (B
row) F, ;Ghendon Y,
; Gibbs A, J; ; Gillespie J, H; ; Lonberg-Ho1m K, ; Mandel B, ; Meln
ick) J, L, ;Mohant
y) S, B, ; Hobby R, C,
; Rueckert R, R, ; Shea 7
y (Schaffer) F, L, and Tyrrel D, 8, J, Intervirology, 1978.10°165-180: M, R,? MacN
Current Topics of Microbiology & Immunology (Current
Top.

Microbiol, [mmunol, ), 1982.
97.1-26].

They attack the upper respiratory tract of humans and cause an acute infection commonly called the common cold that causes symptoms such as colds, coughs, and hoarseness [Stott B, J.

and Kirinbuto:/ (Killington) R
, A., Annual Review of Microbiology (Ann, RevoMicrobiol, )
, 1972.26.503-524]. Infections caused by rhinoviruses are among the most common diseases in humans. Although these disease processes are generally harmless, the common cold temporarily weakens the organism. This may lead to secondary infections with other viruses or bacteria, which can cause serious illness under certain circumstances. Furthermore, the economic damage caused by rhinoviruses is considerable.

In the United States, rhinovirus infections are estimated to result in more than 2 million lost work or school days annually [Davis B, D; Dulbecco R; Eisen, E.
isen) H, N, and Ginsberg (Ginsberg)
berg) H, S, ;? Microbiology (M
icrobiology), 3rd edition, Haber & Row, 1 New York, 1980, p. 1114).

Furthermore, rhinovirus infections have recently increased significantly in large urban areas. Also, whereas many other infectious diseases result in permanent or long-term immunity from the pathogen, the infections caused by rhinoviruses have repeated recurrences. The reason there is no lasting immunity is that there are many strains (species) of rhinovirus. So far,
As many as 100 rhinovirus strains have been isolated, which show no or only slight immunological cross-reactivity with each other [Fax J, P.
iAmerican Journal op Evidencemiology (
Aw+erican J, Epidemiol,)
, 1976.103.345-354; Melnik (Me
lr+1ck) J, L,, Proceedings of
The Medical Biology (Proc, Mad,
Viol, ), 1980, ■, 214-232),
After infection, antibodies against specific virus strains can be detected, but these do not provide any protection against other rhinovirus strains. Repeated rhinovirus infections can occur because of the large number of strains circulating in the population.

The aim of the present invention was therefore to prepare a medicament which provides for the prevention of infections caused by rhinoviruses.

By using hyperimmune sera, it became possible to classify 50 of the 90 rhinovirus serotypes into 16 groups. [Conney M., Fax. J. I', and Kenn.
y) G, E, , Infectious Immunology
(Infect, Imn+un,), 1982.3
7.642-647). Another classification method is based on binding to cellular receptors. Indeed, despite significant heterogeneity in their immunological properties, rhinoviruses behave similarly to each other with respect to binding to receptors on the cell surface.

By competition experiment, HeLa cells (clone R-19)
It was confirmed that there are only two different receptors for each of the 88 rhinovirus strains studied [Abraham G. and Co.
lonno) R, J, , Journal of Virology (J, Virol, ), 1984.51.34o
-344; Colon R, J, Callah
an) P, L, ; and Long W, J
,, Journal of Virology (J, Virol
), 1986.57.7-12]. However, it should be pointed out that these results were obtained on HeLa cells and do not necessarily apply to receptors on natural host cells in the human upper respiratory tract.

A further object of the invention is to prepare oligopeptides which correspond completely or partially to viral proteins and which stimulate an immune response against the complete virus or which bind to cellular receptors and It can be used to block this.

Rhinovirus, as a typical picornavirus, contains single-stranded RNA, which is V P 1 (PID), V
F6 (PIB), VF6 (PIC) and VF6
(PIA) is surrounded by a capsid consisting of four polypeptides known as (PIA) [Medafpa
ppa) K, C,; Muff Clean (McLean)
C6 and Rueckert R,R,
, Pyrology, 1971,
↓↓, 259-270; Terms in parentheses are from Journal of Virology (J, Virol, ), 1984,
2, 957-959]. A single virus particle contains 60 copies of each of these polypeptides. The relative molecular masses in different rhinoviruses are 34-36,000 for VPI and 27-36,000 for VF6.
30000. 24-28000 per VF6 and VF
7-8000 for 6 [Ma
cNaughton) M, R,; in the above document, 19
82). Another feature of rhinoviruses is that they are rapidly inactivated below pH 5 and are sensitive to highly concentrated salt solutions. Furthermore, the majority of rhinoviruses exhibit optimal growth at 33-34°C [Luri
a) S. I.E., Darnell Jr.
,, J, E, ;Baltimore (BalLimore)
D., and Campbell I^, General Virol.
ogy), 1978, 3rd edition, John Wiley & Sons, New York, 308ff).

A single t with a length equivalent to about 7100 nucleotides
i RNA constitutes the genome of the virus.

It can also act as a matrix for viral protein composition at the same time. Most of the nucleotide sequence is first translated into a long polyprotein, from which the complete viral protein is formed by proteolytic cleavage (Butterworth B, E, Virology, 1973, ■, 439-
453; McLean C0 and Rueckert R, R, Journal of Biology D, Vjrol, 1973
, Earthworks, 341-344; Pine Clean C0; Matthews T, J, and Ruckert R
,R,, Journal Op Virology (J, Vi
rol, ), 1976.1, 903-914). The product of this method is the viral coat protein or VPI.
, VF6, VF6 and VF6, as well as numerous proteins such as P2A, P3C, P3B, P3C and P3D.
etc. P3B (commonly known as VPg) is covalently linked to the 5'-terminal nucleotide of the viral RNA. Similar to poliovirus, P2
It can be assumed that A and P3C are proteases that can partially respond to the processing of viral polyproteins [Matsuton M, I? , i supra, 1982; I-Toyoda +1. ,Nicklin
) M, J, H, ;Murray M,
G, i Anderson C, W,
; Dunn J, J, ; St.
udier) P, W.

and Wimmer E., Cel
l), 1986.45.761-770).

Therefore, P3D is the polymerase for viral RNA replication. At present, little is known about the function of protein P2C.

During processing of the viral polyprotein, part of the amino acid sequence is removed and then degraded [McL
ean) C, ;Mattheu T, J
,; and Lueckert R,R,
(cited above, 1976). At present, it is not possible to say whether these peptides have hitherto undiscovered functions.

Recently, our knowledge about rhinoviruses has expanded to include two human rhinoviruses serotype HRV2 (German patent application no. P35 05 148.5; Skern).
) J, ;Simmergruber
uber) W, ;Blaas D,
;Gruendler P, ;
Freundorfer F
,;Pieler C0;Fozzie (
Fogy) 1. and Kuechler
Eo, Nucleic Acids Research (Nucleic
^cids Res, ), 1985.13.2111
-2126) and HRV14C Stanway (Sla
nway) Go, Hughes P,
J, ;? London t)l/do(Mountfor
d) R, C0;-' Minor P, D,
; and Almand J, W., 1984.12.7859-7875; Callahan (
Callahan) P, L; Mizuta = (Mizu
tani) S, and Colonno R
, J. , Proceedings of the National
Academy of Science (Proc. Natl.
Acad, Sci,), 口S^, 1985.82
, 732-736 ; European Patent Application No. 85.401 4
With the determination of the nucleotide sequence of 65.1), the research has been greatly expanded. Furthermore, the structure of HRV 14 has a resolution of 3
This was revealed by X-ray structural analysis of humans [Rossman (
Rossmann M, G; Arnold
d) B; x Erickson J.
V4. ;Frankenberger
er) B, ^, Niggelifis (Griffith) J
, P, ; Hecht tl, J, ; Johnson J, B, ; Kula? −
(Kramer) G,; Luo M,:
%-/Mosser A, G; Rueckert R, R, Sherry
(Sherry) B,; and free end (Vr
iend) G,, Nature (London) (Na
ture (London)), 1985.317
．． 145-153). Additionally, the locations of four epitopes responsive to HRV14 neutralization were identified by mutant strains resistant to HRV14 monoclonal antibodies [Sherry B., Mosser et al.
) A, G, Colonno R, J.

and Rueckert R.R., Journal of Virology (J, Virol,), 1
985, AE, 246-257). All of these findings indicate a close resemblance to poliovirus.

This is true for both nucleic acid or protein sequences and virus structures, and is clear from a comparison of X-ray structural analyzes of poliovirus [Hogle
J, M, ;chi! ! -(Chow) M., and FilIlan D.J., Science (
Science), 1985.229.1358-13
65). Although this indicates a close relationship between rhinoviruses and enteroviruses, one would have expected the two human rhinoviruses to have greater similarities to each other than to polioviruses. However, this fact means that in some genes the homology between HRV2 and HRV14 is no greater than that with poliovirus. One possible explanation for the relatively low similarity between these two rhinoviruses is the fact that HRV2 and HRV14 bind to different cellular receptors and thus constitute the prototype of a particular group. Therefore, other rhinoviruses that bind to the same receptor as HRV14 should also show high sequence similarity to HRV14 in terms of sequence.

Therefore, human rhinovirus strain 89 (HRV89)
belongs to the same receptor group as HRV14 [Abraham (
Abrahaa+) G, and Colon (Colon)
no) R, J.

cit., 1984), which was chosen for sequence studies.

To obtain viral starting material, HeLa cells were infected with HRV89 in an appropriate infection medium and incubated for several hours under optimal growth conditions.

This virus can be obtained from both cells and culture.

The virus purification step yields a virus formulation, the protein pattern of which is shown, for example, in FIG.

The viral RNA was obtained from the virus preparation and purified, for example by phenol extraction.

This was then transcribed into cDNA by reverse transcriptase and oligo dT as primer. Obtained RNA
/cDNA hybrids are extended homopolymerically,
inserted into a suitable plasmid such as pBR322.

The use of reverse transcription of RNA is well described in the literature for the integration of RNA/cDNA hybrids into plasmids and the transformation of bacteria [Kitamura (K.
itamura) N, and Wimmer
) Eo, Proceedings of the National Academy of Sciences (Proc.
, Natl, Acad, Sci, USA) 19
80.77.3196-3200; Nels
on)T.

and Ilrutlag Dl, Method in E
zymology), 1979.68.4l-50i
Zain S, H Sambro
ok) J, ; Roberts J, J
, Keller Its, Fried M, and Dunn A
, Cell, 1979, Earthworks, 851-8
61i Mouth Free (Roychoudhury)
R, and Wu (-U) R1, Method in Enzymolog
y), 1980.65.42-62; Kitamura (Kit
amura) N, Semler B, L,
; Rothberg P, G; Larsen G, R, i Adler
r) C, J, H Dorner A,
J, i Emini (Emini) E, ^, ; Hanekaku (
Hanecak) R,; Lee J, L;
;Van der Werf
S,;Anderson C6W
, ; and Wimmer) E, ; Nature 1-- (London), 1981
．． 291.547-553; van der Welf S,;
Bregegere 'F;
Kopecka H, Kitamura N,; Rothberg P, G; Kourilsk
e) P; Reimer E, and Girard
ard) M.: Proceedings of the National Academy of Sciences (Proc. Nat.
l, Acad, Sci, [l5A), i98
1, Uni, 5983-5987; Namoto (Nas+ot
o) ^,; Omata (抛ata) T, i) Yoda (
Toyoda) Ho, Kuge S, ;llosie tl, ; Kataoka
aoka) Y,; Genba A,
i Nakano Y, and Imra
ura) N,, Ib1d%1982.79.57
93-5797; Stanway
G, , Cann A, J, ; Hoptman (
Hauptmann) R, ;Hughes
) P,, Jellyfish (C1arke) L, D,, Mount'tfold (R, C,, Minor) P, D,, Shield (Schi
ld) G, C0, and Almand
J.W., Nukleitsk Acid Research (Nu
cleic Ac1ds Ree, ) 1983, Earthworks, 5629-5643; Skern
T, Sommergruber
W, , Blaas D, ; Gruendler P, ; Fraundorfer F, ; Peeler
(Pierer) C,? Fogy 1.
and Kuechler B, 1bi
d, 1985).

A suitable host organism, such as Escherichia coli (E.
After transformation of E. coli HB 101, plasmid D
NA was prepared using the “plasmid minimal preparation method” (mint-prep method).
The size of the recombinant DNA was determined using the restriction endonuclease PstI, the fragments were separated by electrophoresis, and the size of the recombinant DNA was determined using the known lambda-Hiud
ll [compared with marker DNA.

To further clone the DNA, recombinant pBR32
The DNA fragment obtained by PstI digestion of the two clones was purified and transferred into a suitable vector, e.g. plasmid pUC.
Incorporated into Q. This recombinant vector is then transferred to a suitable host, such as E. coli (E. coli) JM 1.
Moved to 01. Subclones that were successfully transformed with the recombinant vector were harvested, and their plasmid DNA was isolated and purified.

Three brimer fragments of different lengths [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 long synthetic primer (D
)It was used. With this complementary single-stranded DNA, 3
A reverse transcript of HRV89 labeled with 2P was prepared.

To detect the HRV89 sequence in the recombinant DNA, this plasmid DNA was digested with Pstl and analyzed by electrophoresis. This DNA fragment was transferred from the gel onto a nitrocellulose filter and fixed. These DNs
A-carrying filter with radioactive label 11tHRV89-cDNA
and then discarded this filter. Restriction sites were mapped and sequenced from the thus obtained recombinant DNA 7 fragments complementary to HRV89-RNA. Clones were obtained and these were HRV89
represents the genome of

The sequence of HRV89 genome is shown in FIG. This is shown as DNA obtained from recombinant DNA sequencing. Twenty-one clones (Figure 3) and an additional 5 clones were used for sequencing. This array is 715
2 nucleotides and does not contain the 3'-terminal poly-A. It contains one open reading frame, which encodes a polyprotein with a length corresponding to 2164 amino acids. This open reading frame starts at position AUG at position 619, with a stop codon UA of 42 nucleotides before the start of poly-A.
Terminates at A.

The 5'-end sequence is a clone! Fragment D obtained from lh5 and 10 was used as a primer (see Figure 3); cleavage with PstI yields fragments of fixed length. Through sequencing,
It was found that these two clones both contained the sequence TTAAAC, which was adjacent to oligo-G. This is derived from the integration site in the plasmid. This indicates that these clones are the 5'
- It is concluded that it constitutes the terminus. This sequence corresponds to the first nucleotide of three types of poliovirus. The three polioviruses are Koxalaki virus Bl (Hewlett M, J,
; and Florkiewicz
z) R, Z, , Proceedings of the National Academy of Sciences (Pr.
oc, Natl, Acad, Sci, USA)
, 1980.77.303-307; Stanway (S
tanway) G, ;Cann A, J
,; llauptmann R,
; Hughes P, ; Clark (C1)
arke) L, D, ;Mountholt (Mount
forld) R, C,; Minor P
, D; Schild G, C, and Almond J, 11. , ditto, 1
983), HRV2 [Skern T,
;Sommergruber (SoIlls+ergrub
er) W, ;Blaas D,
;Gruendler P;;Fraundorfer F;
:Pieler C, ;Fogy
) 1. and Keherer Eo,
Ibid., 1985), a and HRVI 4 C7, Starvay G; Hughes (H
ughes) P, J, ;Mount fault (Mount
ford) R, C,; Minor P,
D.; and Almond J. 1
, ibid., 1984).

By multiple comparisons by analyzing the 618 nucleotides between the 5'-end and the start of the long open reading frame, considering the presence of any inserts, 79%
It was found that there is extremely high homology, while HRV
The homology with 14 is 67%, and the homology with type I poliovirus is 62%. This homology is particularly large in several regions. In total, nine different blocks of 17 or more equivalent nucleotides located alternately are present in the 5'-terminal regions of the HRV89 and HRV2 genomes.

