JPH06501850A - Retrovirus-like particles in Chinese hamster ovary cells - 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] 20, for obtaining cells with increased or decreased transcription of the nucleic acid according to paragraph 1. A method, (a) obtaining cells containing the nucleic acid; (b) introducing a transcriptional regulatory element into the cell; (C) increasing or decreasing transcription of the nucleic acid; A method consisting of screening cells that have been

21. Methods for obtaining cells with reduced expression of endogenous type C retroviral sequences The law is (a) obtaining cells with detectable levels of endogenous type C retrovirus sequences; (b) Mesosenary RN of the endogenous type C retrovirus sequence nucleic acid R complementary to A A method comprising introducing a nucleic acid encoding an NA transcript into the cell.

22.0 The method according to item 21, wherein the Torovirus sequence is the sequence of item 3.

23. The method according to item 21, wherein the complementary nucleic acid is the sequence of item 5.

24. Methods for obtaining cells with reduced expression of endogenous type C retroviral sequences The law is (a) obtaining cells with detectable levels of endogenous type C retrovirus sequences; (b) RNA that is complementary to the mesosensor RNA of the endogenous type C retrovirus sequence nucleic acid introducing A into the cell, A method involving stages.

25. The method according to item 24, wherein the type C retrovirus sequence is the sequence of item 3.

26. The method according to item 24, wherein the complementary nucleic acid is the sequence of item 5.

27 Methods for obtaining cells with reduced expression of endogenous type C retroviral sequences The law is (a) Obtain cells with detectable tC type retrovirus RNA A sequence, (b) C cleavage of RNA adjacent to type retroviral RNA sequences or introducing the column into the cell, A method involving stages.

28. The method according to item 27, wherein the Torovirus type 28.0 sequence is the sequence of item 3.

29 Adjacent to a nucleic acid in a proximate position and orientation sufficient to influence the transcription of the nucleic acid. Culture cells containing the nucleic acid according to item 3, in which a transcription regulatory element has been inserted into the DNA. A method consisting of nurturing.

30 Hybridization between the test sample and the probe specific to the sequence in section 3 A method for detecting a retrovirus-like nucleic acid sequence in a test sample, comprising measuring /.

31. Hybridization between the test sample and the probe specific to the sequence of Section 5. A method for detecting retrovirus-like nucleic acid sequences in a test sample, the method comprising measuring retrovirus-like nucleic acid sequences in a test sample.

32. Retrovirus-like nucleic acids in test samples, including the use of polymerase chain reaction A method for determining the presence of expression products of.

33, including the use of immunoassays directed against expression products, A method for determining the presence of expression products of virus-like nucleic acids.

34, the following sequence (SEQ ID NO: 1).

GAATTCAATG TCCTTGTGGG GAAGAACGAT ATT CTAAAACAGGTAACTGA 50GCAATGTGATGCGTG CGCCCGAGTCAACGCATCCAGACTG AAGCTTCCTC 100CCGGGAACCG GTCAGAGGCT ACCGGCCCG AACAC, ATTGG GAGATAGATT 150TCACTGAGAT TAAACCAGGA, ^^1 to TATGGAT ACAAGTATCT ATTA, to TTTTT 200 GTAGACACCT TTTCAGGATG GCTTCAACCCTTCCCCTACTA AAACATGAAAC250A GCCAGATCG TTACTA, AGAA ATTGCTTGAA GAA ATCTTTCCCCGTTATCG 300GATGCCTCAG GTAT TGGGAA CAGAC^^TGG GCCCGCCTTCGTCTCCAG GT 350AAGTCAGTCA GTGGCCACCT TATTGGGG AT TGATTGGAAA TTACATTGTG 400CnATAGAC CCCAAAGTTCA GGACAGGTG AAAGGATGAA TA GAACAATC450^^GGAGACTT TAACA^^^n GTCG CTTGCA ACTGGCACTA GAGAGCTGGG 500TCCT CCTACT　CCCCCTAGCA　CTCTACCGCG　CTCGTAA TACCCCTGGACCA 550CATGGGCTCA CACCCTTT GA GATCCTGTA Ding GGAGTACCTA GCTCCT~TCA 600T AACTTTCT TGATCAAGAT GTCTCAGTT TGCTAACTCCCCTTCTCTCC650AAGCTCATTT AC AGGCCTCC^^CTAGTACAACGGGAGGTCTGG^^A CCC700CTTGCTCAAG CTT, to TAAAGA CCAGAGG GACCATCCCCATCCCCCATTC750CTACCAGAT CGGGACACTG TTTGGGTCCG GCGTCACCAG GC CAAGAACC800TTGAACCCCCG CTGGAAGGGA CCC TACATCG　TTTTGCTTACCACTCCCCACC850GCACT CAAGG TAGACGGCAT TGCAGCnGG ATACATGCT T CACATGTAAA 90 (IGCCAGCCCAA CCCACCGA TT CAGCCACTGCATCAGAA Ding GG AGCCACACC949 at least one retrovirus-like particle encoded by.

Specification Background of the invention of Chinese hamster ovary cell retrovirus-like particles The present invention relates to recombinant cell culture systems and methods, and in particular to recombinant cell culture systems and methods. Concerning the identification and use of nucleic acid sequences encoding lovirus-like particles.

Retrovirus-like particles were collected from thin-film sections of Chinese hamster ovary (CHO) cells. When a piece is observed under a transmission electron microscope, all cells are observed. into a centriole Two types of particles, hemi-shaped particles and budding C-shaped particles, are often present in the cytoplasm. Consistently observed deaths Donahue, PRl et al., Journal O. Bu-no\i-o-rono- (J, Virol,), vol. 62, pp. 722-731 (19 1988), Lieber, M., Science. ce), vol. 182, pp. 56-58 (1973), Lubiniecki ecki), Developments in Bioloncal Standards B (Develp, Biol, 5 standards,), vol. 70, 187 ~191 pages (1989), Ruhinye, Ki et al., Developmental Solutions in By. Oronkar Standardize/Ein, vol. 60, pp. 141-146 (1985) ), Ma-nley, KF, ShiA--+Luyrbu-nefuuru- Biooo',;-(J, Genvirol,), vol. 39, pp. 503-517 ( 1978 5L, Tihon, C et al., Fuji Char-; 2-+B. Ion-(!1at-New Biol, ), vol. 244, 227-231 Page (1973):・0 No infection or spread to other cells has been detected so far. It was: the same magazine and Bojman, FR1 Developmental Biology (Develop, Biol,), Vol. 70, pp. 195-202 ( (1990) Ko. Recombinant CH○ cells can be used to produce biopharmaceuticals intended for human use. Because it is used as a substrate [=Collen, D., Journal τB] ・Pharmacology and Experimental Therapy (U, Phar m, Exp,), vol. 231, pp. 146-152 (1984), Buff-( Patzer), EJ, Biotechnology ), vol. 4, pp. 630-636 (1986), Egrle, JC7 , Progress in Clinical Biology (Prog, C11n, B iol, ), vol. 191, pp. 339-350 (1985). The observed ultrathickness of retrovirus-like particles despite their infectious nature is interesting. biological life Reverse transcriptase activity, an indicator of sexual potential, was observed in retrovirus-like particles in recombinant cell lines. transmission of viruses to recipients of biopharmaceutical products to the extent that they have been shown to be associated with There is growing interest in the possibility of

Conventionally, viruses containing certain types of retroviruses [νon Ruden ) et al., Nyanar of Heirology, Vol. 63, pp. 677-682 (1989 ), Chang et al., Journal of Pyroronour, Vol. 61, 9. 21-924 (1987)], the expression of specific gene products in clear mammalian cell lines. 5 Kasid et al. 243, pp. 1354-1356 (1989), Khok a) et al., Science, Vol. 243, pp. 947-950 (1989*), Izant (Izant) et al., Science, vol. 229, pp. 345-352 (1985). However, not all attempts were successful: Kerr et al. European Journal of Biochemistry (Eur, J, Bi ochem, ), vol. 175, pp. 65-73 (1989)]. Target RNA secondary Structure may influence sensitivity to antisense inhibition. Currently, antisense For the harm to be effective, an excess of antisense compared to the coding mRNA, A. It is believed that SRN A is necessary.

Ribozymes are small RNA molecules that can cleave substrate RNA in a sequence-specific manner. Ru. For example, in vitro, "hammerhead" cutting mRNA at specific sites ” ribozyme was reported by Haseloff et al. [Nature -, vol. 334, 585-591Jj (1988*)]. To these hammers In the case of doribozymes, the target sequence includes the GUA or GUC sequence where cleavage occurs. All you need is to be there. Cleavage specificity and hybridization to bozyme sequences determined by the flanking (boundary) array. i.e. GU, A or GUC Riboza to cut at any 18 bp RNA sequence bordering the replet. time can be manipulated and thus produce extremely precise specificities. Ru.

Also, in the open literature, highly specific cases such as those reported by Koizu et al. An RNA enzyme with endoribonuclease activity has been reported [FEB S. Letters (FEBS Lett, ), volume 239, pages 285-288 (1 988)].

Ribozymes have also been used for in vivo applications. For example, unpublished data can be stabilized in vivo by inserting lipozyme sequences into the tRNA gene. tRNA/lipozyme assembly specific for the HIV-1 tat gene with improved performance The body has been shown to be able to transform the HIV-I LTR promoter by the HIV-1 tat product. It has been demonstrated that it is possible to inhibit the activation of cancer.

The number of retrovirus-like particles previously shown to be present in CHO cells is small. This made identification and characterization difficult. Therefore, one object of the present invention is Novel extracellular type C retrovirus-like particles in culture fluid from recombinant CHo cell lines The purpose is to identify and determine its characteristics. A method for detecting these particles is also covered by the present invention. This is the purpose of

Furthermore, it is an objective of the present invention to increase the expression of these retrovirus-like particles in cultured cells. or to provide a method for reducing it.

° Small or undetectable levels of type C particles or inversions associated with these particles A method of isolating a cell line with transcriptase activity is provided. Such cell lines are manufactured Minimizes regulatory requirements for in-process virus purification and inactivation studies , since it should be possible to eliminate the importance of transmissibility of retroviral particles. provides an ideal parental cell line for subsequent genetic modification and for the production of biological drugs. Provides use as a substrate for

Summary of the invention The purpose of the present invention is to encode extracellular retrovirus-like particles and proviral sequences. This is achieved by novel nucleic acid sequences that have been developed. Furthermore, this purpose Acid-transformed cells, or functionally transform such nucleic acids into exogenous regulatory sequences. providing cells containing the associated host cells and culturing the host cells to introduce retroviruses into the culture. Achieved by a method consisting of accumulating rus-like particles and recovering them from the culture be done.

Unexpectedly, the full-length clones encoding these retrovirus-like particles was identified and recovered. These sequences are derived from the genomic DNA of the CHO cell line. and repetitive proviral sequences conserved in Chinese hamster liver DNA. It turns out that there is.

A polynucleotide protein was used to measure the transcription level of the above DNA, A, in cells. The determinant of the peptide product of the above DNA sequence can also be It is possible to produce a receptor that can specifically recognize this position. Depending on the various applications, Probes and receptors can be labeled with a variety of labels.

Yet another purpose is as a diagnostic reagent for retrovirus in vitro assays. Provide molecules with novel features for use. The sequences provided by the present invention are , homologous to the conserved endonuclease domain of murine leukemia virus However, the sequence contains multiple breaks in the reading frame encoding the endonuclease. I'm here. The above sequence is an endonuclease required for viral integration and replication. The retrovirus-like particles of the present invention are currently not effectively encrypted. It is believed to be non-infectious.

complementary (antisense) nucleic acid sequences, ribozymes, RNA nucleases, DNA Control sequences and exogenous translational regulatory elements are used to develop these particles in cell culture. provides a method for increasing or decreasing the current Increase or decrease of these particles Provided are cells having such expression, and methods for detecting expression levels.

The compositions and methods of the invention can be used, for example, in PCR, immunoassays, or product samples. by using other means to identify these retrovirus-like particles. It can be applied to the detection of contaminants in engineered cell culture products.

Brief description of the drawing Figure 1 shows CHO3-3000-44 using a dual sucrose density gradient protocol. Figure 2 shows purification of particles from cells. By adding micronized sucrose, 4000 Culture medium from twice concentrated CHo 3-3000-44 cells was added to 50% sucrose. The density was adjusted and further layered with successive layers of 40%, 30%, and 20% sucrose.

After centrifugation and equilibration, pool the fractions from the main peak containing R-T (Figure 8). , dialyzed and layered on top of a prepared 10-60% sucrose density gradient, It was centrifuged again and equilibrated. The RT activity of the second gradient is shown in Figure B.

Figure 2 shows the electrons of particles purified by double density gradient from CHo 3-3000-44 cells. Microscope photo. Pool the RT-containing fractions from the second gradient of the double density gradient purification and centrifuge. , underwent dialysis. After dropping on EM dagrid, incative using uranyl acetate Particles were visualized by staining. Three examples of observed particles are shown. its size and The morphology is consistent with previous descriptions of retroviral particles visualized by this method. Ta.

Figure 3. Retrovirus-associated proteins and nucleic acids in density gradient purified particles.

Pools of four fractions each taken from the second gradient of dual density gradient particle purification. (Figure 1.B) for C-type structural proteins and retroviral nucleic acid sequences. I considered its existence. (A) Yaki anti-FeLVp27 serum was added to the C retrovirus structure. Western blot analysis used as a protein probe. p31 hand is king The p64 band represents the core protein, and the p64 band represents the unprocessed pre-gag protein. (B) Extract the nucleic acid from each pool sample and spread it on a membrane, Each was hybridized to the indicated probes. CHO cells and MRC5 Cytoplasmic RNA from cypress (human diploid fibroblasts) was used as a control.

Figure 4 shows the homology of pcHOcNiLlo CDNA with Moo=-MLV genome. Shinnick, T1 Fu-Char, vol. 293, pp. 543-548 (1981) Nucleotide sequence of Ko. is plotted on the horizontal axis, and pCHOC,ML10 nucleotide sequence is plotted on the vertical axis. Each point is Representing 75% identity over a range of 16 nucleotides. Diagonal regions of significant homology Represented as a line. Moloney MLV shows the composition of the RNA chemome to represent the direction. .