Comparison of amino acid sequences reveals that the homologous regions are also continuous in the region encoding the polyprotein (the fifth
figure). It is particularly noteworthy that there is significant homology between HRV89 and HRV2, while surprisingly and contrary to expectations there is low homology with HRVI4. The strongly protected regions are coat proteins and nonstructural proteins (P2A-P3
Includes both areas D). FIG. 18 shows, for example, HRV 2
A sequence comparison of a partial sequence of HRV89 with a partial sequence of HRV89 from the P3A and P3B (VPg) gene regions is shown. Substantial homology in amino acid sequences is readily apparent. Even where there are different amino acids that occur independently, they are generally substituted by amino acids that are chemically closely related, such as arginine (R
) is replaced by lysine (K), valine (V) by isoleucine (1), leucine (L) by isoleucine (1), and so on. P3A(!:P3B(V
The cleavage site between P g) is completely conserved.

This can be easily understood from the homology. On the other hand, there is a clear dispersion within a small region (corresponding to the sequence between nucleotides 5018 and 5029 in HRV2), which results in a dispersion of amino acid sequences in the corresponding region of P3A. The significance of this discovery is currently unknown. At present, the function of P3A is unknown. Therefore, it is not possible to determine whether subtypes of HRV2 exist and have greater homology to HRV89 in this region than shown in this example. This means that HRV89 is H
Despite belonging to the same receptor group as RV14, HRV2
This clearly shows that there is a great deal of similarity. Therefore, based on the sequence, HRV2 and HRV89 are HRV2 and HRV89.
They form a common group that is clearly distinct from V14 and polioviruses. This fact seems to imply that human rhinoviruses are indeed more similar to each other than previously deduced from homology comparisons between HRV2 and HRV14. Thus, the suggestion that rhinoviruses and enteroviruses belong to the same genus of the family Picornaviridae appears to be problematic [Stanway G.;
ghes) P, J, ;Mount fault (Mount
ford) R, C,; Minor (Mtnor) P,
D.: and Almand J.W.
, , supra, 1984).

Viral proteins are obtained from polyproteins by proteolytic cleavage. To determine the cleavage site, the viral coat protein was isolated and the N-terminal amino acid sequence determined (see Figure 6). In this way, we not only defined the cleavage site within the viral coat protein, but also confirmed the open reading frame derived from the nucleotide sequence. By comparing the amino acid sequence from the nucleotide sequence, the VP
4/VP2 cleavage occurs between glutamine and serine;
It is clear that VP2/VP3 cleavage occurs between glutamine and glycine, and VP3/VPI cleavage occurs between glutamine and asparagine. These cleavage sites are exactly the same as HRV2 but different from HRV14 [Stanway G, ; Hughes P, J, ; Mountford R, C, ; Min
or) P, D, and Almand
J.W., supra, 1984).

The present invention is directed to a DNA containing information on HRV89 viral RNA.
This enables the production of A.

However, the invention relates not only to genomic sequences specifically encoding viral proteins, but also to variants obtained, for example, by mutation, degradation, transposition or addition. Included are each sequence that is degenerate by comparison to these illustrative examples. Sequences that hybridize under stringent conditions to the illustrated sequences or portions thereof, e.g. under conditions selecting for homology of 85% or more, preferably 90% or more, and encode proteins with a spectrum of viral activity. included under such conditions. This hybridization was carried out at 65°C with 6XSSC15XDenhardt's
s) Carry out in solution 10.1% SDS.

The degree of stringency is determined by the cleaning process. Thus, about 85%
For selection of DNA sequences with homology greater than or equal to about 90% homology, suitable conditions are 0.2XSSC10.1% SDS/65°C, and for selection of DNA sequences with homology greater than about 90%, suitable conditions are 0.1XSSC10. , 01% SDS/65°C.

As shown, this DNA can be inserted in its entirety or as a fragment thereof into a suitable plasmid vector to achieve propagation of the DNA or expression of the protein itself after transformation of a suitable host organism.

Appropriate hosts, vectors and conditions suitable for these manipulations are already well known to those skilled in the art. Similarly, numerous studies have been published describing the synthesis of foreign proteins in bacteria by genetic engineering [e.g. Harris (II)
arris) T, J, R, ``Genetic1 Replication Engineering''
g) J Williamson (Williamson) R
.

! , 1983, ↓, Academic Press, London.
127ff). For this purpose, the foreign DNA is introduced in the vicinity of the appropriate bacterial control regions (promoter, ribosome binding site) on a plasmid, which allows this information to be expressed (usually as a fusion protein) in high yield.

The corresponding protein is thus obtained. In the field of picornaviruses, there are numerous publications describing the expression of viral genes in bacteria (Keller H,;
) W, H Kurz C,; Fors (F
orss) S, ;Shake-(Schaller
) Ho; Franzel R, ; SLrohsaier K, ; Marquardt 0. ; Zaslausky V, G; and Hofschneider P, H,
, Nature London, 1981.2
89.555-559; Clyte (Ni1eid) D,
G, Yansura D, Small B, Darbe
nko) D,;Moore D,M,
; Grubman M, J; Mkercher P, D;
Morgan D.O., Robertson (
Robertson) B, Il; and Bachrach Il, L, Science (
Science), 1981.214.1125-11
29; Wychowski C,;
van der Werf S
, ;Siffert0. : Crainic Ro: Brunea
u)P.

and Girarl M., EMB O Journal (EIIBOJ), 1983, Earthworks.
2019-2024; Klump W,
;Marquardt 0. and llofschneider P, H
Proc. Nat.
l, Acad, Sci, USA), 1984.8
1.3351-3355; Hanecak
R, ; Semler B, L, ; Ariga tl, ; Anderman
erson) C, W, H & Wiseer
E, ;Cell, 1984.37.10
63-1073; Skern T; Sos+sergruber W;
;Blaas;;Gru;
endler) P, ; Freundorfer (Pr
auunderfer) F,; Peeler (Piel)
er) C0: Fogy 1. and Kuechler E, 1985; Strebel K, Beck (
Beck) E, I Stobma
ier) K, & Schaller
H., Journal of Virology (J, Vir.
(Toyoda H, et al., 1986).

Prokaryotes, e.g. L. 12 strains 294 (ATCCN131 446) is particularly preferred for expression.

Other suitable strains are E, 9dan X11776 (ATCCN
131537). Apart from the above strains, LcoliW
3110 F-, λ-, prototroph, (ATCC 27325), Bacillus strains such as Bacillus subtilis and other Enterobacteriaceae such as Serr.
atta marcescens) and various Shademonas species can also be used.

Generally, plasmid vectors containing replicon and control sequences from species compatible with the host cells can be used with these hosts. This vector usually has a replication site as well as a recognition sequence, the latter allowing phenotypic selection of transformed cells. For example, L. uni is commonly transformed with pBR322, a plasmid of P. elegans origin [Bolivar et al., Gene, 197
7.2.95].

pBR322 contains genes encoding ampicillin and tetracycline resistance and thus provides a simple means for identifying transformed cells. This pBR322 plasmid or other plasmid must also contain a promoter itself or be mutated to contain a promoter that can be utilized by the microorganism for the expression of its own protein, this promoter being recombinant. D.N.
The β-lactamase (benicillinase) and lactose promoter systems [Chang et al., Nature] are most frequently utilized for the preparation of A.
, 1978.275.615; Itakur
a) etc., Science, 1977.1
98.1056; Goeddel et al.
1979.281.544] and tryptophan (t
rp) promoter system [Goedel et al., Nucleic Acid Research (Nucleic Acid Res)
), 1980, main, 4057; European Patent Application-A
-0,036,776]. Although there are promoters that are most commonly used, other microorganisms have also been developed and
and is used. The genomic sequences according to the invention can be used, for example, under the control of the left-hand promoter of the λ-bacteriophage (PL). This promoter is one of those known to be particularly powerful and controllable. Control is possible by the λ-repressor, whose flanking restriction cleavage sites are known.

A thermosensitive allele of this gene can be inserted into a vector containing the viral DNA-sequences. Increasing the temperature to 42° C. inactivates this repressor and the promoter is expressed to its maximum concentration.

The total amount of mRNA produced under such conditions should be sufficient to yield cells with approximately 10% P of promoter origin in the newly synthesized RNA.

Thus, clone banks can be established in which functional viral DNA sequences are placed in the vicinity of the liposome binding site at varying distances from the λ-PL promoter. These clones are then tested and the one with the highest yield is selected.

Expression as well as translation of viral DNA sequences may also take place under the control of other control systems, which in untranslated form may be considered homologous to the organ.

Thus, for example, the chromosomal DNA from lactose-dependent E,:21J contains the lactose or 1ac-operon, which allows the degradation of lactose by secreting the β-galactosidase enzyme.

I! ac-control element is λ-bacteriophage pl
It can be obtained from ac 5, which infects mu uni. The 1ac-operon of this phage can be obtained from the same bacterial species by transduction.

The control system that can be used in the method of the invention is obtained from plasmid DNA that is unique to the organism. This i! The ac-promoter operator system is induced by IPTG.

Similarly good effects can be obtained using other promoter operator systems or parts thereof, such as arabinose operator, colicin E1 operator, galactose operator, alkaline phosphatase operator, xylose A operator,
” promoter, etc.

This gene is preferably expressed on the expression plasmid pER103 (
E. Rastl-Dwor
kin) et al., Gene, 1983.21.
237-248: European Patent Application No. 83112812.9
Reference: Deposited DSM2773 December 20, 1983)
or a plasmid derived from it, e.g. pRH
loo (Figure 7). All of these vectors contain regulatory elements that lead to high expression rates of the cloned genes.

The present invention relates to the integration of the total DNA of human rhinovirus types 2 and 89, parts of said DNA and various combinations of fragments of these DNAs into suitable plasmid vectors, the plasmid vector itself, and the host to be transformed thereby. It includes organisms and the synthesis of related proteins, the proteins themselves, and their uses.

As already mentioned, so-called expression vectors are usually used for the preparation of proteins in a host organism by genetic manipulation. These expression vectors contain so-called regulatory sequences, which are capable of responding to optimal transcription and translation of non-corresponding DNA. These regulatory sequences include a strong promoter, preferably a regulatable promoter, and include one or more specific cleavage sites for restriction enzymes in the direction of transcription of the promoter, the sites being located at appropriate distances from the promoter. It's dark.

Some expression vectors have a starting codon (start codon)
is placed before the first specific cleavage site in the downstream region of the promoter. If a vector without a start codon is used, the DNA to be expressed is first provided with a start codon and then inserted. Furthermore, the DNA to be expressed in the vector must be accompanied by a so-called stop codon.An important point in creating an expression vector with a structural gene is that this structural gene must be inserted in the phase containing the start codon. be.

Non-corresponding DNA can be inserted into expression vectors using known methods. In some very rare cases, the beginning of the DNA has the same recognition site as the specific cleavage site of the expression vector, and in many cases it is necessary to make the cleavage sites compatible. This is achieved, for example, by terminating or digesting the overhanging end of the terminal strand, or by adding a suitable linker. If the chosen expression vector does not have a start codon, one can be added at the same time as a linker. In all of these operations, it is important to recognize that the non-matching DNA begins in the phase with the start codon.

The problem of the invention can be solved particularly advantageously by using the vector pRH100 and expression plasmids derived therefrom for DNA or DNA fragments of HRV2 or HRV89. This vector was derived from pBR322, and its essential structural characteristics are ±iEetly- and -+7':A (Serr
atia marcescens), which constitutes an effective signal sequence to initiate protein synthesis, and 3-β-indoleacrylic acid (=IAA: inducer of the trp-operon).
It can be activated by Behind this trp-promoter there is a synthetic ribosome binding site and a start codon ATG. Immediately after the start codon is a 5acl cleavage site (see Figure 8). For purposes of the present invention, the single stranded end of this cleavage site is removed by a single strand specific nuclease. The plasmid thus modified is a DNA or a fragment thereof (plant-end), such as HRV2.
- cDNA plant-end Xho11 fragment 9
69/1 (Figure 9), an expression plasmid is formed which produces the protein without the fusion in a host cell transformed with the plasmid, such as a prokaryotic or eukaryotic system. . This type of protein expression has the advantage compared to fusion proteins that the expressed protein retains its original structure and makes it possible to subject the protein to immunological or enzymatic tests.

Knowing the complete NA sequences of HRV2 and HRV89, the present invention allows each given DNA fragment to be isolated from a suitable expression plasmid, such as the expression plasmid pRH.
100 to allow expression of the DNA.

However, it is necessary that the fragment be at the correct distance from the regulatory sequences and in the correct reading frame with respect to the start codon ATG. The predetermined fragment can be obtained by complete chemical oligonucleotide synthesis or by European patent application 0
192175 or German published patent 3628658.3
can be generated by fragmenting a plasmid according to the method.

It is also possible to combine these two methods.

Thus, for example, the DNA molecule shown in FIG.
930 and 1581 by a1I and Bs5HII
Fragments can be obtained by double digestion at the . Next, to express mature VP2, 2
Two oligonucleotide fragments are required, these are 818-931 and 1582-1 from FIG.
Represents 600+TAG nucleotides. These oligonucleotides can be prepared using known methods such as an Applied Biosystems Model 381A DNA Synthesizer (A
pplied Biosystems Model 3
81A DNA5ynthesizer). After ligation of these fragments (Fig. 12) and insertion into the "plant" 5acl cleavage site of plasmid pRH100, the expression plasmid pRH969/2 is obtained, which is suitable for the expression of mature VP2 (Fig. 15).

As already mentioned above, this method is suitable for HRV2 or HRV8
It can also be applied to 9 other viral proteins.

Using the three Hindnl cleavage sites in the DNA of HRV89, an expression plasmid containing nucleotides of HRV8902251-4703 can be obtained. To do this, the DNA molecule encoding the complete viral protein region is cut and isolated with the old nd[[, approximately 38
The 60 bp long fragment H-H is purified and then cut with 5phx.

If necessary, linkers are added to one or both ends of fragment S-H representing nucleotides 2273-4703. This linker inserts the fragment into the appropriate linearized expression vector in the correct reading frame.

The choice of linker depends on the available restriction sites within the expression vector. Start codons such as ATC and TAA
It is particularly advantageous to use linkers which contain a stop codon such as eg in phase with the DNA sequence to be expressed. However, this is the case when the expression vector or the sequence to be expressed does not already have these codons.

Appropriate expression vectors can be found, for example, in the catalog Ch. 27-493.
Vector pK available from Pharmacia as 5-01
K233-2 (4602bp) (Franc (^mann
)E.

and Brosius J., Gene (G.
ene), 19.85, ee, 183-190).

This vector is double digested with NcoI and old ndI[I, and the larger fragment is isolated and purified. In order to be able to insert this fragment ()S-H into a linearized vector, it has the following formula: Nc
o l-3ph I') must have an anchor.

CATGGTGT'GCATGGTTTCTGCATG
Fragment S-H provided with a linker to CACACGTACCAAAGA is conjugated with a linear vector. The reagents required for this operation are known to those skilled in the art and are used in a known manner.

The expression plasmid pKK89/2A (Figure 16) thus obtained can be regulated by IPTC induction and allows the expression of the mature protein (additional amino acids from the vector) shown in Figure 17 for example in strain JM105. . This protein is expressed when the ATG to Met at position (optimal distance from the regulatory region) 545 is utilized as ribosomally initiated. However, it is also possible to use the ATG for Met at position 548 as a starting signal, which would give a protein three amino acids shorter.

In addition to prokaryotes, eukaryotic microorganisms such as yeast cultures are the most commonly used eukaryotes, but it is also common to obtain numerous other species. For expression in Saccharomyces, for example the plasmid YTp'M St inchcomb, Nature (N
ature), 1979.282.39; Kingsman et al., Gene, 197
9, Yu, 141; Tschumper et al., 1980.10.157] and plasmid YE
p13 (Bwach, etc., Gene)
, 1979.8.121-1333 is advantageously utilized. Plasmid YRp7 contains the TRPI gene, which is a selectable marker for enzyme mutants that cannot grow in tryptophan-free media (e.g., A
TCCNα44076”).

The presence of a TRpl defect as a feature of the yeast host genome is an effective aid in the detection of transformation when cultured without tryptophan. This situation corresponds to plasmid YEp 13
I also like it very much. This is the yeast gene LEU2
, which can be used to complement the LEU-2-minus mutant strain.

A suitable promoter sequence for yeast vectors is 5' of ADHI.
-Flanking 'nM area [Amera- (As+n+erer) G, , Methods of Enzymology
+++ology), 1983.

101.192-201), 3-phosphochrycerate-kinase [Ilitzen et al.
Journal of Biological Chemistry (
J. Biol, Chell.), 1980.2
55.2073) or other glycolytic enzymes [Kawasaki (
Kawasaki) & Fraenkel
), Biochemical & Biophysical Research
Communications (Biochem, Bioph
ys, Res, Comm, ), 1982.108.
1107-1112), including enolase, glyceraldehyde-3-phosphneet dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase and glucokinase.

By forming a suitable expression plasmid, termination sequences associated with these genes can be inserted at the 3'-end of the sequence to be expressed in the expression vector, thereby ensuring polyadenylation and termination of the mRNA. Ru.

Other promoters that have the advantage of having transcription regulated by growth conditions include alcohol dehydrogenase-20) intochrome C1 acid phosphatase, a degrading enzyme associated with nitrogen metabolism, and glyceraldehyde-3, mentioned above.
The promoter region of phosphate dehydrogenase and enzymes responsive to maltose and galactose processing. Yeast mating type loci such as the gene BARI,
Promoters regulated by MEI, 5TE2.5TE3 and 5TE5 can be used in temperature-controlled systems using mutant strains [Rhines Ph, D.

Thesis, Oregon State University, Eugen Oregon (1979); Herzwitz & Oshima (t
lerskowitz and Oshima), Molecular Biology of the Yeast Saccharomyce genus (The Molec
ular Biology of the East
5accharoa+ces) Part ■, 1981
．． 181-209, Cold Spring Harbor Laboratory
*These mutants affect the expression of the yeast quiescent mating type cassette. as a result,
Acts as an indirect mating type dependent promoter. but,
In general, any plasmid vector containing a yeast-compatible promoter, origin of replication, and termination sequences is suitable.

In addition to microorganisms, cultures of multicellular organisms are also suitable as host organisms. In theory, any of these cultures could be used, whether they were obtained from an animal or an animal. However, what is of most interest are tissue cells, and the proliferation of tissue cells in culture (cell culture) has recently become a routine method [Tissue Culture, Academia. Tsukupress, Kruse & Batterson edition (Kruse & Batterson)
Patterson), 1973]. Examples of useful host cell lines of this type are VERO and ) IeLaiB cells, hamster ovary (CHO) cells and WI38, BH
K, CO3-7 and MDCK cell lines. Expression vectors for these cells are generally (if necessary)
It includes a replication site, a promoter located in front of the gene to be expressed, along with any necessary liposome binding sites, RNA splicing sites, polyadenylation sites, and transcription termination sequences.

When using mammalian cells, control functions in the expression vector are often obtained from viral materials. for example,
Commonly used promoters are derived from polyoma, adenovirus 2 and especially simian virus 40 (SV40). The SV40 early and late promoters are particularly useful.

This is because both of these can be easily obtained from the SV40 virus as a fragment that also contains the viral replication site (Fiers).
et al., Nature + - (Nature), 1978.27
3.113), 5V40(7) small or large fragments can also be used.

However, these include sequences with a length equivalent to about 250 bp,
This extends from the 1lindln cleavage site to the 8gll cleavage site at the virus replication site.

Additionally, it is possible and desirable to use control sequences or promoters that are generally linked to a given gene sequence. However, these control sequences must be compatible with the host cell system.

The replication site can be provided with a corresponding vector structure for integrating exogenous sites, such as SV40 or other viral sources (eg polyoma, adeno, vSv, etc.). It is also possible to provide the chromosome replication machinery of the host cell. The latter size is usually sufficient if the vector is integrated into the chromosome of the host cell.

Transformation of the cells with a vector can be performed in a number of ways. For example, using calcium, washing cells with magnesium and adding DNA to cells suspended in calcium.
Alternatively, the cells can be co-precipitated with DNA and calcium phosphate. For subsequent gene expression, these cells are transferred to a medium that selects for transformed cells.

After transformation of the host, gene expression and fermentation or cell culture is carried out under conditions in which the protein is expressed, and the products are extracted by conventional chromatographic methods for separation.
Materials containing viral proteins with or without leader and tailing sequences are thus obtained.

This protein can be expressed by a leader sequence at the N-terminus (pre-protein), which can be removed in some host cells. Otherwise, the leader polypeptide (if present) must be detached to give the mature protein. Alternatively, this mature protein can be produced directly in microorganisms. In this example, the precursor sequence of the yeast mating pheromone MF-α-1 is used to ensure correct maturation of the fusion protein and precipitation of the product into the growth medium or periplasmic space. This DNA sequence is capable of binding MF-α-1 at the predicted cleavage site.

Apart from the use of the DNA according to the invention for the preparation of relevant proteins in bacterial or eukaryotic cells, amino acid sequences from nucleotide sequences, i.e. in whole or in part, can be synthesized, for example, on an automatic peptide synthesizer [e.g. ) model 990].

These oligopeptides, as well as genetically engineered proteins, can then be used to stimulate an immune response against the whole virus or to bind to or block cellular receptors. Studies using oligopeptides to stimulate immune responsiveness to poliovirus have been published [Emini
E,'A, ;Jameson B,
A, & Wimmor E,, Neich+
-(Nature), London, 1983.30.6
99-703). Similar research has also shown that foot virus (
foot uirus) and mouse virus (mou
th utrus)
Bittle) etc., Nature, etc.
ondon, 1982.298.30-33; Buff (Pf
aff) et al., EMBOJ, 19820), 869
-874]. The present invention also encompasses oligo- and polypeptide components of the HRV89 protein, which may be combined with other oligos or polypeptides as a result of synthesis or for a given use, such as a vaccine.

The invention further relates to monoclonal antibodies and their production, antibodies directed against structural and non-structural viral proteins (natural and denatured). The binding of a monoclonal antibody to the cellular receptor for HRVI4 and its blocking have already been disclosed by Colonno et al. [European Patent Application No. 0169146]
.

The property of antibodies specifically binding to antigens can be put to practical use in in vitro quantitative and qualitative determinations (immunoassays) and antigen purification (immunoafinity chromatography). The serum of an immunized animal usually contains a number of different antibodies, which react with the same antigen with different affinities and at different binding sites. The serum also contains antibodies against other antigens. However, effective use of antibodies for antigen determination and purification requires high specificity and reproducibility.

Homogeneous antibodies meeting these requirements are obtained by the hybridoma method disclosed by Kahler and Milstein. In principle, this method consists of fusing antibody-secreting B-lymphocytes, for example cells from the lungs of an immunized animal, with tumor cells. The formed hybridoma cells have the ability to permanently reproduce by dividing, and form a uniform type of antibody.
It also has the ability to secrete. By culturing in a selective medium in which unfused tumor cells die, hybridoma cells can proliferate and, by appropriate manipulation, obtain clones, i.e., populations of cells derived from a single hybridoma cell and genetically equivalent. It becomes possible. Monoclonal antibodies produced by these cells are then isolated.

The present invention relates to monoclonal antibodies against rhinovirus strains HRV2 and HRV89, hybridoma cells producing such antibodies, methods for their preparation and use.

It also relates to monoclonal antibodies against the viral proteins according to the invention, hybridoma cells producing them, methods for their preparation, and uses thereof. Preferred are hybridoma cell lines and monoclonal antibodies secreted by them that react specifically with the viral proteins or parts thereof. Monoclonal anti-H
The method for preparing RV2 and anti-HRV89 antibodies involves immunizing mice with rhinovirus or its viral proteins,
B-'J lymphocytes from animals thus immunized are fused with myeloma cells, and the resulting hybridoma cells are cloned and then cultured in vitro or injected into mice to generate antibodies. Characterized by isolation from culture.

The invention further relates to immunoaffinity chromatography columns and immunoassay test kits containing these antibodies.

Using the method of the invention, mice (eg, Barb/C mice) are immunized using known methods. In a preferred embodiment, the rhinovirus is injected roughly weekly or in extended cycles over several weeks, e.g., 5-12 weeks, to form a sufficient number of antibody-producing B-IJ lymphocytes.

B-lymphocyte-containing organisms, such as tile cells, are removed from the immunized mice and fused with myeloma cells that, as a result of the mutation, will not grow in the selective medium. These myeloma cells are known and, for example, X63-Ag3,
63-Ag8.6.5.3, MPC-11, NSL-
Ag4/1, MOPC-21NS/1, MS-1 (AT
CCTl818) or 5P210. In a preferred embodiment, the tile cells of the immunized mice are transformed into cell line NS.
-1 (ATCCTl818) myeloma cells.

This fusion can be carried out using a known method of mixing B-lymphocytes and myeloma cells with the addition of a cell fusion agent such as polyethylene glycol, Sendai virus, calcium chloride, or lysolecithin. The fusion is preferably carried out in the presence of polyethylene glycol having a molecular weight of, for example, 1000-4000. After fusion, the resulting hybrid is cultured in a known manner in a selective medium (HAT medium) supplemented with hypoxanthine, aminopterin and thymidine. Unfused myeloma cells cannot grow in this medium and die. The same applies to normal lymphocytes.

Supernatants from hybridoma cultures can be tested for the content of specific antibodies according to known methods, for example by radioimmunoassay or agglutination. It was found that the above method yields hybridoma cells that secrete antibodies specific to rhinovirus. Hybridoma cells that produce antibodies with a given specificity are created by cloning.
selected from a mixture of various hybridoma cells resulting from fusion. This culture consists of a known method called limiting dilution, starting from a single growing cell.

For mass production, hybridoma cell clones producing antibodies with a given specificity are cultured in vitro in known media or by injection into mice for replication. In a preferred embodiment, hybridoma cells are injected into mice pretreated with Blistane, ascites fluid is collected, and separated by precipitation from the fluid with ammonium sulfate solution. Monoclonal antibodies according to the invention, such as antibody 8F5, are directed against rhinovirus proteins and can be used for therapeutic purposes.

Antibodies against rhinoviruses and obtained with these hybridoma cells can be used in known methods for the preparation of immunoaffinity chromatography columns. In a preferred embodiment of the invention, a suitable carrier material (suspended in a buffer solution) is combined with the antibody solution, any unbound fractions are washed away and empty sites of the carrier are blocked. Specific antibodies obtained by hybridoma cells are used in known manner for the production of test kits. These test kits may be based on various methods, such as radioimmunoassays, latex agglutination, dot tests, competition or sandwich radioimmunoassays, enzyme immunoassays, immunofluorescence or immunochemical enzyme tests. These kits contain, in addition to the usual antibodies of various origins, antibody conjugates with enzymes or fluorescent carriers (e.g. HRV2 or HRV89 labeled with radioisotopes such as Izs, or e.g. horseradish peroxidase; (including complexes with enzymes such as alkaline phosphatase), as well as enzyme substrates, suitable buffers, gels, latex, polystyrene or other fillers and carriers.

Further features of the invention are described in the following examples, which are illustrative of the invention and are not intended to limit it.

Within the scope of the present invention, when we speak of properties and/or biological activities with respect to proteins (polypeptides), we mean whether the proteins (polypeptides) in question stimulate an immune response in biological tests and/or against rhinoviruses. The present invention is particularly relevant to the following points.

A DNA molecule encoding at least one viral protein of rhinovirus strain HRV89; -corresponding to the entire viral RNA or a part thereof of the viral RNA of rhinovirus strain HRV89; part 4) Lt stambac VPI, VF6, VF6, VF
6, P2A, P2BS P3C, P3A.

D that encodes a viral protein consisting of P3B and P3C; 1) that encodes a viral protein in which at least two of the above proteins are combined in any predetermined combination; -or that encodes an individual viral protein itself.
NA molecule.

The invention also relates to DNA molecules encoding degenerate forms (synonymous forms) and alleles of the above DNAs.

D corresponding partially or completely to the arrangement shown in FIG.
NA molecules are preferred.

The present invention further describes the use of rhinovirus strains HRV that are hybridized with one of the specific DNA molecules or synonyms of these molecules under stringent conditions to give a homology of 85% or more.
This DNA molecule according to the invention can be expressed in a suitable expression carrier, such as a microorganism, preferably a prokaryote,
It can be integrated into a plasmid that can be replicated in eukaryotic or mammalian cells. The invention also relates to DNA molecules encoding at least one of the proteins of the invention. Furthermore, the present invention relates to transformed host organs, preferably prokaryotic, eukaryotic or mammalian cell lines, especially mucosal, which contain the genetic information encoding the viral proteins of the invention.

Preferably, this genetic information is contained in a carrier capable of replicating within the host organ in question.

The invention also relates to expression plasmids such as pRH969/
1, pRH969/20) and pKK89/2A, which contain by insert the DNA of a polypeptide, which polypeptide has at least one biological activity of a viral protein of rhinovirus strain HRV2 or HRV89, in particular VP1, VP2, VP3, VP
6.P2A.

A polypeptide having an amino acid sequence for P2B, P3C, P3A, P3B or P3C, preferably an amino acid sequence according to FIG. 4 or FIG. 14 or a part thereof, and also at least one of the viral proteins of rhinovirus strain HRV2 or HRV89. polypeptides containing at least two of said viral proteins in association in any predetermined combination and order, and the amino acid sequences shown in FIG. 4 or FIG. Partly derived from and/
or polypeptides that bind to and/or block cellular receptors for rhinovirus strains HRV2 or HRV89 and corresponding polypeptides.

The invention also provides a polypeptide having at least one biological activity of the viral proteins of rhinovirus strain I (RV89) and/or encoded by one of the above DNA molecules, in particular VPI, VF6, VF6, VF6. ,
P2A, P2B.

Amino acid sequence of P2C, P3A, P3B or P3C,
Preferably, it also relates to polypeptides having the amino acid sequence shown in FIG. 4 or a part thereof.

A polypeptide corresponding in whole or in part to at least one of the viral proteins of rhinovirus strain HRV89, a polypeptide comprising at least two of said viral proteins in combination in any predetermined combination and order and as shown in FIG. polypeptide derived from the amino acid sequence or a part thereof and/or rhinovirus strain F (R
Polypeptides that bind to and/or block cellular receptors for V89 are also objects of the invention.

The DNA molecule according to the invention is derived from rhinovirus strain HRV8.
9 viral RNA was isolated, and the complement DNA for it was isolated.
or by constructing a viral CDNA/RNA hybrid into a suitable replicable vector and transforming a suitable host organ with this vector.
It is prepared by enzymatically decomposing part or all of the DNA, and optionally by chemical DNA synthesis, binding reaction with a suitable linker, or by a combination of enzymatic and chemical reactions.

The polypeptide according to the invention can be used in a suitable host organ, preferably in prokaryotic, eukaryotic or mammalian cells, especially in E.
This is obtained by transforming the duplex with the genetic information of the invention encoding the viral polypeptide of the invention, then expressing said information and isolating the viral polypeptide of the invention.

The polypeptides according to the invention can be used therapeutically, for example to stimulate the immune system, to bind and/or block cellular receptors for rhinovirus strain HRV89, and to produce monoclonal antibodies or polyclonal antisera. .

The polypeptides of the invention can also be used as pharmaceutical compositions, which compositions contain an effective amount of at least one polypeptide of the invention together with a pharmaceutically inert excipient or carrier.

The invention further relates to a hybrid cell line obtained by immunization with HRV20)HRV89 or a part of its viral proteins, and also to the antibodies obtained from said hybrid and the uses of said antibodies, e.g. It is also relevant for use for diagnosis or for the purification of proteins that bind to these.