Figure 5 shows the start and stop codons of the pcHOc, ML10 sequence. -Methionine codon (M) in three reading frames of the MLV genome and collinear strands and a stop codon (0). Is there multiple division of the coded sequence in all three reading frames? exist. No significant reading frame was observed on the opposite mirror.

Figure 6 shows C-type proteins in the genomic DNA of CHO cells and Chinese hamster liver. Plot analysis of lovirus sequences. Parental cells and recombinant CHo cell lines, and cha HindIII-digested DNA isolated from Innies hamster liver (Rune hit) 2 μg) was fractionated on an agarose gel, blotted onto a membrane plate, and retrovirus hybridized with the rus probe. CHO-Kl is an ancestral cell line, All other cells are derived from cCHO-dhfr-, which is derived from the parental cell line (CHO -DUXBll) and recombinant cell line CHO3-3000-44 (CHO-4 4) was transfected with an expression vector and then derived from it. Ko The number control is a restriction enzyme-cleaved plasmid, and the insert amount is equal to that of the Chinese hamster. Kel was loaded to be equivalent to the displayed copy number per kenome of the diploid kenome. . Using a labeled HindIII fragment of λDNA as a molecular weight marker (MW) did. The size of the molecular weight markers (in kilobase pairs) is indicated on the right side of each panel. Each professional The inferred size of the HindIII fragment of the King genome that hybridizes with the Shown on the left side of each panel. (A) Type C related sequences represent pcH purified particle RNA. By hybridization with a partial cDNA clone of Oc, ~IL10 Detected (see text). Copy number control is EcoRI-digested pcHOc, M This is the L10 plasmid. pCHOC, ML10 clone used as probe A 0.6 kb genomic HindIII fragment was predicted from the sequence. (B) CHo TAP family 1r-related sequences are pcHIAP, YL9 cell cDNA clone. [Anderson, KP et al., Journal of Pyrology, Volume 64, Pages 2012-2032 (1990) Ko. Related sequences were observed in purified extracellular particles containing CHO (see Figure 3). copy - Number control is HindIII digested pcHIAP YL9 plasmid.

detailed description C2 retrovirus-like particles are not generally described in the literature cited above. It is a budding particle with retrovirus-like morphology and immunological properties. center Intracytoplasmic type A particles that often accompany corpuscles are essentially excluded from this definition. . As used herein, a type C retrovirus nucleic acid sequence encodes a particle as described above. It is defined as a sequence that can be multi-divided into possible cryptographic sequences.

As used herein, homology with respect to type C retrovirus sequences is defined as maximum percent homology. To arrive at Candidate sequences that are identical to residues in the sequences identified as Sequence No. 1 and SEQ ID No. 2 is defined as the percentage of residues in The homology for type C retrovirus particles is that is defined in the same way as for particles encoded by sequences such as . Candidate placement Preferably, if the sequence is a fragment of a particle encoded by SEQ ID NO. has at least ~75% homology with type C retroviral particles defined herein. However, it is not necessarily limited to that.

Included within the scope of the term type C retroviral particle as used herein are Particles with the amino acid sequence encoded by the number 1, glycosylated or glycated Derivatives of non-lycodylated particles, homologs of the sequence encoded by SEQ ID NO: 1 amino acid sequence variants and homologous in vitro generated SEQ ID NO. Variants and derivatives of the encoded amino acid sequence according to SEQ ID NO. It is capable of producing two particles that exhibit biological activity in common with the encrypted particles.

The biological activity of type C particles is similar to type C retrovirus encoded by SEQ ID NO: 1. Defined as immunological cross-reactivity with at least one of the epitopes of the particle.

As used herein, immunological cross-reactivity refers to polypeptides induced against known active analogs. Qualitative bioactivity between clonal antiserum and type C retrovirus-like particles with this activity This means that the candidate polypeptide can competitively inhibit the biological activity of the candidate polypeptide. Such resistance Serum can be used in goat or rabbit, e.g. with complete Freund's adjuvant preparation of known active analogues. Injected subcutaneously with an incomplete Freund's adjuvant, then intraperitoneally or subcutaneously with an incomplete Freund's adjuvant. immunization by the usual method of boosting with immunization.

A “foreign” element is one that is foreign to or homologous to the cell. means that the element is located at a location within the host cell where it is not normally found.

Provided herein are novel nucleic acid compositions. The present invention provides an endogenous C-type protein in CHO cells. The following DNA sequence (sequence) encoding the RNA A transcript of the torovirus-like particle Number 1) is included.

G^^TTCAATG　TCCTTGTGGG　G^^GAACGAT　ATT CTAAAACAGGTAACTGA 5 (IGCAATGTGAT GCGT GCGCCCGAGTCCAACGCATCCAGACTG AAGCTTCCCT C100CCGGGAACCG GTCAGAGGCT ACCGGCCCG AACACATTGG GAGATAGATT 150TCACTGAGAT TAAACCAGGA AAAATATGGAT ACAAGTATCT An AATTTT　200GTAGACACCT　TTTCAGGATG　GGT TGAAGCCTTCCCCTACTA AAACATGAAAC250AGCCA GATCG TTACTAAGAA ATTGC Ding Ding GAA GAAATCTT TCCCCGTTATCG 300GATGCCTCAG GTATTGGG^ ^CAGACAATGGCCCGCCTTCGTCTCCAGGT 350 AAGTCAGTCA GTGGCCACCT TATTGGGGAT TGA TTGG^^A TTACATTGTG 400CTTATAGACCCCAA AGTTCI~GGACAGGTAG AAAGGATGAA TAG^^CA ATC450, ^AGGAGACTT DingAAC, AAAATT GTCGCT TGCA ACTGGCACTA GAGAGCTGGG 500TCCTCC TACT　CCCCCTAGCA　CTCTACCCGCG　CTCGTAATA CCCCTGGACCA 550CATGGGCTCA CACCCTTTGA GATCCTGT, 4 guns GGAGTACCTA GCTCCTATCA 6 00 pieces ^^ CTTTCT TGATCAAGAT GTCTCAGTTTT TG CTAACTCCCCTTCTCTCC650 to 1GCTC, 8TTT AC AGGCCCTCCAAACTAGTAC^^CGGGAGGTCTGGAAA CCC700CTTGCTCAAG CTTATAAAAGA CCAGAGGG ACCATCCCCA　TCCCCCATTC750CTACC, ^GAT CGGGACACTG TTTGGGTCCG GC G CACCAG GC CAAGAACC800TTGAACCCCCG CTGGAAGGGA CCC TACATCG　TTTTGCTTACCACTCCCCACC850GCACT CAAGG TAGACGGCAT TGCAGCTTGG ATACATGC TT CACATGTAA＾900GCCAGCCCAA CCCACCGAT T CAGCCACTGCATCAGAATGG AGCCACACC949 pieces The invention further provides such NAs and NAs possessing hybridization specificity. and fragments of RNA sequences.