ABTS: 2,2'-azinodi(3-ethylbenzthiazoline sulfonate) AMP': Ampicillin resistance BCIP: 5-bromo-4-chloro-3-indolyl-
Phosphate BME: β-Mercaptoethanol BPB: Bromophenol Blue BSA: Bovine Serum Albumin ccc Plasmid: “Circular Covalently Closed” Plasmid DE-81-Paper: Diethylaminoethyl Cellulose Paper DMEM: Dulbecco Modified Eagle Medium dNTPs: Equimolar solutions of all four deoxytriphosphates DTT Nidithiothreitol EDTA: Ethylenediaminetetraacetic acid FC3: Calf serum g: Earth promoted HAT-medium: Hypoxanthine thymidine medium HIF
e2: heat-inactivated calf serum HRV2 or 14: human rhinovirus type 2 or 4 IAA: β-indole acrylic acid kb; kilobase kd: kilodalton Cournot (enzyme): DNA polymerase ■ glue Renault fragment LB medium: Luria Bertani medium containing 1% milk casein (batatotributone) pancreatin digest, 0.5% yeast extract and 1% NaCl in distilled water was heated in an autoclave at 120°C for 20 minutes. M: Aqueous solution containing 1 mole of the substance in question per 11 MEM: Minimum essential medium mM: 17! ml of an aqueous solution containing 1 mmol of the substance in question: ml NBT: nitroblue tetradolium n to nanometer OD h. . : Optical density at 600 rows PBS : 1
137 mmol NaCl per replicate.

2.7 mmol KCl, 8 mmol Nag HP Oa , 1.5 mmol K HtP
Oa, containing 0.5 mmol MgClI, .6HtO and 1 mmol CaC1z, p) 17.4 aqueous buffer PBS def: P, BS Same as S, but without Ca and Mg salts.

PEG: Polyethylene glycol PFU: “Plaque-forming unit” rATP: RiboATP RPMI medium: Roswell Park Memory Institute
al In5 titute) medium SDS sodium nidodecyl sulfate TE: lj! 10 mmol of Tris HCj
! (pH 7,4) and an aqueous buffer mixture containing 1 mmol EDTA T, -PNK:T, -Polynucleotide Kinase Tris: ) Lishydroxymethylaminomethane TWEEN20: Polyoxyethylene (20) Sorbitan Monolaurate U: Units μC1: Microcurie Mg: Microdulum μl: Microliter μM: Aqueous solution containing 1 micromole of the substance in question per Il VPI, 2.3.4: Virus coat protein VPI, 2
．． 3.4 VP○: Viral precursor protein XC of coat protein VP2 and VF6: Xylenesia Tool Example I Preparation of HRV89 HeLa cells [strain HeLa-Ohio
io), 03-147, Flow Laboratories (pl
ow Laboratories), England (E
ngland)) was cultured in suspension at 37°C. This suspension medium [Thomas et al., Proc.
c, Exp, Biol.

Med, ), 1970.133.62-65; Stott et al., Journal of Genetic Virology (J, Gen, Virol, )
, l 970, E, 15-241 is Shoklic (Jo
(Gibco 072-1300) and 7% horse serum (Seromed 0135). Inoculation density is 5-10XIO' cells/ml
The capacity was 500mf. Cell density 1 x 10
'/ml, the suspension was diluted under sterile conditions at 300g for 10
Centrifuged for minutes. The supernatant was discarded and the cells were incubated with infection medium (MEM modified for suspension culture, 2% horse serum and 2% horse serum).
resuspended in 100 mji (containing mM MgC122). Carefully pipetted several times with a 20 mf pipette to evenly distribute the cells in the infection medium. This was then made up to 500ml. Bring the cell suspension to 34°C
Cells were infected with HRV89 (twice plaque purified) at a rate of 0.1 virus per cell. HRV89 strain is from the American Type Culture Collection (ATCCV).
R-1199>. The strain used is HRV89
(ATCC Catalog NaATCCVR-1199A
s/CP). The control serum used was HRV 2 (catalog patient ATCC VR
-1112As/CP), which showed no neutralization. Virus was harvested after 60 hours at 34°C.

Virus was obtained from both cells and cell fragments, as well as from the culture medium. For this, the medium was separated from infected cells and cell fragments. Separation was performed by centrifugation at 1500 g for 10 minutes. Pellets were frozen at =70°C.

Combine cell pellets from suspension cultures 121 and 40 m
7M buffer of A - (20n+M Tris/HC
I (pH 7,5), ZmM MgCl (1 g), placed on ice for 15 min, then dounced.
ce) Disintegrate with a homogenizer and centrifuge the mixture at 6000g for 30 minutes. The pellet was washed once again with 10++1 portions of 7M buffer. Combine the two supernatants and make 110
The virus was pelleted by centrifugation at ,000g for 3 hours. This virus pellet is then transferred to Ion/! K of
TMP buffer (50111M KCj!, 50mM
Tris/HCj! -(pH7,5), 5+l1
MgCf, 2mM mercaptoethanol, 1
(mM puromycin, 0.5mM GTP) and 1
After adding 50 mcg of DNase 1 (Sigma, ribonuclease free),
Incubated for hours.

The virus was incubated with polyethylene glycol (Merck, PEG6000) at a concentration of 7% and 450+a at 4°C.
It was precipitated from the infection medium by stirring with NaCl of M (Korant B, D, et al., Virology, 19720) ± Engineering, 7l-86). After 4 hours at low temperature, the virus was centrifuged at 1500 g for 30 minutes and the pellet was collected at 75 mcg.
The mixture was incubated on ice for 1 hour and then frozen at -70°C.

Viruses obtained from the cells and culture medium were combined and incubated at 37°C.
Incubate for 5 minutes in cold TE buffer y-(10+
M Tris/H (1 (pH-7, 4), In+M
EDTAI was added and cooled, then sonicated in a water bath for 5 minutes.

Next, it was centrifuged at 6000g for 30 minutes. , 7% PEG
Add 920 ml of TE buffer containing 6000 and 450+ wM NaCl to the supernatant and carefully stir this
℃ for 4 hours, and the resulting precipitate was mixed with 6000 g of
It was pelleted for 0 minutes. This pellet is 100o+j!
virus in 7M buffer of PEG6000 and NaCj! was added and precipitated as above and then pelleted. The pellet was resuspended in 40 tal of 7M buffer, the suspension was centrifuged at 6000 g for 30 minutes, and the virus was pelleted at 110.000 g for 3 hours. The pellet was dissolved in 1 ml of 7M buffer, 50 μg of DNase T was added and incubated for 1 hour at 4° C., and then 1 μg of TE buffer was added. For further purification, the virus suspension was centrifuged under a sucrose gradient (10-30% W/W in TE buffer) at 110,000 g for 4 hours at 4°C. Virus-containing fractions were determined by absorption with 260rII11 and diluted with 7M buffer to a final concentration of 10% in sucrose. Next, it was centrifuged at 85,000g for 8 hours. The obtained virus pellet was taken up in 1 mA 7M buffer and stored at -70°C. To check the purity of the virus formulation, aerophoresis was performed on a 12.5% polyacrylamide gel in the presence of 0.1% SDS [Laemn+1i U, Nature (N
ature), London, 1970, 277.680
-685). The protein hand was then stained with Coomassie brilliant blue. ) [A typical image of the protein pattern of a formulation of RV89 is shown in FIG.

Example 2 Cloning of cDNA-RNA hybrids RNA extraction from oHRV8'9 formulation. NTES buffer y-C 100mM Naf
J, 10mM Tris/HCj2 (pH9)
, 1mM EDTA, 0.1% SDS:] and extracted with phenol. Chloroform was added to avoid phase separation. 20μβ of 5M NaC! and 20μ
β 3M sodium acetate (pH 5,6) was added to the aqueous phase and the viral RNA was precipitated twice with equal volumes of ethanol.

VPg covalently linked to the 5'-end of viral RNA
To remove the RNA, the RNA was then digested with 1 mg/ml Brotainase K (Merck) in NTBS buffer for 15 minutes at 37°C.

To avoid contamination with ribonucleases, the proteinase mother liquor (50 mg/mf) was pre-incubated at 37°C for 1 hour.
Incubated for 5 minutes. After digestion with Brotainase K, the solution was extracted with phenol/chloroform as above and ethanol was added to precipitate the RNA. A small amount of RNA was then transferred to TAB buffer (10 mM sodium acetate, 40 mM Tris/
2 in H1l (pH 8,2), 2mM EDTA)
% agarose gel. After staining with ethidium bromide, the complete HRV89-RNA band was visualized (Figure 2). A weak diffuse band below the band indicates slight degradation of the RNA.

HRV89- with oligo-dT as 0 primer
Reverse transcription of RNA 4ug(7)HRV 89-RNA at 10μ! H of
3C and 5 μE of 10X RT-Buffer [IX
RT buffer = 100mM KCj2,10+aM MgCj! z, 50 mM Tris/HC1 (pH 8
, 3>), 5 μg of oligo-aT(12-18) (Phanmacia P-L Biochemicals
P-LBiochea+1 cals) ), 10
#C3[α”P) -dCTP (3000Ci/ms
+oj! ;Am-Jam International (Am
ersham International, England) and 20nw+ol dATP.

dGTPSdTTPSdCTP was added and the whole was incubated for 2 hours at 42°C in a total volume of 50 μl. After adding 2μβ of 250mM EDTA (p118), extraction with phenol/chloroform, the aqueous layer was transferred to a column packed with Biogel P 3.0 (or Sephadex G-25) using Pasteur. ) pipetted through. TE buffer was used for elution. The resulting cDNA-RNA hybrid was thus separated from excess [α32p″l dcTP and precipitated after addition of 1/10 volume part of 3M sodium acetate (pH 5,6) and 2 volumes of ethanol.

oHRV89 RNA-cDNA hybrid homopolymer extension (tailing) HRV89 RNA-CDNA vibe 'J' (7
) extension, Roy cord free and Wu [:Roych
udhury and IAu (cited above, 19
80:1 according to the method. This HRV89
RNA-cDNA hybrids were prepared in 50μβ of TT buffer (200mM potassium cacodylate, 25mM
Tris/HCj' (pH 6, 9), 0.5
2% mof containing 25 U of terminal transferase (Fublumashi 1 PL Biochemicals) in mM Mn (Jg, 2 mM dithiothreitol).
(ct32P3 d CTP (5Ci/mmoJ)
[Deng G, R, & Wu Ro, Methods of Enzymology.

Enzy+*o1. ), 1983.100-B, 96
-116]. 2 μl 0.25 MEDTA (pH 8)
was extracted with phenol/chloroform and the reaction mixture was then chromatographed on Biogel P30 as above and the oligo-dC-carried RNA-cDNA hybrid was precipitated with ethanol.

Integration (“annealing”) of the 0 oligo-dC-carrying HRV89 RNA-CDNA hybrid into plasmid pBR322 and Escherichia ':1') (E
scherichia colt) HB 101 transformed oligo-dC-carrying RNA-CDNA hybrid
100IJIl of NTE buffer [100mM
NaCl, 10mM Tris/HCj! (p
H7,6), 1mM EDTA), and this was added to pBR322 plasmid [previously cut with Pst and polymerized with oligo-dG residues: Bethesda Research Laboratories (Bethesda Research L
aboratories)) and heated first at 65°C for 5 minutes and then at 42°C for 2 hours, gradually cooled to ambient temperature overnight and stored at 4°C.

For transformation of cells, strain HB 101 (03M1
607) was cultured in 50 Ill of LB medium (containing 10 g of tryptone, 5 g of yeast extract, and Log of NaCl in 1β) [Mandel Hl et al., Journal Op Molecular Biology (J9M)].
o1. Vlol, ), 1970.53.159-16
2) To obtain cells suitable for transformation (competent cells), pellet the bacteria and incubate with 25mj
TR buffer (KCj of 150+*M!, 50−M
of CaCff1l, 1w+M of Tris/HCI (
pH 7), 3n+M MgClt), kept on ice for 30 minutes, centrifuged again, and once again diluted with 2IIII of TR.
It was suspended in buffer and kept on ice for 1 hour. This mixture containing pBR322 containing the inserted HRV89 RNA-cDNA hybrid contains 100 μβ and 20
Add 0 μl of cell suspension and also 5 μl of IM Ca
Then, 2 ml of L
Medium B was added and the resulting mixture was incubated at 37°C for 15 minutes. This cell suspension was applied to LB-agar plates (1.5% agar in LB medium) containing 10 gg/ml tetracytalin (Sigma) and then incubated overnight. Tetracytalline-resistant clones were tested for ampicillin sensitivity on ampicillin agar plates (100 μg ampicillin/llll1; Sigma).

Characterization and Isolation of Replicating DNA Molecules Tetracycline-resistant and ampicillin-sensitive bacterial clones were cultured overnight in 5 tall of LB medium (10 gg tetracytalline/++1) and plasmid DNA was extracted using the “plasmid minimal preparation method (mint- preparation
technique) ” [Birnboim (Bir)
nboim) H, C, etc., nuclear aids
Research (Nucleic Ac1ds Res,)
% 1979 S7.1195-1204) and the size of the replicated DNA was determined by digestion with the restriction enzyme Pstl. This plasmid DNA is 50+
25all RE buffer containing M NaCl [6
μlM MgC1z, 1 (1++M Tris/
2 U of the restriction enzyme Pstl (Bethesda Research Laboratories) in HC replication (pH 7,5), 6 mM mercaptoethanol].
Research Laboratories)) and 5 μg Noribonuclease A for 2 hours at 37°C. The probes were then separated electrophoretically on a 1.4% agarose gel.

The size of the insert could be determined by staining with ethidium bromide and comparing it with the λ-old ndlll marker DNA. This size was 300-3.300 bp.

To isolate large amounts of DNA inserts, plasmids were obtained from 200 rsl cultures of tetracycline-resistant and ampicillin-sensitive bacterial clones and digested with Pstl, as described above. ? j! ! ! ! DNA fragments were separated on a preparative agarose gel as described above. Cut the band and transfer the DNA to 0.05XTBE buffer (IXTBE buffer = 100mM Tr
is/borate (pl (=8.3), 2mM BDTA
) and precipitated with ethanol.

o L uni strain JMIOI by pU09 vector
Subcloning plasmid pUC9 (Vieisa J,
et al., Gene, 1982.1, 259-
268) is ampicillin resistance gene, plasmid pB
Replication origin region derived from R322 and fac of E, uni
Contains part of the Z gene. There is a small NA fragment in the 1acZe5 region that contains several restriction sites, so that cloning of DNA at one of these cleavage sites blocks the facZ gene region.

Colonies containing the DNA insert are therefore
-Ga1 = 5-bromo-4-chloro-3-indolyl-β-D-galactoside; Bethesda Research Laboratories) Appears as white colonies on indicator plates, while those without inserts are very blue. [Rffther U, Molecular Gene & Genetic (Mo1. Gen,
Genet, ), 1980.178.475-478
). To subclon the DNA insert in pUCQ, the replicated pBR322 clone (approximately 7 μg) was transformed into Pst
The DNA insert was separated from the vector DNA after digestion with l and separation on an agarose gel (1.2% to 1.4%).

The DNA was recovered from the gel by electroelution and ethanol precipitation as described above. The isolated DNA insert was added to 0.4 μg of pUC9 vector (cut with Pstl and pretreated with bacterial alkaline phosphatase).
1a+M A in 20 μl RE buffer containing
Incubate for 1 hour at 15°C in the presence of TP and 3 U of Ta-ligase (Bethesda Research Laboratories) and store at 4°C [Vieiza (-Viei
sa) J8 et al., supra, 1982), and at the same time, a 2°C strain JMI O that is competent for transformation.
1 cell [New England Biolabs (New
f! nglandBiolabs)) was prepared as described above. 200. The competent cell suspension of crJ was mixed with 20 μE of pUC9 ligase reaction-mixture and the resulting mixture was incubated at 0° C. for 1 hour.

After incubation with heat (90 seconds at 42°C), the cells were incubated with 10 μl of 200 mM isopropylthiogalactoside (Sigma), 50 μl of a solution of 20 mg of X-Ga1 in 1 μl of dimethylformamide, and 1 vg.
of LB medium and the resulting mixture was incubated at 37° C. for 1 hour. 200μ of this cell suspension
1 batch of ampicillin LB-agar plate (100
μg/+wz) and incubated overnight at 37° C. in an incubator. Positive transformants were identified as white colonies and examined for DNA inserts using the "plasmid minimal preparation method".