Includes novel nucleic acid compositions that are complementary (antisense) to the above sequences. D below The NA sequence (SEQ ID NO: 2) is complementary to SEQ ID NO: 1 and is endogenous in the CHO cells described above. The RNA transcript complementary to the RNA transcript of the C type retrovirus-like particle is coded G. GTGTGGCTCCATTCTGATG CAGTGGCTGA ATCGG TGGGT　TGGGCTGGCT　50 TACATGTGA　AGCATG TA DING CCAAGCTGCAA TGCCGTCTACCTTGAGTGCG 100GTGGGAGTGG TAAGCAAAACGATGTAGGGT C CCTTCCAGCGGGGTTCAAG 150GT Ding CTTGGCC Ding GG TGACGCCGGACCCAAACAGTGTCCCCG ATCTGGTA GG 200AATGGGGGAT GGTGGGATGG TCCCTCTG GT CTTTATAAGCTTGAGCAAGG 250GGTTTCCAG A CCTCCCGTTG TACTAGTTGG AGGGCCTGTA A ATGAGCTTG 300GAGAGAAGGG GAGTrAGCAA A ACTGAGACA TCTTGATCAA GAAAGTT^^T350GA TAGGAGCT AGGTACTCCA TACAGG person TCT CA^^G GGTGT GAGCCCATGT 400GGTCCAGGGG TATTA CGAGCGCGGTAGAGT GCTAGGGGGA GTAGGAGGA C450CCAGCTCTCT AGTGCCAGTT GCAAGCGACA ATTTTGTTAAAGTCTCCTTG 500ATTGTTCTAT TCATCCTTTCTACCTGTCCT GAACTTTGGG GTC CAATGTAAT TTCCAATCAA TCC to TATAAGC5501 CCAATAA GGTGGCCACT GACTGACTTA 600CCT GGAGACG　AAGGCGGGGCCCATTGTCTGT　TCCC^^T ACCTGAGGCATCC650GATAACGGGG AAAGATTTC T TCAAGCAAT Ding TC Ding TAGTAACGATCTGGCTG 70 0TTTCATGTTT AGTAGGGAAG GCTTCAAACCCATC CTGAAAA GGTGTCTACA 750＾AAAATTAATA GAT ACTTGτA TCCATATTTT CCTGGTTTAA TCTCAG TGAA　800ATCTATCTCCCAATGTGTTCCGGGCCGG TA　GCCTCTGACCGGTTCCCGGG　850＾GGAAGCTT CAGTCTGGATG　CGTTGACTCG　GGCGCACGCA　TC ACATTGCT　900CAGTTACCTG　TTTTAGAATA　TC GTTCTTCCCCCACAAGGACATTG^^TTC949 This complementary DNA and complementary RNA transcripts, and hybridization/ion specificity. Fragments of such complementary NA and RNA sequences having Included.

The present invention also provides amino acid sequence variations of type C particles encoded by SEQ ID NO: 1. Includes variants. The amino acid sequence variant of this particle is the particle for its ligand. Increased binding affinity, modified infectivity/transmissibility, increased stability, purification, and preparation of It is created with various purposes in mind, including:

Amino acid sequence variants of the retrovirus-like particles of the invention include insertions, substitutions, or deletions. They belong to one or more of the three classes of deletion mutants. Usually these mutations The body uses site-directed mutations of nucleotides in the DNA that encode the particles. , after obtaining the DNA encoding the mutant, the DNA is transformed in recombinant cell culture. Created by expressing. However, about 100 to 150 amino acid residues In the case of fragments having Amino acid sequence variants of type C retrovirus-like particles of It is a predicted variant not found in the gene. Variants are typically naturally occurring Exhibits the same qualitative biological activity (eg receptor binding) as the analogue. However, that too Even if the mutant or derivative is unable to bind to the receptor regardless of whether (a) C of the present invention (b) known as a diagnostic assay reagent for type retrovirus-like particles or their antibodies; If insolubilization cannot be achieved by this method, anti-retro (C) at least one type C retrovirus as a reagent for purifying viral antibodies; As long as the S-like epitope remains active, the As an immunogen to produce antibodies or as a component of an immunoassay kit (labeled and prepared) useful as a total reagent for natural particles or as a standard for unlabeled type C particle measurements). Ru.

The site for introducing a change in amino acid sequence can be predicted, but the mutation itself cannot be predicted. There's no need. For example, to optimize the execution of mutations at a particular site, you can or a saturation mutation (in which case all 20 possible residues are inserted). Targeting and testing of expressed particle variants for the optimal combination of desired activities Screen. Such screening is within the knowledge of one of ordinary skill in the art. be.

Amino acid insertions are usually carried out to the extent of about 1 to 10 amino acid residues. Replacement is standard Only one residue is introduced. Deletions are made in the range of approximately 1-30 residues. deletion or The insertion is preferably a deletion of a pair of adjacent residues, i.e. two residues, or a deletion of two residues. This is done by inserting a residue. This may include substitutions, deletions, insertions, or any The discussion below introduces or combines combinations of to arrive at the desired assembly. It should be clear enough.

Insertional amino acid sequence variants may contain one or more proteins that are foreign to type C retrovirus-like particles. The above amino acid residues are introduced into the planned site of the target particle, and the previously existing residues are It changes its position.

Insertion mutants generally involve adding a foreign protein to the amino or carboxyl terminus of the particle. protein or polypeptide fusion. Such mutants are called retrovirus-like and a sequence other than that normally found in the particle at the insertion site. It is called fusion with de. Several groups of fusions are included.

The novel polypeptides of the invention can be obtained in the C type by immunoaffinity techniques known per se. Useful for particle diagnosis or purification.

The preferred fusion of the type C particles of the present invention is that they have retroviral activity or not. However, it involves the fusion of a signal sequence and a Cheng Atsushi particle sequence that are heterologous to the binding partner.

Noval sequence fusions are used to produce more rapid secretion of particles. natural particles Once the signal is replaced by a heterologous signal and the resulting fusion is recognized (i.e., the host when processed and cleaved by cells), retrovirus-like particles are secreted be done. Signals are chosen based on the intended host cell, including bacterial, yeast, and mammalian cells. virus sequences, etc. Natural LHR nognal or herpes gD sugar Protein signals are suitable for use in complemented mammalian expression systems.

Substitution variants include at least one of the sequences encoded by the sequence SEQ ID NO: 1. A residue is removed and a different residue is inserted in its place. Such a placement Modifications are generally carried out according to conventional methods when it is desired to successfully modify the characteristics of a particle. It will be implemented.

Novel amino acid sequences, and isosteric analogues (amino acids or ) are also included within the scope of the present invention.

Substantial changes in function or immunological properties may occur in (a) If-transformed region polypeptides; (b) structure of the backbone (e.g. /-t or helix conformation), (b) (C) their effect on maintaining the bulk of the side chains; by choosing residue substitutions that differ significantly.