Preparation of pUC9 plasmid with insert of 0 replication DNA The generated subclone of E, :21JJM101 transformed with pUC9 and containing the DNA insert was cultured in 200 ml of LB medium (containing 100 μg of ampicillin/lag). and isolated this plasmid DNA [Btrnboin+ H,
C. et al., supra, 1979), the plasmid DNA was then dissolved in 100 μl of TE buffer, and the solution was diluted with Sephacryl-1000.
Chromatography was performed on a column (IX20 cm) with TE buffer.

Both fractions containing the purified plasmid were identified by absorption, then combined and lyophilized. This plasmid was taken up in 500 μl of TE buffer, incubated at 65° C. for 5 minutes, extracted again with phenol/chloroform, and precipitated with ethanol.

0 plasmid pHRV89-100. pHRV89-1
36, Preparation of Primer Fragments from pHRV89-193 and by Synthetic Methods Clones 100.136 and 193 containing the corresponding DNA inserts were pUc
9 and 200 ml of I, B medium (100 ml).
ampicillin/IIIt) and the plasmid was isolated as described above. clone 100
(54 nucleotides) obtained by digesting the primer fragment (54 nucleotides) with the restriction enzymes Ncol and Pvu (designated as fragment A in FIG. 3).

For this, purified plasmid 200 from clone 100
20 μg in the presence of 50 n+M NaCl containing 30 U of Ncol (New England Biolabs).
Incubate in 0 μl RE medium at 37° C. for 15 hours. After completion of the reaction, ethanol precipitation was performed, and the cut plasmid was diluted with 20 U of PvuI (New England Biolabs) in the presence of 50 mM NaCl.
00μm! of RE buffer at 37°C for 15 hours. Digestion pattern with restriction enzymes is 1.
Examination was performed by electrophoresis on a 4% agarose gel. Nc
ol/PvuI [fragment 100 μβ OG? solution (1% Ficoll, 1mM E
D.T.A.

Add this DNA to 0.01% Orange G) and prepare 15
% polyacrylamide gel (acrylamide/Bisac 1-noramide = 111, gel thickness 1.2 mm), l
Separated in xTBE buffer. 54bpNcoI
/PvuII fragments were cut after staining with ethidium bromide, the fragments were electroeluted in 0.5x TBE buffer, and the DNA was precipitated with ethanol. This DNA fragment was then added to 100 mM T, pH 8, containing 200 U of bacterial alkaline phosphatase (Bethesda Research Laboratories).
ris/HCI 50 u 1 at 65°C.
Incubated for hours. After reaction, 0.5 M at pH 8
2.5 μl of EDT A was added, the aqueous phase was extracted twice with phenol/chloroform, and the DNA was precipitated with ethanol. Dissolve this DNA in water and add 20 μCi
γ-(”P)-ATP [specific activity 5000Ci/mm
o l H Amerjam International (Ame
rsham International)) and 5U of polynucleotide kinase (Pharmacia P-
Buffer (10mM MgCffz, 50mM Tris/HC) containing 50μβ buffer (L Laboratories)
1 (pH 8), 5n+M dithioerythritol] for 30 minutes at 37°C. This 3tP-labeled fragment was then precipitated and 1mM
Taken up in 30 μβ of 30% dimethyl sulfoxide containing EDTA and 0.01% xylene cyanol-bromophenol blue.

This solution was incubated at 90°C for 2 min, cooled in water and applied to a 15% polyacrylamide gel (acrylamide/bisacrylamide - 59:1 gel thickness 1.2 mm) in TBE buffer to separate the two DNA strands. were separated by electrophoresis (200V for 15 hours). Using autoradiography, we confirmed these two strands,
Cut and electroelute. One dot-blot shows the strand hybridized with HRV89-RNA.
)” Determined by testing, where dimensions 2 x 2 cf.
Two types of nitrocellulose strips [Schleicher & 5chi
ill), B A 85.0.45 μm) with H,O
20×SSC (I×5SC=15
0111M NaCl, 15mM sodium citrate, pH 7.4) and air-dried.

Approximately 1 μg of HRV89-RNA was applied as a dot to each strip, then dried and incubated at 80° C. for 2 hours. The strip was then wetted with 2X SSC and 111Il of H buffer (400mM Na(
1,49mM P I FES (pH 6,4), 1m
EDTA, 80% formamide) with 4 μg of denatured salmon sperm DNA (Sigma, incubated for 2 min at 100°C and cooled to 0°C) in plastic film for 1 h at 42°C. . The two nitrocellulose strips were then separately treated with 0,5n1(7)H buffer, 4 μg of denatured salmon sperm DNA and 1 aliquot of isolated strands (20000 cp
It was sealed with a plastic film together with m) and hybridized overnight at 42°C. After this incubation, filters were washed twice in 2X SSC for 10 min at 50°C,
IXSSC 2 degrees, 0.1% SO5 at 50℃
After washing for 30 minutes, radioactivity was measured.

Primer fragments from HRV89-136 and HRV89-193 were thus isolated. pH
The fragment from RV89-136 (shown as fragment B in Figure 3) contains DraI and lindlI [
It lies between the restriction sites (122 nucleotides) and can be obtained by digestion with these restriction enzymes. Fragment C is p
It was isolated from HRV89-193 and has a length corresponding to 139 nucleotides, lying between the two RsaI restriction enzymes (Figure 3).

Strand separation and hybridization were performed as described above. Primer D was chemically prepared using Applied Biosystems App.
aratus) using established methods.

HRV89-RNA using 0 primer fragment
Reverse transcription of clone 100 (fragment A), as above
136 (Fragment B) and 193 (Fragmen)
HRV89-R isolated or chemically synthesized from C)
5paI01 of single-stranded DNA complementary to NA was extracted from an aqueous solution with ethanol at 0.25pmol! t's HRV8
9-RNA was co-precipitated.

This precipitate was taken up in 20 μl of I] buffer, coupled to a capillary, incubated at 72°C for 10 minutes, and then 50°C.
The mixture was cooled gradually to 35°C and then placed on ice. This solution was then diluted in 100 μl of RT buffer for 14
0U of reverse transcriptase [Anglian Biotechnology Corporation Cambridge (Anglian B
iotechnology Co, +Cambridge
e)), 8 U of ribonuclease inhibitor (RN
asin, Bethesda Research Laboratories), 0
．． 2mM dATP, dCTP, dGTP and dT
T P, [α”P)dCTP of 30 pCt and 5
2 at 42°C in the presence of mM dithioerythritol.
Incubated for hours. The obtained reverse transcript was treated as described above.

Detection and Restriction Mapping of HRV89 Sequences in 0 Replicated DNA Plasmid DNA from replicates was isolated in 3 ml cultures [Birnboim fl, C
.

et al., supra, 1979]. This DNA is digested with restriction enzyme Ps
This probe was incubated with tI at 1.4%
Analyzed by electrophoresis on agarose gel. This gel was stained with ethidium bromide. Next, this DNA
was transferred from the gel to a nitrocellulose filter [Southern ε0M.

Journal of Molecular Biology (J.
Mol, Biol, ), 1975.98.503-
517) and immobilized on nitrocellulose by incubation at 80°C for 2 hours. This filter
50% formamide, 1x Denhardt's solution
7 ([)enhardt) D, T9, Biochem, Biophys, Res, Co
mm, ), 1966.23.641-646), 900
mM NaCj! , 50mM sodium phosphate (pH
7,4) 7 ml of ε0Mg in 511M EDTA
of denatured salmon sperm DNA for 2 hours at 42°C in a plastic bag. Radioactive HRV89-cDNA was prepared as described above. However, this reaction mixture contained 50 μCi of [α”P)dCTP. This (7) cDNA-HRV89-RNA hybrid was denatured by heating at 100°C for 90 seconds. For hybridization, this filter was Incubated with radioactive HRV89-cDNA for 18 h at 42 °C as in 2×SSC and 0.1
Washed twice in %SDS for 30 minutes at 50°C, air dried,
-70℃ [Kodak (Kodak)
R-5, 8-40 hours on intensified film] The presence of a time-paired band indicated the presence of replicated DNA complementary to HRV89-RNA.

For mapping, DNA inserts were digested with restriction enzymes (New England Biolabs and Bethesda Research Laboratories) under the manufacturer's specified incubation conditions. The results of limit mapping are shown in Figure 3.

Plasmids pHRV89-80 and pHRV89-8
2 contains a long sequence of A residues in close proximity to oligo-C derived from chain extension resulting from the terminal transcriptase reaction, which forms part of the 3'-terminal po'J-A of HRV89-RNA. do. The other plasmids are pHRV89-80 and pHRV8 due to the specific cleavage sites of the individual restriction enzymes.
9-82. DN less than 500bp
The A insert was not analyzed by restriction enzyme mapping, but was immediately subcloned and sequenced with pUS9 (see Example 3). Identification of the remaining clones was performed using the method of Grunstein and Hogness.
M., & Hogness, D.S., Proc. Natl., Acad.

Sci, USA), 1975, ■, 3961-396
5) using 'nick transl
This was achieved by colony hybridization using DNA inserts that had been previously mapped with the ``Ation)'' probe.

The szp-labeled DNA probe was obtained with a 6-two translation kit (manufactured by Amersham International, England; Mersham kit lk5000).

However, [α-”P) d CTP (3000Ci/
ml1o1) according to the manufacturer's instructions. Labeled DNA was separated with TE buffer using Biogel P30 in a Pasteur pipette. Fractions corresponding to the excluded volume were combined, heated at 100° C. for 2 minutes, and immediately placed in ice water. Hybridization was performed as described above. 50
mj! Cultures were prepared (overnight in LB medium with 10 μg 7 ml tetracytalline) from colonies showing positive hybridization signals. The DNA inserts were separated from the plasmid DNA with PstI. These inserts were then digested with various restriction enzymes and characterized by sequencing. A clone was thus obtained, which represents the genome of HRV89.

Example 3 A cDNA clone of HRV89 was prepared using the method of Maxam and Gilbert (Maxa-8, & G11bert W.
,,Methods in Enzymology
s.

Replication nzymo1. ), 1980.65, 499-56
The improved method of 03 or the M13-chain termination method of Sanger et al.
ethod) (Sanger F, et al., Proceedings of the National Academy of Sciences) (Proc, Natl, Acad,
Sci, USA), 1977.1 Sat, 5463-
5467) was used for sequencing.

To sequence according to Maxam and Gilbert,
The DNA insert was subcloned in puc 9 as described above and competent E, 11JJMIOI cells were thereby transformed. Positive transformants were isolated as white colonies and added to LB medium (100 μg/rsll).
ampicillin). 10-20/J
gc) DNA was digested in 100 μl overnight with restriction enzymes under standard reaction conditions. This enzyme has a cleavage site in the polylinker region of pUC9 in the plasmid, such as BamHI, EcoRI, Acc I,
1lindnr, etc. The restriction fragments were then dephosphorylated. For digestion with restriction enzymes, 5μ
l of 2M Tris/HC1(p)18) and 10
0 U of bacterial alkaline phosphatase (Bethesda Research Laboratories) was added and incubated at 65°C for 3 hours. After adding EDTA to 20mM, the mixture was extracted twice with phenol/chloroform and D
NA was precipitated with ethanol. Next, add this DNA to 5
0ttl of 50mM Tris/HC/! (pH
8), 1 (leM MgCj!x, 25 μCi of [T-
”P) ATP (500 (7Ci/vamoIH Amershaa+I
international)) and 4U of T4-polynucleotide kinase (Pharmacia P-L
Biochemicals) for 30 minutes at 37°C, and the labeled DNA was precipitated with ethanol.

3 in one chain! The P-labeled replicated DNA was run on an agarose gel (1,2) as described above using other restriction enzymes.
~1.4%) followed by electroelution and ethanol precipitation. The enzyme performs the cleavage in the polylinker region of pUC9.

DN by a modification of O. Maxam and Gilbert's method
A Sequencing This sequencing reaction was carried out with the following modifications by Maxam and Gilbert (cited above, 1980).
.

- No carrier DNA was added.

- The DNA solution was divided into the following aliquots.

Guanine (G) specific reaction 7.5μl; Guanine and adenine (G/A) specific reaction 10μl; Cytosine and thymine (C/T) specific reaction 10μl; Cytosine (
C) 6 μl of specific reaction.

-25 μl of 96% formic acid was added to the (G/A)-reaction mixture. This was then incubated for 4.25 minutes at 19°C. 200 μl of hydrazine stop solution and 750 μl of 96% ethanol were added to stop the reaction.

This (G/A)-reaction was then processed in the same way as the other three reactions.

- instead of hydrazine, hydrazinium hydroxide (
Merck & Co.) was used in the (C/T) and (C)-reactions and the zero reaction time was 7.5 minutes.

-Piperidine reaction was incubated at 95°C for 30 minutes. After lyophilization, the fragments were buffered in 3-20 μl of buffer (80% deionized formamide, I XTBE, 0.5 μl).
0.05% bromophenol blue and 0.05% xylene cyatool) at 95°C for 90 seconds and rapidly cooled to 0°C.

For sequencing, 6% polyacrylamide gel (40c
mX 20cmX O, 4mm) with 8M urea and I
Used with XTBE. This is done before applying the probe.
Electrophoresis was performed at 0 W for 1 hour. Between 1 μβ and 3 μl of each reaction mixture was applied to the gel.

Typically, two gel loadings were performed at different times.

The first pass of the reaction mixture was carried out until the bromophenol blue marker reached the end of the gel, and the second pass was continued until the xylene cyan tool marker reached the end of the gel.

This gel was then washed with 10% acetic acid and 10% methanol (
Fix for 20 minutes in about 21℃, transfer to 3MM-filter paper,
Dry on a gel dryer at °C.

This gel was then screened for 170 minutes using an XAR-5 screen (Kodak) without any thickening film.
(approximately 18-36 hours).

OM13 sequencing DNA inserts with a length equivalent to 1000 bp or more were prepared using the shotgun method [Deininger P, L, Analytical Biochemistry (Anal, Bioches)]
+, ), 1983.129.216-223).

Replicating clones were digested with appropriate restriction enzymes and DNA inserts were run on a preparative agarose gel (1,2-1
．． 4%), followed by electroelution and ethanol precipitation.

The DNA insert was treated with 1 mM ATP and 3 U of T4 DNA ligase (New England Biolabs).
The cells were incubated for 155 hours at 14° C. in 20 μl of RE buffer. The circularized DNA was buffered in TE buffer (IOIIM Tris/H (J! (pH 7,5).
), 1 mM EDTA) to a final volume of 500 μl, sonicated on ice for 4 x 5 seconds (maximum energy, MSE ultrasound output device), and precipitated with ethanol.

The ends of the resulting DNA fragments were incubated in 20 μl of RE buffer at 14° C. for 2 hours. The buffer contains 50 gM of each dATP.

dCTP, dGTP, and dTTP and 2U of Klenow fragment (Boehringer Mannheim)
) included. After separation on an agarose gel (1.4%), DNA fragments with lengths corresponding to 400-900 bp were recovered by electroelution and ethanol precipitation. These DNA fragments were inserted into the M13mp9 vector (which was linearized with the restriction enzyme Sma'I).
and treated with bacterial alkaline phosphatase in the presence of 1 mM ATP and IU of T4-DNA ligase (insert to vector = 5:1), the treatment was carried out for 15 hours at 15°C.

E, competent for transformation! Strain JM
I01 cells were incubated with the mixture from the ligase reaction at 0°C for 2 hours.
After a second heat shock, the cells were transferred to 40 μl of 100 m
MIPTO, 40 μl of a DMF solution containing 2%
, 8g agar) and then slightly preheated H
plate (10 g tryptone, 8 g NaC in IIt)
1.12 g of agar). After the agar had hardened, the plates were incubated at 37° C. overnight.

To isolate single-stranded phage DNA, colorless plaque 1
．． 5III ME, JJ M 101 culture (
A static culture of 1 mβ diluted in 100 ml of 2XTY medium (16 g of tryptone, 10 g of yeast extract, 5 g of NaCβ in 11) was transferred to a test tube. After 6 hours of incubation at 37°C, the bacteria were removed by centrifugation and the phages were precipitated from the supernatant by adding 200 uJ of a solution containing 20% PEG3000 and 2.5M NaCl over 15 minutes at ambient temperature. The precipitate was pelleted and the entire supernatant was removed. This pellet was dissolved in 100 μl of TE buffer and mixed as above.

The dimensions of each -zero14 DNA were compared to those of the wild-type phage D.
Determined by electrophoresis on a 0.8% agarose gel for NA. This sequencing was performed using the chain termination method [Sanger F. et al., supra, 1977].
This was done without any changes.

Analysis of Sequencing Data Sequencing data was analyzed on a Cyber 17 Q computer. The programs used were the Staden program (Staden R) and Nucleic Assos Research (Nucleic A).
c1dsRes, ), 1980S8.3673-36
94) and the Isono Improvement Program (Isono K
,, Ibid., 19820) Retired Emperor, 85-89).

This viral protein is obtained by proteolytic cleavage of the polyprotein. To identify these cleavage sites,
The viral coat protein was isolated and the N-terminal amino acid sequence determined. For this, protein from 2 mg of HRV2 was transferred to a 12.5% polyacrylamide gel [Laemli
(Laemmlf) U., supra, 1970)
Separated by electrophoresis above. The gel was stained with saturated Coomassie brilliant blue solution of 50 mM Tris/H (pH 7.4) and the protein bands were excised.
Time electroeluted. AB-470A
Protein sequencer [Applied Biosystems Inc. Foster City, California, Nynisny]
e+ss, Inc.

Foster C1ty, CA, USA). For sequencing, 2 μmol of VPI and VF6 and lnmoff of VF6 were used. The amino acid thus obtained was analyzed by HPLC (high performance liquid chromatography). The N-terminal sequences of individual proteins are shown in FIG.

Example 5 All enzymatic reactions of Boosmid RHloo were performed under the conditions specified by the manufacturer.

7 μg of plasmid pER103 (Ova Dworkin-Rastl, etc.)
Gita (Gene), 1983.21.237-24
8; European Patent Application A-0155613] was linearized with the restriction endonuclease 1lindnl in 50 μl of reaction medium. After incubating for 1 hour at 37°C, 50 μl of 2X CIP buffer was added (2XC
IP buffer y −= 20mM Tris (p)1
9.2), 0.2mM EDTA), 2U of calf intestinal alkaline phosphatase (CIP) was added to remove the 5'-terminal phosphate residue, and the incubation was continued for 4
It was carried out for 30 minutes at 5°C. Add this reaction to 4 μl of 0.5 M
EDTA solution and 10 μ2 IMTris (pH 8
,0) solution was added to stop. Protein was removed by extraction twice with phenol and once with phenol/chloroform. This DNA is pos, s 0
．． The aqueous phase was precipitated after addition of 1 volume of 3M sodium acetate solution and 250 μl of ethanol. this DNA
Centrifuge the precipitate and wash once with 70% ethanol solution.

This DNA was dried and the pellet was washed with TE buffer [
10mM Tris (pH 8,0), 1
20 pl (mM EDTA).

Synthetically prepared oligonucleotide d (AGCT
TAAAGATGAC, CT) and d (CATCT
A 1 Mg batch of TTA) was added at 1 Mg in a 10 μl reaction mixture.
Phosphorylation was performed by adding 0 U of T4-PNK and 1+M rATP. The reaction was carried out at 37°C and lasted 45 minutes. This was stopped by heating at 70°C for 10 minutes.

A 5 μl batch of plasmid solution and phosphorylated oligopeptide were mixed together and heated at 70° C. for 5 minutes. The solution was then cooled to 0°C and added with 2 μl of 10x ligase buffer (500 mM Tris (pH 7,5),
100mM MgC1z, 200mM DTT, 1m
M rATP, 500μg/ml BSA), 2μ
1 of water and IOU of T4-DNA ligase were added.

The reaction lasted for 40 hours and was carried out at 4°C. This is 70
Heating was stopped at ℃ for 10 minutes.

2 μl of this ligase reaction was digested with IOU of restriction endonuclease 5acI (New England Biolabs) for 3 hours at 37° C. for a total solution of 30 μβ. The reaction was stopped by heating at 70° C. for 10 minutes.

5μβ of this reaction mixture was combined with IO
U of T4-PNK was added and binding was performed at 14°C for 16 hours.

200 μg of competent E, : ]l, I) (B
The IOI was combined with 10 μl of this ligase reaction. The bacteria were kept on ice for 45 minutes and then heated to 42° C. for 2 minutes to allow DNA uptake. This bacterial suspension was then incubated again at 0°C for 10 minutes. Finally, transform the transformed bacteria at 50 μg/m
1 on LB agar containing ampicillin.

Randomly select 12 of the formed bacterial colonies,
Plasmids were isolated from these on a microscale [Birnboim H, G, et al., Nucl.
es, ), 1979, ? , 1513-1523),
The resulting DNA was cut with restriction endonuclease 5acl, and the DNA was resolved on an agarose gel (1%; IXTBE buffer). Transfer of the DNA as a linear molecule with a length corresponding to approximately 4400 bp confirmed that the 5acl recognition site had been inserted into the plasmid.

One of these plasmids was randomly selected. L again
JHBIOI was transformed with this DNA using the corresponding minimal preparation method. One colony of the obtained transformed bacteria was selected and cultured on a large scale. These isolated plasmids were cut with restriction endonucleases EcoRI and BamHI, the DNA was analyzed on a 1% agarose gel, and small fragments were isolated from the gel by electroelution. Eco with a length equivalent to about 46 obp
The RI-Baa+HI DNA fragment was purified by Sanger (Sanger F, et al., Proc, Natl, Ac
ad.

Sci., 1977.74.5463-5467). The plasmid thus analyzed was pR
It was named Hloo (Figure 7).

For example, the DNA shown in FIG.
HR obtainable according to European Patent Application No. A-192175, present as an insert in UC9
Starting from a fragment of the V2 genome, this DNA insert was digested with the restriction enzyme 11indlI [(Biolabs)
and EcoRI (Boehringer Mannheim) in 100 μl Eco-old buffer y (7,5
mM Mg(J!, 75mM Tris/HC1(p
H7,5) was cleaved by 15 h time-type digestion with 15 U of EcoRI and old ndI[r in 50 mM Na(1) at 37°C. This insert D
NA was separated from the linearized vector on a 1% agarose gel. For this, 100 μl was combined with 11 μl of 10× agarose gel “loading buffer” (1,1% bromophenol blue, 0.1% xylene cyanate, 35% Ficoll, 0.5% 5DS). pHRV
The EcoRI/Hindll [fragment (approximately 1.1 kb) of 2-969 was isolated from an agarose gel using DE81 filter paper [)7-/Whatman] [Dretzen G,
M, etc., Analytical Biochemistry (An
al, Biochem, ), 1981.112.2
95-298). After separating the DNA fragments on the agarose gel, slots were cut before and after the DNA to be isolated and strips of DE81 paper were inserted into these slots. Electrophoresis was continued (do not cover the gel with buffer) to completely bind the desired DNA fragment to the front DE81 strip. rear DE8
One strip prevented contamination by large NA fragments. DE8 with bound DNA fragment
1 filter paper in a 1.5mj2 Eppendorf
orf) in a test tube (in which the tube has an outlet at the bottom and polyaromatic wool is placed on top)
, 500 μl wash buffer y (0,2mM
NaCl, 25mM Tris/H(J (pH 8
, 0), IM EDTA), and the washings were transferred by brief centrifugation (approximately 1 second) into a second Ethopendorf container placed below. The bound DNA was then washed twice as above, then in 200111 elution buffer y.
-(1mM NaC4!.

25mM Tris/HC1 (pH 17,5), 1
The DNA was eluted from the ED81 filter (ethidium bromide remained adsorbed) by incubation for 15 minutes in n+M EDTA). 400 μl of the eluate was centrifuged in an Esopendorf centrifuge (15000 g) for 10 minutes to remove all filter paper debris. Carefully transfer the supernatant to a new Eppendorf vessel, add 800 μl of 96% ethanol, and let the mixture settle at -20°C (approximately 2 hours), wash twice with 70% ethanol, dry, and add 800 μl of 96% ethanol.
l (10mM Tris/HC1 (pH 8), 1
2 at 37°C in a buffer of 0mM MgC1z).
IOU was cleaved with XhoII (Boehringer) over time.

The mixture was incubated at 70° C. for 2 minutes to inactivate the restriction enzymes and then slowly cooled to ambient temperature. To these 50 μl, add 6 μl of 10× Tarnow buffer (660111MTris/HCj2 (pH
7,5), 66mM MgC replication z, 3a+Md
NTPs, 1mM DTT, 0.1mg/ml BS
A), 2 μl of Cournot enzyme (4 U/ml; Amar-Jam) and 2 μl of H,O were added, and the mixture was heated to 22
Incubated for 30 minutes at °C.

The reaction was stopped by the addition of 2 μE of 0.5 M EDTA and the aqueous phase was mixed with the same volume of phenol/CHCj!
once with s/isoamyl alcohol (25:24:1);
Extract once with the same volume of CHCJ3, then 7μ! 1
Add 0x agarose gel buffer. 1092
In order to separate the XholI fragment with a length corresponding to bp from the resulting oligonucleotide, a fragment with a “plant-end” structure (969/1) was isolated from DE81.
It was isolated from a 1% agarose gel using filtering. The expression vector used, pRHloo, was essentially pBR32
2 derivative, and its denaturation results in a fragment with a length equivalent to 123 bp at base 436210 (Bco of pBR322).
RI site) and 29 (old ndl of pBR322 [
r site), and this fragment is inserted between the Serratia marcescens (Serrat.
iamarcescens) tryptophan promoter region, synthetic ribosome binding site and start codon^
It has a TG (see Figure 8).

The formation of the expression plasmid pRH969/1 is shown schematically in FIG. Approximately 10 μg of expression vector pRH1
00 to 2 with 100 U of 5acI (Bio Lab).
00μm (6mM Tris/HC1 (pH 7,5
), 6mM BMB, 6mM MgC (replicated) overnight at 37°C. The plasmid DNA was precipitated in the usual manner with ethanol, washed, dried, and precipitated with 200#Il (200mM NaCl, 1mM
ZnClt, 30mM pH 4.75 sodium acetate, 5% glycerol) and treated with 150U of Jaena nuclease. First mix this mixture with 2
One batch was incubated at 14°C for 2 hours while the other batch was incubated at 37°C.
Incubated for 30 minutes at °C. Add this reaction to 5 μl of 0
．． Stop by adding 5M EDTA and phenol/C
Extracted with HCl,/isoamyl alcohol (25:24:1) or CH (J3), and the two mixtures were combined. The linearized plasmid was precipitated with sodium acetate and ethanol in a conventional manner, then washed and dried. This DNA was then diluted with 100 μl of a solution (100 mM Tri
s/HC1 (pH 8)) at 37°C with 40 U of alkaline phosphatase (Boehringer Mannheim).
and incubated for 1 hour. After adding EDTA to 20mM, it was diluted twice with phenol/CHC1x/isoamyl alcohol (25:24:1) and CHCl in the usual manner.
2 and the DNA was precipitated with ethanol.

Approximately 50-175 ng pRH of each mixture for ligation
100 (linearized with SacI and treated with maven nuclease and alkaline phosphatase) and approximately 1-2μ
The Xho■ fragment of subclone pHRV2-969 (treated with Klenow) was precipitated with ethanol, dried and added to 10 μl of freshly prepared 1× ligase buffer [50 mM Tris/HC1 (pH 7,5
), 10mM MgC1'z, 20mM DTT
, 0.1mM rATP and 50μg/ml BS
I took A). Ligations were performed with IU of 74-ligase (Boehringer Mannheim) for 70 hours at 14°C. To obtain competent cells for transformation, a modification of the method of Mandel and Higa was used (Mandel, M. &
Higa A, Journal Op Molecular
Biology (J, Mol.

Biol, ), 1970.53.159-162), E
.

0.5 ml of the “-night culture” of the two bacterial strains HBIOI was added to 50
mj! of LE medium (Log/J tryptone, 5 g/l
yeast extract, *Og/l NaCl),
OD of about 0.4. . and then pelleted for 5 minutes at 5°C and 4°C.

Next, this bacteria (ice-cold) was added to 0. IMMgCj
! z 25 mj! The cells were carefully resuspended in the medium, placed on ice for 5 minutes, and re-centrifuged for 5 minutes at 5°C and 4°C. The pellet was water-cooled with 0.1 M CaCl.

Resuspended in 20 ml, placed on ice for 4 hours, and centrifuged for 5 minutes at 5°C and 4°C. Transfer the cells to 1x storage buffer
(0, IM CaCl2/glycerol = 4/1%
v/v) 2.5 tal, placed on ice for 20 minutes, divided into 100 μβ aliquots, and shock-frozen in liquid nitrogen L/·-80
Stored at °C. 5 μl of the above ligation mixture was added to 100 μl of competent cell suspension, thawed on ice, the cells were incubated on ice for 1 hour and 2 minutes at 42° C., and finally on ice for 5 minutes. Before applying the cells thinly, add 900 μl of LB medium, incubate for 10 minutes at 37°C, and transfer 200 μl of the cell suspension to LB medium.
Agar plates (1.5% agar in LB medium containing 100 mg/j! of ampicillin) were applied and incubated overnight. A total of 35 Ampr clones were isolated from which plasmid DNA was extracted from the “plasmid minimal Preparation method (Plasmid m1ni-preparat
iontechnique)'' [Birnboim (Bir)
nboim et al., cited above]. By old ndnI digestion of this plasmid DNA, eight clones with inserts corresponding to the size of the Xhon fragment could be detected. In this formation, plant-end connections are required at both ends, so theoretically there are two
There are two possible orientations and a restriction analysis was performed. 3.5U
Bal E/10mM Tris/HCj with 100ng of plasmid DNA! (pH 7,5)
, 1011MMgC1z and 10n+M BME and 25mM NaCj2.6mM T
ris/HCj! (pH 7,5), 6mM MgC
8UBssHII in 1z, 2 OmM BME
/100ng plasmid DNA (2 at 50°C)
time) and 150mM NaCj! , 6MM T
ris/HC1 (pH 7,5), 6+M MgC
1'z, 20 UBamHI in 2hM BME
/ 6 clones (Nos,
156.165.289.301.307 and 319
) is identified as having the correct insert orientation,
Two clones (Nos, 1 and 224) were identified with opposite insert orientations. 969 XhoI
To check whether the I fragment coincides with the starting codon ATG, the base sequence of the region between the non-coding and coding parts was determined directly on the CCC-plasmid using the 1-bear 1-sequence primer. This sequence primer is complementary to the first 17 bases of the noncoding portion of the Trp promoter region (see Figure 8).

To do this, plasmid DNA was obtained from the above clones using the "minimal preparation method" and 100 μl of T
In addition to E buffer, 100 μl of 5M LiCl was mixed and incubated at 0° C. for 5 minutes. After centrifuging Eppendorf centrifuge 1 (15,000 g, 5 minutes at 4°C),
400 μ of supernatant! of 96% ethanol, centrifuged again at ambient temperature for 2 minutes, and the pellet was washed once with 70% ethanol and dried. Next, add this plasmid DNA to 11
A batch of 5 μl aqueous plasmid solution was dissolved in 5 μl of 1× denaturing solution [2671 NaOH
, 0,267mM EDTA).

After incubation for 15 minutes at ambient temperature, 1 μl of 1 bear 1 sequence primer and 2 μl of 2MCH,C00NH
, (p) 14.5) was added (50 ng/μi) and the mixture was incubated for 5 minutes at ambient temperature. 75 μl
After adding 96% ethanol and precipitating at -70°C (15 minutes), the precipitate was pelleted at 15,000g for 5 minutes, washed with 70% ethanol, dried, and the plasmid DNA hybridized with this primer was prepared in 8. The temperature was set at 5 μβ Hz O. Of these, 8.5 μβ was treated with 1 μl of 10× binding buffer (100 mM Tris/HCA
! (pH 8), 50mM Mg01z), 3μ
35S-AT P (1000Ci/ mmoj2
; Ammarjam) and 1 μl of Klenow enzyme (4U
/μi; Biolab) was added. The remainder of the sequencing step was performed using the chain termination method of Sanger et al.
Science knee (Proc, Natl, A
cad, sci.