Certain deletions, insertions, and substitutions do not fundamentally change the characteristics of type C particles. Probably. However, it is difficult to predict the exact effect of a substitution, deletion, or insertion before implementing it. Even in cases where it is difficult to Of course, we can evaluate its effectiveness. For example, site-specific mutations in the nucleic acid encoding the particle Generate mutants by standard mutagenesis and express mutant nucleic acids in recombinant cell culture If desired, for example, a polyclonal anti-type C retrovirus column can be obtained from cell culture. by immunoaffinity adsorption (by at least one remaining immunoepitope) purify it (to adsorb the mutant). Then use a suitable screening assay. screen the activity of cell lysates or purified particle variants for desired characteristics. . Changes in particle traits, such as retroviral activity, can be detected by standard assays. Measure. Understanding the in vivo functions of the particles of the present invention-1 If so, other assays may be useful for such screening. oxidation reduction, or thermal stability, hydrophobicity, susceptibility to protein hydrolysis, or modification of protein properties, such as the tendency to aggregate into molecules or multimers, by methods known to those skilled in the art. Verify by.

Digested fragments obtained when elucidating the amino acid sequence of C-type particles and SEQ ID NO: 1 or or 2 with no more than ~75% sequence homology with any particle encoded by Protein fragments that have the following are excluded from the scope of deletion mutants.

Covalent modification of retrovirus-like particles is within the scope of this invention. like that The modification involves targeting the target amino acid residues of the recovered protein to the selected IIIJ chain or by reacting with an organic derivatizing reagent that can react with the terminal residues, or by reacting with a selected organic derivatizing reagent. can be introduced by utilizing post-translational modification mechanisms that can function in recombinant host cells. Ru. The resulting covalent derivatives can be used in immunoassays or in recombinant retrovirus-like particles. Identify residues important for biological activity for the generation of immunoaffinity-purified antiparticle antibodies. useful for planning. Such modifications are within the knowledge of those of ordinary skill in the art. , without unnecessary experimentation.

Nucleic acids encoding type C retrovirus-like particles of the present invention can be obtained by in vitro methods. or easily obtained from cDNA libraries. particle Manual operation or automatic bag of DNA encoding! Synthetic production by either Techniques well known to those of ordinary skill in the art, but especially practiced with reference to the teachings contained herein. It is possible. An example of the current state of technology related to polypeptide synthesis is Maniatis et al. [Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory (Molecul ar Cloning-, 4Labor-atory Manual, Co1 d.Sping Harbor Laboratory) (1984), and Horvath et al., An Automated DNA Synthesis. Sesizer Employing Deoxynew Freoside 3°-Phosphor Amidai (An Auto-rated DNA 5ynthesizer) Employing Deoxynucleoside 3-Phospho ramid amakashi sword j, me Sods in Enzymolog (11methods in Enzymolog) y), Vol. 154, pp. 313-326 (1987)].

As an alternative to obtaining the DNA encoding the particles from a source other than the hamster, C. The entire DNA sequence of a preferred embodiment of type retrovirus-like particle (SEQ ID NO: 1) is given below. Since the cDNA library of a particular animal contains homologous sequences, particles or fragments thereof (usually about 20 bp or longer, generally hybridization screen using labeled DNA encoding approximately 50 bp or more) cloned by restriction enzyme analysis and nucleic acid sequencing. All you need to do is analyze the clones and identify full-length clones. If the full length clone is not present in the library, suitable fragments are recovered from various clones and Full-length clones are assembled by ligation at common restriction sites between the pieces. other The DNA encoding retrovirus-like particles derived from animal species such as Probe libraries from species with hamster sequences or insert genes. ・Obtained by synthesis with hydro. Other retroviruses with known sequences The DNA of rus-like particles can be obtained using similar routine hybridization techniques. available. In the present invention, SEQ ID NO. A nucleic acid sequence is provided that hybridizes to a fragment of a DNA sequence. The fragment is approx. Greater than 10 bp, preferably 20 to 50 bp, greater than 100 bp Even. Also hybridized under stringent conditions to fragments of SEQ ID NO: 1 or SEQ ID NO: 2. nucleic acid sequences that form, and such hybridization reactions to these sequences. Nucleic acid sequences that retain specificity are also within the scope of the present invention.

Also, for DNA of SEQ ID NO: 1 or 2, or type C retrovirus-like particles. the genome gene Σisidron, and the 5゛ or 3゜fra spreading to neighboring genes. 2 (i.e. approximately 5000 bp, in any case larger) Also included within the scope of the invention are nucleic acid probes that can hybridize under stringent conditions to Ru.

Identification of the chemome DNA of the six-tipped particles can be achieved by labeling them with a detectable group (e.g. radioactive phosphorus). Probe a specific genome library with the selected cDNA or fragment thereof to It can be carried out in an open manner to recover clones containing the . Niuo if necessary - Piecing together the complete gene by Kingko. Typically such a professional The particles do not encode sequences that are less than 75% homologous to the particles of the invention. , their length ranges from 10 to 100 bp. Other retrovirus-like particles Homology with and size can be determined without unnecessary experimentation.

Hyprint DNA technology can be used to obtain expression. DNA sequence restriction map to determine the suitable site for cutting. This method cuts the array and creates a suitable It is introduced into a vector that has control, transcriptional regulatory regions, and regulatory signals. DNA sequence Once the insects are obtained, the particles can be purified and antibodies can be made against them.

Cloning of DNA sequences for prokaryotes in general to construct vectors useful in the present invention used for. For example, E. coli (E. coli) K 12 strains 294 ( , 8 TCC No. 31446) are particularly useful. Other microbial strains that can be used include Cherichia coli B and Enerychia coli X1776 (ATCC No. 3 1537) etc. These examples are for illustrative purposes only and only It is not limited to. Alternatively, methods such as polymerase chain reaction In vitro cloning methods are preferred.

Polypeptides of the invention can be directly synthesized as N-terminal methionyl analogs in recombinant cell culture. or fusion with a heterologous polypeptide, preferably the N- with other polypeptides that have knobal sequences or specific cleavage sites at their ends. Expressed as a fusion. Assemble prokaryotic secretion expression vectors for e.g. particles In some cases, a natural C-type particle signal is used in conjunction with a host that recognizes the signal. . When the secretion 1 node is recognized by the host J2, the host signal peptid A enzyme cleaves the C-terminally fused leader polypeptide to produce the desired mature protein. Torovirus-like particles can be obtained. Producing retrovirus-like particle signals In host prokaryotes that do not process the signal, e.g. alkaline phosphatase , peptidases, PP or thermostable enterotokin II leaders. Replace with a prokaryotic signal selected from In yeast secretion, natural signals are The mother invertase, alpha factor or acid phosphatase leader may be substituted. Mammal For expression in mammalian cells, other mammalian secreted protein signals are also suitable; , natural signals are sufficient, and as virus secretion leaders, e.g. The pesgD signal is preferred.

The novel particles of the invention can be expressed in any host, but are preferably synthesized in a mammalian host. do. However, host cells such as prokaryotes, fungi, yeast, insects, etc. may also be used for expression.

Representative prokaryotes include strains suitable for cloning, and E. coli W31. 10 (F-・λ-prototrophic strain, ATCCN0.27325), others, Serratia - Intestinal microorganisms such as Serratia marcescens Fungi, bacilli, and various Pseudomonas species. Preferably the host cell is Minimal amounts of proteolytic enzymes should be secreted.