USA), 1977, Dan, 54.63-5467). In the expression plasmid pRH969/1 from clones 307 and 319 the insert is AT
It was found to be consistent with G.

To detect the expression of plasmid-encoded proteins in L2i, the maxi-cell line (maxi-cell
l system) was used. This was developed by Sancar et al., Journal of Bacteriology (J, Bacteriol), 197
9.137.692-693). This L unimaxie cell line C3R603 (CGS (: 5830; F-,
thr-1, LeuB6, proA20) phr-
1゜recAl, argE3, thi -1, uv
rA 6 , ala-14, /acY1, galK20
)xy'-5, n+tf-1, gyrA 98 (
nag A 98), rps31, tsx-3
3. λ-1supE44) does not have a mechanism to repair defects caused in DNA by UV irradiation.

For experiments, the radiation dose is optimized so that the chromosomes are often affected, but the relatively small plasmid DNA, present in a few copies per cell, is left largely undamaged. The damaged chromosomal DNA is degraded by the cell, while the plasmids unaffected by UV radiation continue to replicate and make up the bulk of the remaining DNA. After all intact replicating cells are killed with the antibiotic D-cycloserine and the endogenous mRNA is used up, only the plasmid-encoded genes are transcribed and translated in the remaining cells. After transferring the bacteria to a sulfate-free medium, the protein formed is radiolabeled and thus detected by incorporation of 35S-methionine.

L uni strain C3R603 cells were cultured as described above (9
Clones 307 and 319 (with reverse orientation of 69/1)
or the appropriate expression plasmid pRH969 from 224
Transformed with /1. The transformed cells were incubated for 60 min at 37°C in 15 mj' of expression medium without tryptophan.
The cells were cultured until the optical density at 0 nm was 0.5 to 0.7. The expression medium used was as follows (amounts are per 11 medium).

a) 10g of (NH4)! Kuz of HPO4,3,5g
pO, and 0.5 g of NaCIf were dissolved in 500 ml of water and the solution was brought to pH 7.3 with NaOH and then brought to 800 ml.

b) 21 g of casamino acids (acid-hydrolyzed, vitamin-free; Merck & Co.) were dissolved in 100 ml of water.

c) 50ml of 20% glucose solution.

d) 1mf IM Mg5O,.

e) 1mj! 0.1M CaCl,.

f) 1 mg of thiamine-HCl and 20 mg of thiamine-cysteine were dissolved in 1 ml of water.

Immediately before use, these autoclaved and sterile-filtered solutions were mixed at 10,100 O! Toshi・100mg
/j! of ampicillin was added. Transfer 10 m2 of harvested bacterial culture to an open 13.5 cm veterinary dish and apply U for 5 seconds from a distance of 50 cm with slight rotation.
It was irradiated with a V germicidal lamp (15W) and further incubated at 37°C. This culture was mixed with 100 μg/nil D-
Mixed with cycloserine (fresh 100x concentrated solution in 501 phosphate buffer, pH 8) and incubated for 14-16 hours at 37°C on a shaker. The bacteria were harvested by centrifugation (5 minutes at ambient temperature 5), washed twice with 5 ml of Tlershey's salt solution, and 20 μg/ml of 3-β-indoleacrylic acid (=
The cells were suspended in 5 ml of Virgie's medium containing IAA;) inducer of the liptophan operon) and shaken at 37°C for 1 hour.

O bargie salt solution (per 11): 5.4 g Nacl 15 mg CaC1z・2
HzO87mgK HzP Oa 3.0 g
KCl0.2gMg (12.6H20 12.1g Tris+HCj2 (pH7.4)
1,1 g N HaCII 0.2 mgFe
Cl3 .6 Hz OO Virgie medium (per Virgie's salt solution 100+j!): 20) Onl's 20% glucose 1.0 mj! 2% proline 0.5n11 2% threonine 1.0 + wIl 2% arginine 1.0mj! 1% leucine 0.1mj! 0.1% thiamine-HCj2 about 15μ
Ci/ml of 383-methionine (1000Ci/f
f1lIIo1; Amar-Jam) was added to each culture, and the cultures were then incubated for an additional A hour at 37°C. The cells were then harvested by centrifugation (5 minutes at ambient temperature 5) and 200 μl of SDS sample buffer (10 m
M Tris/HCJ (pH 7,4), 125m
M BME, 2% SDS, 10% sucrose, 0.0
The cells were lysed in 5% bromophenol blue]. This sample was incubated at 95°C for 5 minutes and
Centrifuged separately. The supernatant was checked for excess protein. SD
Add 20-60 μl of cell lysis supernatant in S sample buffer to 1
Separated according to size on a 0% 5DS-containing polyacrylamide gel [Laemmli U., Nature, 1970.27]
7.680-686).

This 1.5 mm thick gel is 10% of the length of about 10 cm.
It consisted of a separating gel and a 3% collection gel with a length of 3 cm.

Electrophoresis was performed in the collection gel at approximately 3 W (constant) and in the separating gel at 12 W (constant) for a total of 4.5 hours. B
SA (66kd), ovalbumin (45kd)
, pepsin (34,7 kd), β-lactoglobulin (
A protein mixture consisting of lysozyme (18,4 kd) and lysozyme (11,3 kd) was also separated and used as a size marker. After electrophoresis, the marker protein gel lane was cut out from the remaining gel, incubated for 30 minutes in a staining solution (50% methanol, 10% acetic acid, 0.1% Coomassie Brilliant Blue 0250), and then incubated in a destaining solution (
The mixture was shaken overnight in a 5% methanol solution (10% acetic acid), placed in a glycerin bath (7% acetic acid, 2% glycerin) for 30 minutes, and dried in a gel dryer at 60°C for about 1.5 hours on fat-free 3MM filter paper. (See lane M in Figure 11).

Proteins in cell lysates undergo electromigration (E
From the gel to the nitrocellulose filter (Schleicher and Schleich)
, B A 85 ; 0.45 μ] [Burnette W, N1, Analyt.

Biochem, ) 1981.112.195-
203].

This transfer was performed using a transfer buffer y-[:20mM Tris,
15QmM glycine, 20% methanol p, a,
; pH 8,8:], 0.1% empigen [εm
pigen ; Marchon Italiana (March
on Italiana) S, P, A, ] at 60 V for 5 h in a Bio-Rad protein plot device.
I went there [Mandrell R, Snake.

et al., Journal of Immunological Methods (J
, ImmunollMeth, ), 1984.67.
1-11].

Expression of proteins exclusively encoded by this plasmid was detected by incorporation of 35S-methionine.

Figure 11 shows the Kodak X-Omat
The autoradiogram of the "Western plot" (lane 82 B3) is shown after exposure on S X-ray film. Lane B2 (corresponds to lane A2 of Western plot) and Lane B4 (corresponds to lane A4)
shows the two signals of insert 969/1 expressed and the signal of β-lactamase encoded by the plasmid gene for ampicillin resistance, while lane B3 (corresponding to lane A3) with 969/1 (inverted orientation) shows the 40 kd has no signal, but β
- Has a signal for lactamases. Due to the fact that several termination codons are present at the beginning of the encoded region in reverse orientation, no protein is expressed at all.

Example 8 Production and Isolation of Monoclonal Antibodies Description of Solutions and Media Used: oloox Aminopterin 1.8 mg of aminopterin was dissolved in 50+ml water with the addition of IM NaOH and then added to 100 mff in the dark. (stored at 120 tons).

0100x OPI mix 7.5g oxalic acid and 2.5g pyruvate in 450m
1000 U of insulin (several mI2
0.1M HCj! (dissolved in). Replenish this mixture with water to 500 mI! shall be.

oloox HT mix 0, 6805 g (200 mff of D-hampoxanthine
The solution was dissolved in water and adjusted to pH 10 with NaOH.
00 mJ aqueous thymidine solution (0,1937 g; 200
(in m1) and supplemented to make 500 mA+.

o HT medium 500ml Dulbec improved Eagle medium CDulbec
co'S Modified Eagle's Medi
um; DMEM: Flow Laboratories (F
low Laboratories), high glucose] 6mji! :200mM L-glutamine 6mj! :
100x HT mix 6ml: 100x OPI mix 50ml: HIFC8 (heat inactivated calf serum) 0.6
mj! Gentamicin (50
MG / MF) OHAT Medicine 100mj2: HT Medicine 1m1100X Aminopterin O ABTS Solar 100 mg / Nu 2,2' -Azinodi (3 -ethyl benzoch anodolinzorinzuruhonate) aqueous solution PBS 137mm NACL 2.7mm KC L GMM NA28 P 04 1.5mM K82P0゜0, 5mM MgC1z ・6 H3C1mM C
aCj! z・2H2゜o PBS def: Same as PBS, but does not contain Ca salt and Mg salt.

o PBS washing solution PBS containing 0.05% Tween 20
defyuo 20%FKS DMEM 445mA Dulbecco's improved Eagle medium (Flo Laboratories) 50 ml HIFC3 (heat-inactivated calf serum) 511
11 Henicillin/Streptomycin (10,000U
/ml) ORPMI compl.

5001lil RPMI 1640 medium (Flow Laboratories; Nr, 12-12-54) 50 Fe2 5mj! 200+wM L-glutamine 5 ml
Penicillin/Streptomycin (10,'OOO
U/n+jlio PEG-4000 Solution Dissolve 20 g of PEG in 28*J2 PBS def and incubate in a water bath until the PEG is dissolved (approximately 30 minutes).

ORPMI wash solution 500 ml RPM11640 medium 5 ml
Penicillin/Streptomycin (10,000U/
+IIn 5 ml 200 mM L-glutamine HAT thymocyte medium The thymus of a 3-4 month old Ba 1 b-c mouse, removed under sterile conditions, preferably free of connective tissue, was transferred to a sieve; through a sterile syringe plunger (slow circular motion, rotating the sieve in a circular motion for approximately 1 minute);
300 g of cell suspension in RPMI wash solution 2Snl;
Centrifuge for 10 minutes at ambient temperature. Remove the supernatant by blotting with a Pasteur pipette and remove the pellet from the 2011If
(7) Resuspend in RPMI compJ, and further
Dilute to X10” cells/1I11.

This thymocyte-containing medium was incubated with CO in a small culture flask.
! Store at 37°C in an incubator (can be maintained for about 14 days). To obtain HAT thymocyte medium, the cells are removed by centrifugation, washed in RPMI wash solution as above, centrifuged again, and the cells are removed into HAT medium.

Purified HRV2 emulsified in complete Freund's adjuvant
5-10. Cgg was injected intraperitoneally into 2-3 month old Ba 1 b-c mice. The same amount of HRV 2 (emulsified in incomplete Freund's adjuvant) at 27.49.100
．． Post-dosing was given 117 and 118 days later. One day after the last inoculation, the lungs were removed and the cells were divided into myeloma cells and
For example, it was fused with NS-1 (ATCCT I B 1 B). This substantially followed the method of Galfre et al. (G
Alfre G. et al., Methods in Enzymol, 1981
, Uni, 3-46).

The lungs were removed under sterile conditions and placed in a Petri dish at a distance of approximately 5 m.
1 of RPMI wash solution. The lungs were ground in 5 ml of RPMI wash solution and passed through a sieve using a syringe plunger (slow circular movements). Finally, the cell suspension was placed on ice to sediment any cell clumps and connective tissue debris. The finely suspended cells were pipetted, transferred to a 501 ml round bottom centrifuge tube, supplemented with freshly prepared RPMI wash solution to 50n+1, and centrifuged at 300 g for 5 minutes at ambient temperature. The supernatant was removed and the pellet was washed with 5 Ill of lysis buffer (155-111 M NHtCl, 101
Contains 1M KHCO2 and 1mM EDTA; p1
7.2) and incubate for 2 minutes at 0°C. This suspension was added to 50 mj by supplementation with RPMI wash solution.
! The mixture was sealed and carefully stirred using a Pasteur pipette. Any lipid-containing compounds and fats adhered to the pipette and could be easily removed in this way. The suspension was centrifuged again at 300g for 5 minutes at ambient temperature. 10 mj of the obtained pellets! Resuspend in RPMI wash solution of approximately 5X10? mouse myeloma cells, e.g. NS-1 (50 cells from four 75*J tissue culture flasks)
% fusogenic). These cells were first collected in 50+sl
Wash 3 times with RPMI washing solution and centrifuge at 300g (
5 minutes at ambient temperature) to remove all serum. A mixture of lung cells and myeloma cells was suspended in 50*J of RPMI wash solution and centrifuged as above. The supernatant was carefully removed completely, and the resulting pellet was immediately mixed with a PEG4000 solution temperature-controlled at 37°C, and the test tube was incubated for 2 minutes.
The pellets and PEG 4000 were carefully applied to the edge of the platform until mixed (“grittycon”) until mixed.
The mixture was then incubated for 2 minutes at 37° C. with slow shaking, immediately followed by the dropwise addition of 20+11 RPMI wash solution.

At this time, each droplet was made to flow down the wall soil of the container separately.

An additional 30 III liters of RPMI wash solution was carefully added. After centrifugation of the a+ cells (5 min at 300 g at ambient temperature), the supernatant is removed by aspiration and the resulting pellet is collected using a Pasteur pipette at 10 n/! of HAT thymocyte medium.

This suspension was aliquoted in 1 ml aliquots into microtiter plates (96 wells per plate) and incubated at 37°C in 5% CO (plates do not need to be moved in an incubator). Approximately 0.1 mj2 of HAT medium was added 3 days later. After an additional 3 days, 0.05 mA of HAT medium was added. 10 after starting fusion
On day 70% of the medium was removed and 0.2 ml of HT medium was added. On day 12 post-fusion, the plates were observed microscopically and the medium was harvested for ELISA and neutralization tests.
Approximately 0.3 mJ of 20% FC3-DMEM was added to each well. Select positive hybrids by the above two tests, separate these hybrids from 1:3 to 1:10,
That is, transfer the contents of the wells to 3 to 1 microtiter plates.
0 wells.

After growth, the hybrids were tested again and the one showing the highest titer was selected. These hybrids were subjected to end-point dilution (en
d point dilution) twice. Meanwhile, using a microscope, it was confirmed whether the hybrid cells were derived from a single cell. Cloning was performed in the presence of thymocytes and 20% FC3. Each clone was grown to 50% confluence in 75 cni cell culture flasks. Cells from this type of cell tissue culture flask were injected into mice that had been pretreated 10 days earlier with an intraperitoneal injection of Bristane (0,5mj2). Ascitic fluid was collected from the abdominal wall 2 to 6 weeks later.

For each well of the EL TSA microtiter plate, carbonate buffer y-(15mM NazSOi/35mMNa
Virus dilution in HCO3 (1-2 μg/ml H
50 ttll of RV 2 ) were incubated overnight at ambient temperature. 100μ# PBSdef.

was added and incubated at 37°C for 1 hour to achieve saturation. This PBSdef contains 1% BSA. Empty the microtiter plate and fill the wells with 50
Filled with μl of hybrid supernatant. Pure HAT medium was used as a negative control. Then incubate for 1 hour at 37°C, empty the plate again and add PB
Washed three times with S wash solution (PBS def, +o, o 5% Tween 20). Then PBSdef,
11 of peroxidase-conjugated antiserum (RAM = “rabbit anti-mouse”) in (+1% BS^ and 2% Tween 20)
50 μβ of 500 dilutions were given to each well. After incubation (1 hour at 37°C), wells were washed 5 times with PBS wash solution as above.

To develop the ELISA test, add 50 μβ developer, (1 ml of 50
mM citrate buffer (pH 4), 10 μi AB
TS solution and 5 μl of 30% hydrogen peroxide) were added to each well. A positive result is indicated by a green color.