The expression host is labeled with DNA encoding the hybrid ligated into the expression vector. quasi-transformed. Such vectors typically possess a replication site (this is not necessary if chromosomal integration is occurring). Invention vector can provide phenotypic selection in transformed cells, as described in more detail below. Contains a marker sequence that can be used. For example, Enerychia coli is typically Plasmid pBR322 derived from Lychia coli sp. [Bolivar ) et al., Gene, vol. 2, p. 95 (1977)]. do. pBR322 contains ampinoline and tetracycline resistance genes. and therefore, for either cloning or expression purposes, transformed cells provides an easy means to identify. Expression vectors are also used for transcription and translation. Sequences that are useful for regulation, such as promoters and Nyain-Dalgarno sequences ( (for prokaryotes), or promoters and enhancers (lli for mammalian cells) ).

The promoter may be, but need not be, inducible. expression It is assumed that the vector does not require any expression control, replication sequences, or selection genes. but their absence makes it difficult to identify transformants and achieve high levels of particle expression. development may be hindered.

Examples of promoters suitable for use in prokaryotes include the β-lactamer Ze and lactose promoter system [Chang et al., Fuji Char, vol. 275, 615 (19784) and Goeddel et al., Nature, vol. 281. , p. 544 (1979)], alkaline bosph 7tase, tryptophan (t rp) Promoter system E Keddel, 1-Nucreinota Antz's Reser Nuc^cids Res, vol. 8, p. 4057 (1980) and European Patent Application No. 36776], a hive such as the tac promoter Lid Promoter [H, de Boer et al., Bronodings ・Of the National Academy of Sciences of the USA (Proc, Natl, Acad, Sci, LIS^), Volume 80, 21 ~25 pages (1983) Second prize. However, other functional bacterial promoters - is also suitable. Their nucleotide sequences are generally known and those skilled in the art This allows you to use a linker or adapter to create an LHR library (Si ebenlist et al., Cell, vol. 20, p. 269 (1980)] Functionally link them to the DNA encoding them and add any necessary controls. A restriction site can be provided. In addition, the promoter used in bacterial systems is a C-type promoter. Functionally linked to DNA encoding torovirus-like particles / Yain Dar Contains the Garnot (S, D,) sequence.

Not only prokaryotes but also eukaryotic microorganisms such as yeast or filamentous fungi are desirable. many Although other strains of Saccharomyces cerevisiae are widely available, aro-myces cerevisiae) is the most widely used eukaryotic microorganism It is. Plasmid YRp7 is a good expression vector in yeast [Stinkcomb (Stinchcomb) La, Fujicher, vol. 282, p. 39 (1979) , Kingsman et al., Gene, vol. 7, p. 141 (1979) ), Tschemper et al., Scene, vol. 10, p. 157 (198 0 years)]. This plastic is for example ATCC No. 44076 or PEP4 1C Jones, GeneticS, 8 Vol. 5, p. 12 (1977) It already contains the trpl gene, which provides a selectable marker for yeast mutants. fermentation The presence of trpl deficiency as a feature of the mother host cell genome results in the presence of tryptophan. Provides an effective environment for detecting transformation in the absence of growth.

Facilitating sequences suitable for use in yeast hosts include 3-phosphoglycerate kinase [Hitzeman et al., Journal of Biological Sciences Chemistry (J, Biol, Chem), vol. 255, p. 2073 (19 1980), or enolase, glyceraldehyde-3-phosphate dehydrogena -ase, hexokinase, pyruvate decarboxylase, phosphofructokiner ze, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triose phosphate isomerase, phosphoglucose isomer and other glycolytic enzymes such as glucokinase [Hess Advances in Enzyme Regulation (J, Adv, E r+zyme Reg, ), vol. 7, p. 149 (1968), Holland (11 o11and), Biochemistry, Volume 17, 4,900 pages (1978)].

Inducible promoters with the added benefit of transcription controlled by growth conditions Other yeast promoters that are routers include alcohol dehydrogenase 2, socytochrome C1 acidic phosph-7tase, a degrading enzyme involved in nitrogen metabolism, metalloti onein, glyceraldehyde-3-phosphate dehydrogenase, and maltose This is the promoter region of enzymes involved in the utilization of sugar and caractose. Further R (European Patent Publication No. 73657) used in yeast expression. We report vectors and promoters suitable for this purpose.

Expression control sequences are also known for eukaryotes. Virtually all eukaryotes The gene is located approximately 25-30 bases upstream from the transcription start site. territory rich in children It has a range of areas. Found 70 to 80 bases upstream from the transcription start site of many genes. Another sequence is the CXCAAT region (X is any nucleotide). Big The 3' end of a partial eukaryotic gene is an additional polyA tail at the 3° end of the coding sequence. AAT is an AAA sequence that can be a signal. All of these sequences are mammalian inserted into a product expression vector.

Promoters suitable for controlling transcription from the vector in mammalian host cells include: For example, Po11oma virus, SV40, Azone virus, M~iV (St. ), retroviruses (e.g. HIV LTR), type B various sources such as hepatitis viruses, most preferably +-itomechaloviruses. or a heterologous mammalian promoter (e.g. β-actin promoter). It is easily obtained from The early and late promoters of SV40 are Conveniently included as an SV40 restricted piece that also contains the replication start point of the virus. Fiers et al., Fujicher, vol. 273, p. 113 (1) 978);・. The immediate promoter of the human cytomechanical seven viruses is Hi Greena i-way, conveniently obtained as a dIHE restriction fragment. way), PJ et al., Scene, vol. 18, pp. 355-360 (3°, 1982) Transfer of DNA encoding the retrovirus-like particles of the present invention by higher eukaryotes Transcription is enhanced by inserting enhancer sequences into the vector. Enhan A promoter is a cis-acting element of DNA that acts on a promoter to enhance its transcription. usually about 10 to 300 bp). Enhancers can be direction and position dependent. 5′ [Laimins, L , Producings of the Academy of Science Sciences of the USA, vol. 78, p. 993 (1981) Ko and 3' Lo. Lusky, M. L. et al., Molecular and Cellular Bi. Ology (Mo1. Ce1l Bio,), vol. 3, p. 1108 (1983) ], an intron [Banerji, JL.

Cell, vol. 33, p. 729 (1983)] and the code sequence itself [Oz. Os-borne, T. F., et al., Molecular and Cellular ・Biology, Volume 4, Page 1293 (1984)]. Nowadays , numerous enhancer sequences are present in mammalian genes (globin, elastase, albumen). (alpha-fetoprotein, insulin). However, as standard uses enhancers from eukaryotic viruses. Examples of these include replication development. SV40 enhancer on the late side of the starting point (100-270 bp), Cytomega poliovirus early promoter enhancer-1 on the late side of the origin of replication. Examples include maenhancer-1 and adenovirus enhancer.

Also eukaryotic host cells (yeast, fungi, insects, plants, animals, humans, or other The expression vector used in cells with nuclei derived from cell-based organisms does not affect mRNA expression. Contains sequences necessary for transcription termination that can have an effect. These areas encrypt the particles transcribed as a polyadenylation segment into the untranslated part of the mRNA be done. The 3° untranslated region also contains a transcription termination site.

Expression vectors may contain a selection gene, also called a selection marker. mammalian cells Examples of suitable selectable markers are dihydrofolate reductase (DHFR), demino Kinase (TK), or neomycin. such a selection marker Upon successful transfer into a mammalian host cell, the transformed mammalian host cell becomes selective. They can survive even when placed under selective pressure. Two different categories that are widely used There is a method for selecting Golly. The first category is based on cellular metabolism. For this purpose, mutant strains lacking the ability to grow independently of supplemented media are used. two examples CHODHFR-cells and mouse LTK-cells are shown. in these cells lacks the ability to grow without the addition of nutrients such as thymidine or hyboxanthin.

These cells lack specific genes required for the complete nucleotide synthesis pathway Therefore, they cannot survive unless the lost nucleotides are replenished into the supplemented medium. supplement the medium An alternative approach is to add intact DHFR or T to cells lacking the corresponding gene. This method changes the requirements for proliferation by introducing the K gene. D Individual cells that were not transformed with HFR or TK genes were Can't survive.

The second category of selection methods is the preference method that describes the selection method used for any given cell type. It is a sexual selection method and does not require the use of mutant cell lines. In this method, the standard Use drugs that prevent host cells from multiplying. Cells successfully transformed with a heterologous gene The cells express proteins that confer drug resistance and therefore survive the selection conditions. I can stay. An example of such dominant selection is neomycin [Southern n) et al., Journal of Molecular and Applied Genetics Kuss (J.

Molec, Appl, Genet, vol. 1, p. 327 (1982)] , mycophenolic acid [Mulligan et al., Science, 209 Vol., p. 1422 (1980)] or Sugd. en) et al., Molecular and Cellular Biology, vol. 5, 410-4. p. 13 (1985) using drugs such as j.

In the three examples above, the preferred drug G418 or neomycin xgpt (mycophenolic acid), or hygromylene. Using bacterial genes under the control of eukaryotic cells to transmit.

Suitable eukaryotic host cells for expressing type C retrovirus-like particles include SV40 Transformed monkey kidney CV l line (CO5-7, ATCCCRL1651), human embryonic kidney system (293 cells or 293 cells subcloned for suspension culture growth) ) F Graham, FL, et al., Journal of General, Pyrology 0. GenJirol, ), volume 36, page 59 (1977)], Litter/% Muster kidney cells (BHK, ATCCCCCLIO), Chinese ham Star Ovarian Cells - DHFR (CHO) Urlaub and Cha Chasin, Boundaries of the National Academy - of Sciences of the USA, vol. 77, p. 4216 (1980) ), Mouse Sertoli cells (TM4) [Mather, JPl, Biology of Reproduction (BiolRep-rod.), Volume 23 , pp. 243-251 (1980)], monkey kidney cells (CVIATCCCCL70 ), African green monkey kidney cells (VERO-76, ATCCCRL-1587 ), human cervical cancer cells (HELA, ATCCCCL2), canine kidney cells (MDCK SATCCCCL34), buffalo rat hepatocytes (BRL3A, ATCC CRL1442), human lung cells (W138, ATCCCCL75), human liver cells cell (HepG2, HB8065), mouse breast cancer (MMT O60562, AT CCCCL51) and TRI cells [Mather, J., P. et al. Anna of the New York Academy of Sciences ls N, Y, Acad, Sci,), vol. 383, pp. 44-68 (1982 2003) Ko et al.

To construct a suitable vector containing the desired coding and control sequences, standard Use standard ligation techniques. Cut the isolated plasmid or DNA fragment. It can be cut, processed, and reconstituted in the desired format to create the necessary plasmids. Turn on.

For analysis to confirm the correct sequence in the assembled plasmid, mix The product was used to transform Escherichia coli 12 strain 294 (ATCC 31446). and where appropriate, treat successful transformants with ampicillin or tetracycline. Select based on resistance to stress. Plasmids were prepared from transformants, restriction analyzed, and modeled. and/or the method of Messing et al. [Nuclay 1. Ri・a ,.

Nucleic: ~cids Res, Volume 9, Page 309 (1981) or the method of Maxaw et al. 65, p. 499 (1980).

W=transform cells with the expression vector of the present invention, and induce and transform the promoter. Modifications suitable for selection of transformants or amplification of genes encoding desired sequences. Culture in normal nutrient medium decorated with

Host cells used to practice the invention may be cultured in a variety of media. commercially Available media such as Ham's Flo (Sigma), Minimum Essential Medium (MEM, Sigma), RP~ll-1640 (Sigma), and Da Lubetzko's modified Eagle's medium (1) ME, Sigma) is suitable for culturing host cells. It is. Norm and Wallace / Morology, vol. 58, p. 44 (1979) Koh, Barnes, et al. Sato E Analyte I Cal/<Iochemistry<Anal , Biochem, ), vol. 102, p. 255 (1980): U.S. Patent No. 4 No. 76704, No. 4657866, WO90103430, WO3710 No. 0195, U.S. Patent Re, 30985, U.S. Patent No. 4,927,762, and 45606.55 is used as a culture medium for host cells. It can be used as If necessary, add hormones and 2' or is other growth factors (e.g. insulin, transferrin, epidermal growth factor, etc.) , salts (e.g. sodium chloride, calcium, magne/rum, phosphates, etc.), buffer solution (e.g. HEPES, etc.), nucleotides (e.g. adenonone, thymidine, etc.) , antibiotics (e.g. gentamicin, etc.), trace elements (the final dose is usually (as a range of inorganic compounds), and glucose or its corresponding energy – Replenish sources. Other duties! The necessary supplements may be added at suitable concentrations.

Culture conditions such as temperature, pH, etc., are those previously used in the host cell chosen for expression. It is obvious to those of ordinary skill in the art.

Host cells as used herein include cells in in vitro culture, and cells in host animals. Includes cells in the body.

"Transformation" refers to the use of DNA as an extrachromosomal element or by chromosomal integration. Either means introducing DNA into an organism so that it can be replicated. Other theories Unless otherwise specified, the methods used herein for the transformation of host cells are raham), F., and van der Eb1. Eight [Vi-rol.

gy), Vol. 52, pp. 456-457 (1973). However, nuclear Other methods of introducing NA into cells such as injection or protoplast fusion can also be used. Because it uses prokaryotic cells or cells that contain a strong cell wall structure If so, the preferred method of transfection is as described by Cohen, FN ``Bronde'' is a calcium treatment method using calcium chloride reported by , et al. ings of the national academy of sciences ・USA.

69, p. 2110 (1972)].

``Transfection'' refers to transfection, whether or not the coding sequence is ultimately expressed. It refers to the introduction of DNA into host cells. For example Ca PO, method and electroporation Many methods of transfection are known to those of ordinary skill in the art, such as the present invention. inn Transformation of the principal cells is the result of successful transfection.

rPcRJ (polymerase chain reaction) is a technique for amplifying DNA pieces. increase 3° and 5' ends of the A segment (sense strand or antisense strand) The oligonucleotide primer corresponding to the strand-check) was hybridized under suitable conditions. bridging and using Taq polymerase enzyme or equivalent enzyme, A copy of the DNA, A, sandwiched between both primers is synthesized.