Cell culture supernatants from primary hybridoma colonies were tested for the presence of virus-neutralizing antibodies by microneutralization as follows. Briefly, 50 ttl of hybridoma supernatant was given to each well and 50 μ#MEM was added in 5% Cot.
(Minimum essential medium; Flow Laboratories) with 34
Incubated for 1 hour at °C. This MEM is 100O
PFU HRV contained 2.30 mM HgC1z, and 2% HI Fe2. Next, 3 x 1
0' HeLa cells (strain: HeLa-0hio; 0
3-147; Flow Laboratories) was added to each well, the plate was incubated for 488 hours at 34°C in 5% CO2, and stained with 8 solutions of 0.1% crystal violet in 20% ethanol. Wells with a complete cell layer were considered positive. Under the above conditions, 1/10 of ascites
At 0.00 dilution, 5% neutralization could be detected. natural) I
A monoclonal antibody was selected from the group of monoclonal antibodies directed against RV2, which, apart from having neutralizing properties, was able to demonstrate by Western methods that it binds to the antigen, namely the denatured form of the viral capsid protein VP2 (see Figure 11). Such an antibody was named 8F5. By using this monoclonal antibody, HRV
2 or in Western plot V
In addition to being able to detect expression products containing F6, this antibody has also been demonstrated in the poliovirus system, for example for VPl [Wychowski et al.
It can also be used to more clearly characterize the immunogenic site of HRV2 in VF6, as in C. et al., supra, 1983'l.

A monoclonal antibody with the above properties, such as 8F5, was used to test the antigenicity of the expressed protein pRH969/1 from clones 307 and 319.

As already mentioned, this antibody has the advantageous property for expression studies that it recognizes VF6 (and consequently 969/L) not only in its native state but also in its denatured state.

This type of monoclonal antibody is rare, and until now it was based on the poliovirus [Wychowsky
i) C. et al., supra, 1983). The filter with bound proteins is placed in a “blocking solution”.
)'' i.e. PBS (137sM NaCl,2,
7mM KCI, 8mM Na, HP Oa, 1
．． 5mMKHzPOa, 0.5mM MgC1z
'6H20,1mMCaC(lt ・2 HzO
(containing 1% BSA), 1% Tween 20 and 10% heat inactivated calf serum (HIFC3)) 50 mβ overnight at ambient temperature. This was then incubated for 3 hours with antibody 8F5 (mouse ascites supernatant diluted 1/200 in blocking solution) in 4 ml of blocking solution.

Here, the filter was incubated in a sealed state with household film (fixed on a seesaw mechanism). The filter was then thoroughly washed with running water (approximately 20 minutes) and washed three times with PBS501I11 containing 1% Tween 20 for 15 minutes. In the final step, the filter was treated with alkaline phosphatase-conjugated anti-mouse IgG in blocking solution of 501111.
(diluted approximately 1/3000 in blocking solution) for 3 hours at ambient temperature. The filter was again thoroughly washed with running water and washed three times with 50 ml of PBS containing 1% Tween 20 as described above. Dyes, nitro blue tetrazolium (NBT: 165 μg/mf) and 5-
Bromo-4-chloro-3-indolyl-phosphate (
BCrP: 82.5 μg/mf), 10 mj
! of phosphatase buffer (100mM T
ris/H1d! (pH 9,5), 100mM
Staining was performed in NaC&, 5mM MgCItz). This alkaline phosphatase-conjugated anti-mouse IgG
(H+L) and the dyes NBT and BCIP were taken from the Protoblot TB system from Promega Biotec. This staining reaction was stopped after about 5 minutes by washing under running water. Figure 11 shows the maxi cell line C5R60.
The results of the expression of 969/1 in 3 are shown.

Lanes A1-A4 show Western plot experiments after staining with NBT and BCIP; in lane A1;
Denatured HRV2 particles (approximately 50 ng of HRV2(7)) were also split as a control. As understood from FIG. 11, monoclonal antibody 8F5 selectively selectively affects VF6 (
The high or low molecular weight bands, which are very difficult to distinguish, represent proteolytic degradation products of vpo (precursor of coat protein) or VF6. In lanes A2 and A4, plasmid p
H, near 1J strain C3R60 transformed with RH969/1 (derived from clones 307 and 319)
Three cell lysates were split. In both lanes, approximately 40 kd (40,2 kd
) A specific signal of the expression product 969/l could be detected as a band. As a negative control, in lane A3, lysates of L 2i strain C3R603 cells transformed with the plasmid from clone 224 were analyzed. As mentioned above, this plasmid contains insert 96
It has a 9/1 reverse orientation. No signal is obtained from the Western plot in lane A3 due to the concomitant loss of specific antigenicity and the fact that multiple termination codons are present at the start of the conversely encoded region.

Lane M shows Coomassie blue staining of marker proteins (see above). Lanes B2-B4 are lanes A2-
An A4 autoradiograph is shown (see Example 2 above for accuracy).

An expression plasmid containing the XhoII fragment from FIG. 9, e.g. plasmid pRH969/1, was linearized at the single Bs5HI [cleavage site and 2 .4.6.8 and 10 minutes processed. The thus shortened linear plasmid was recircularized and transformed into Muuni cells. This expressed protein was then tested for its ability to bind monoclonal antibody 8F5. By comparing the sequence of a plasmid expressing a protein that can bind with that of a plasmid expressing a protein that cannot bind, it was determined that the immunogenic site of HRV2 on VPZ for monoclonal antibody 8F5 is located at nucleotides 1274 to 1309 of HRV2.
It was determined that the amino acids present between the amino acids encoded by (see FIGS. 9 and 13).

[Brief explanation of the drawing]

FIG. 1 shows electrophoretic separation of coat proteins of HRV89 on a 12.5% polyacrylamide gel in the presence of SDS, with no vp4 seen. This is because it moves from the gel together with the front. Figure 2 shows electrophoresis of I(RV89-RNA) on a 1.2% agarose gel in the presence of SDS. The positions of ribosomal RNA markers are indicated on the right. Restriction map of the HRV89 genome. 21 overlapping clones used for sequencing are shown. Specific cleavage sites for several restriction enzymes are shown. Arrows (A-D) indicate primers for reverse transcription. The restriction fragments used are shown. Figure 4 shows the sequence of the cloned HRV89 genome and the amino acid sequence obtained therefrom. Cleavage sites of the polyprotein are indicated by arrows. Black arrows N) indicate experimental It is the cleavage site required for
The white arrow (8) indicates the predicted cleavage site in the polyprotein based on homology with other picornaviruses. FIG. 5 compares the homology (%) in the amino acid sequences of each gene among HRV89, HRV20) HRV14 and poliovirus type 1. FIG. 6 is the N-terminal amino acid sequence of the coat protein of HRV89. Figure 7 shows the construction plan for expression plasmid pRH100. Figure 8 shows the tryptophan promoter/operator area,
Represents the synthetic ribosome binding site (RB S) and disclosed codons of the expression vector pRH100. Brimer (1) used for sequencing expression plasmid pRH969/1
7 wildebeest) is also given. Lowercase letters and numbers at the bottom are p
The sequence of BR322 is shown, with uppercase letters indicating the sequence inserted between pBR322 nucleotide numbers 1 and 29. Figure 9 shows H between nucleotide numbers 700 and 2000.
The sequence part of RV2-cDNA is shown, and the short thick arrow indicates V
The F6 sequence is represented, with thin arrows indicating the most important deadline sites in this part. Figure 10 shows the construction plan of the expression plasmid pRH969/1 (NCR-non-coding region, untranslated region HtrpP
10-tryptophan promoter operator region). Figure 11 shows 969/969 in maxi cell line C3R603.
Lanes A1-A4 show Western experiments after staining with NBT and BljP; in lane AI, denatured HRV2 particles (approximately 50 n
g HRV2) was also split as a control. In lanes A2 and A4, plasmid pRH969
Cell lysates of P. uni strain C3R603 cells transformed with P./1 (obtained from clones 307 and 319) were analyzed. In lane A3, strain C
Cell lysates of 3R603 cells were analyzed as a negative control. These cells were clone 224 (969/1
(transfer orientation of the insert). Lane M is Coomassie b.
Lanes B2-B4 show autoradiographs of lanes A2-A4. Figure 12 shows the insertion of plasmid pRH969/1. Figure 13 shows the binding site of 8F5 on the viral coat protein VPZ. Numbers 1 to 261 on the top line are VP2
shows the amino acid sequence of HRV
The DNA sequence of 2 is shown. The DNA and amino acid sequences of the binding region are shown, where each deletion (mutation) is given precisely. FIG. 14 shows the sequence of the Ucloned HRV2 genome and the amino acid sequence derived therefrom. The cleavage site in the polyprotein is indicated by an arrow. Solid arrows indicate experimentally determined cleavage sites, while hollow arrows indicate cleavage sites in the polyprotein predicted based on homology with other picornaviruses. Figure 15 shows the construction plan for expression plasmid pRH969/2. Figure 16 shows the construction plan for expression plasmid pKK89/2A. Figure 17 is the polypeptide expressed by pKK89/2A. FIG. 18 is a comparison of the amino acid and nucleotide sequences of the genes for P3A and P3B (VPg) of HRV2 and HRV89. The white arrow indicates the preliminary cleavage site estimated from comparison with other vicorinaviruses. FIG. 19 is a comparison of the nucleotide sequences of the 5' untranslated regions of HRV2 and HRV89. FIG. 20 is a comparison of the amino acid sequences in the viral protein regions of HRV2 and HRV89. -c4c'. Stretch [-'s ψ ray shi--1 ('N ('
Nr+IIJI Ll-”＞＞＞〉Q
4Q4 Mountain Mountain 〉 B Luxury〉 〉 10E-4 〉-One bacterium -- 1 〇〉 〃 ζ 0 Utsuro 0 − α〕Q4
Qs - 2: ψ 0 +1 -F+QQ- 0 Inventor Dietil Blaas Aussudo Inventor Ernst Cue Aussudo-23-6 0 Inventor Rielka Fratsel Aussudo Inventor Manfret Zuo Ossrun Nidradoria A 1090 Vienna Liechtensteintrasse 119 The State of Tria A1190 Vienna Gelgengatsedria A1020 Vienna Ober Donaurasse 5 The State of Doria A1140 Vienna Mawerbachschese 46 A5 3. Relationship with the person making the amendment Applicant 4, Agent

Claims (34)

[Claims] (1) A DNA molecule corresponding to part or all of the viral RNA of rhinovirus strain HRV89 or a degenerate form thereof. (2) A DN that has the characteristics of at least one or a part of the viral protein of rhinovirus strain HRV89 and encodes a viral protein, a part thereof, or an allele thereof having the amino acid sequence 1 to 2164 shown in Figure 4.
A DNA molecule characterized by containing the A sequence and its degenerate form. (3) Viral proteins VP1, VP2, VP3, VP
4, P2A, P2B, P2C, P3A, P3B or P
DN encoding 3C, a part thereof or an allele thereof
A DNA molecule according to claim (1) or (2), characterized in that it contains the A sequence and has the characteristics of at least one or a part of the viral proteins of rhinovirus strain HRV89. and its degenerate form. (4) A DNA molecule and a degenerate form thereof according to claim (3), characterized in that at least two of the viral proteins are bound together in any predetermined combination. (5) Hybridize with the DNA molecule according to any one of the above claims under strict conditions that make it possible to detect 85% or more homology, and at least a viral protein of rhinovirus strain HRV89. A DNA molecule characterized in that it encodes a viral protein having one or some of its properties. (6) A replication cloning vector comprising the DNA molecule according to any one of claims (1) to (5). (7) Encodes a viral protein having the amino acid sequence shown in Figure 4 or Figure 14, a part thereof, or an allele thereof, and rhinovirus strain HRV89 or H
A replicable expression vector capable of expressing a DNA sequence characteristic of at least one of the viral proteins of RV2 or a portion thereof and degenerate versions thereof in a transformed host organism or cell culture. (8) The DNA sequence corresponds to claims (1) to (
5) A replicable expression vector according to claim 7, characterized in that it corresponds to a sequence described in any of claims 5). (9) The replicable expression vector according to claim (7), which is vector pKK89/2A. (10) Vector pRH969/1 or pRH969
/2. The replicable expression vector according to claim (7). (11) Characterized by being able to express a viral protein, allele, or part of the viral protein of rhinovirus strain HRV89 or HRV2 encoded by the DNA sequence and having the amino acid sequence shown in FIG. 4 or FIG. 14. A transformed host organism or cell culture. (12) The above DNA sequence is from claim (1) to
Transformed host organism or cell culture according to claim (11), characterized in that it corresponds to one of those mentioned in paragraph (5). (13) The above DNA sequence is from claim (7) to
A transformed host organism or cell culture according to claim (11), characterized in that it is contained in a replicable expression vector according to any one of paragraph (10). (14) ¥E. Claim No. (11) which is coli yen
, (12) or (13). (15) The DNA molecule according to any one of claims (1) to (5) or claim (7)
encoded by the DNA sequence contained in the replicable expression vector according to any one of items 1 to 10, and having at least one characteristic of the viral protein shown in Figure 4 or Figure 14; A polypeptide characterized by (16) A polypeptide characterized in that the amino acid sequences 1 to 2164 correspond to at least one of the viral proteins shown in FIG. 4, or a part or allele thereof, and do not contain other proteins. (17) Viral proteins VP1, VP2, VP3, V
A polypeptide characterized in that it contains an amino acid sequence of P4, P2A, P2B, P2C, P3A, P3B, or P3C, or a part thereof, and does not contain other proteins. (18) The polypeptide according to claim (17), wherein at least two of the viral proteins are linked together in any predetermined combination. (19) A derivative of a polypeptide having the amino acid sequence 1 to 2164 shown in FIG. polypeptide. (20) A method for preparing the polypeptide according to any one of claims (15) to (19), comprising using a suitable host organism or a suitable cell culture, preferably using a suitable host organism or a suitable cell culture. The host organism or cell culture according to any one of claims (11) to (14) is used in claim (1).
Genetic information encoding the polypeptide according to any one of items 5) to (19), preferably claim item (19).
7) Transformed with the genetic information contained in the replicable vector according to any one of items 10), cultured in the thus transformed host organism or cell culture under appropriate conditions, and expressed. The above method, which comprises isolating and purifying the polypeptide. (21) A hybrid cell line characterized by secreting a monoclonal antibody against one of the polypeptides according to any one of claims (15) to (19). (22) completely or partially specifically neutralizes the action of the polypeptide according to any one of claims (15) to (19), or one of these polypeptides; A monoclonal antibody characterized by specifically binding to. (23) A monoclonal antibody characterized by having antigen neutralizing properties and binding to a denatured form of the antigen. (24) Claim No. (15) of the monoclonal antibody according to Claim No. (22) or (23)
) to (19) for quantitatively and/or qualitatively measuring one of the polypeptides described in any one of the items. (25) Claims (22) or (23) for purifying one of the polypeptides described in any one of Claims (15) to (19). Use of monoclonal antibodies. (26) Use of the monoclonal antibody according to claim (22) or (23) for treatment. (27) The polypeptide according to any one of claims (15) to (19), which comprises the monoclonal antibody according to claim (22) or (23). Test kit for measurement. (28) A host animal is immunized with one of the polypeptides according to any one of claims (15) to (19), and B-lymph cells of the host animal are fused with myeloma cells. A method for preparing a monoclonal antibody, which comprises subcloning a hybrid cell line that secretes the monoclonal antibody according to claim (22) or (23), and culturing it in vivo or in vitro. (29) Use of the polypeptide according to any one of claims (15) to (19) for treatment. (30) Use of one polypeptide according to any one of claims (15) to (19) for the treatment of rhinovirus infection. (31) Use of one of the polypeptides described in claims (15) to (19) for the prevention of rhinovirus infection. (32) Use of one of the polypeptides according to claims (15) to (19) for stimulating the immune system. (33) Use of one of the polypeptides according to claims (15) to (19) for binding to and/or blocking the cell receptor of rhinovirus strain HRV89. (34) Contains an effective amount of at least one polypeptide according to any one of claims (15) to (19) in addition to a pharmaceutically inert excipient and/or carrier. Pharmaceutical composition according to any one of claims (29) to (33), characterized in that it is suitable for use with the above-mentioned polypeptide.