Furthermore, the particles of the invention can be produced by homologous recombinant methods or by introduced into cells that already contain DNA encoding the desired product It is anticipated that recombinant production methods that utilize regulatory elements will allow for the production of live harps. example strong promoter/enhancer elements, suppressors, or exogenous transcription The regulatory element can be used to sufficiently influence the transcription of the DNA encoding the desired particle. into the genome of the intended host cell in a contiguous position and orientation. This element is a book Although not encoding the particles of the invention, the DNA is present in the genome of the host cell. next Screening cells that produce the particles of the invention or, if desired, expression levels. increase or decrease.

Novel polypeptides can be synthesized by ammonium sulfate or ethanol precipitation, acid extraction, anionic ion or cation exchange chromatography, phosphocellulose chromatography -, immunoaffinity chromatography, hydroxyapatite chromatography and recombinant cell culture by known methods such as lectin chromatography. collected and purified.

Gene amplification and/or expression can be performed based on the sequence provided, e.g. Conventional Southern blotting or dot plot using probes (DNA Both can be measured directly in the sample by analyte analysis). A variety of labels can be used, but most Generally radionuclides are used, especially 32P. However, biotin-modified nuclei Other techniques, such as those used to introduce otides into polypeptides, can also be used. .

The biotin can then serve as a binding site for avidin or antibodies. , which can be labeled with various labels such as radionuclides, fluorescent agents, or enzymes, etc. Ru.

Alternatively, DNA double helix, RNA double helix, and DNA-RN Special features including A-hybrid double helix or DNA-protein double helix, etc. Antibodies that can recognize different double helices may be used. Furthermore, the antibody is labeled and double-labeled. If the helix is bonded to the surface, the double helix will form during the formation of the double helix on the surface. Assays can be performed to detect the presence of antibodies bound to.

Alternatively, a Northern blot [Thomas mas), Pronouders of the National Academy of Sciences Sciences of the U.S.A., vol. 77, pp. 5201-5205 (1980 year)], dot plot, in situ hybridization, or tissue section immunohistochemical staining, and cell culture or cell culture to directly quantify gene product expression. Gene expression can be measured by any immunological method, such as measurement of body fluids.

Immunohistochemical staining techniques typically prepare cell samples by dehydration and fixation. , and then reacted with a labeled antibody specific for the bound gene product. That sign is are usually visually detectable, such as enzyme labels, fluorescent labels, luminescent labels, etc. . A particularly sensitive staining technique suitable for use in the present invention is Hsu et al. American Journal of Clinical Pathology (Am, J, C11 Path, Vol. 75, pp. 734-738 (1980)]. It is.

Antibodies useful for immunohistochemical staining and/or assay of sample fluids are monoclonal. , or polyclonal. For convenience, the DNA provided herein Antibodies can be generated against synthetic peptides based on their sequences. Then such a synthesis Peptides can be used as immunogens for the generation of antibodies by well-known techniques. alternatively Natural gene products and/or parts thereof can be isolated and used as immunogens .

In one embodiment of the present invention, by introducing an antisense or ribozyme gene, or screening and selection techniques to identify endogenous type C retrovirus-like sequences. Cell lines with low levels of expression are isolated. The CHO cell line is suitable for the application of these methods. Although particularly preferred, these methods are routinely used in any of the host cells mentioned above. .

Cell lines with low or undetectable expression of endogenous type C retrovirus genes Two methods for isolating are described. The first method is to use endogenous type C retrovirus genes. To specifically inhibit gene expression, antisense RNA gene or lipozyme It consists of introducing the gene into the parental cell line. Alternatively, screening and Low expressing subclones are isolated using selection and selection techniques. Both of these methods are original and will be explained in more detail below.

The nucleic acid sequences of the invention can be used to target specific antisense RNA genes or lipozyme genes. By being able to introduce cells that express small amounts of C-type particles, it is possible to introduce cell lines that express small amounts of C-type particles using conventional methods. make it possible to do it by hand. Inhibition of specific gene products using antisense technology The harm is complementary (antisense) to the metsenger RNA of the gene product in question. Consists of introducing a gene into a target cell that encodes a certain RNA transcript . To the antisense R in the cell, base pairs with the possibly complementary mRN, A. , thereby inhibiting the translation or prosenoning of menseno. Final The result is a reduction in the expression of specific gene products. antisense RNA High levels of expression are achieved using current technology. Science, Vol. 243, pp. 1354-1356 (1989), Koka et al., Science , vol. 243, pp. 947-950 (1989), Izant et al., Science, 2 29, pp. 345-352 (1985), Fono Ruten et al., Journal. of Pyroronau, Vol. 63, pp. 677-682 (1989iE), Chan et al. Journal of Hiroconau, 61 sounds, pp. 921-924 (1987) L iIk optimization of the expression level of antisense sequences is within the routine skill of those skilled in the art; Level of expression of "sense" sequences in target cells and potential production of transformed cells This is done by taking into account the possible opposite results. For example, nucleic acid plot analysis can be performed on CHO cells. Several hundred copies of type C-related DNA sequences and moderately abundant type C RNA transcripts within Therefore, in order for this method to be effective, Jun Takamizu's development of antisense RNA is necessary. need the present.

Alternatively, it is complementary to the Messenzya-RNA of type C retrovirus-like particles. RNA can be introduced into a host cell to prevent its translation.

An alternative method to introducing genes into antisense RN is the use of ribozymes. Ru. As mentioned above, lyphozymes are small molecules that can cleave substrate RNA in a sequence-specific manner. type of RNA molecule. A particularly preferred lipozyme for carrying out the invention is the goby This is the ``hammer to 2 de'' rehozyme reported by Roff et al. [Nature, 334 Vol., pp. 585-591 (1988)]. In this cited document, in hydro Discloses the use of Har/Marhead ribozyme to cleave mRNA at different sites are doing.

In the case of these hammer henodriphocymes, the target sequence is the GUA or or GUC sequence. Cleavage specificity of the cleavage enzyme Determined by the flanking sequences that hybridize to the sequence. That is, G.U.A. or any 18 bp RNA sequence adjacent to the GUC triplet. refozymes can be manipulated to produce highly precise specificity. I can do it. Alternatively, as reported by Koizumi et al. 2FE B where an RNA enzyme having bonuclease activity can be used to carry out the present invention S. Letters, Vol. 239, pp. 285-288 (1988).

Using the sequence of the present invention, specificity is obtained for several regions encoding type C mRNA. Antisense or ribozyme genes can be assembled and transfected with standard transfection. into host cells using the John's protocol. Through subsequent genetic manipulation, these recombinant clones using unique selectable markers to ensure uninterrupted cell availability. A parental cell line suitable for assembly of the cell lines is isolated. Facilitate isolation of clones if desired Antisense or ribozyme genes can be amplified using the following amplification methods. is desirable.

Cell lines are then evaluated for expression of endogenous type C products. Detection of expression is structural Immunoassay of proteins, enzymatic assay of reverse transcriptase activity, plotting of RNA transcripts analysis and by examining cell sections with an electron microscope for the presence of type C particles. I can give.

In a separate tube, the routine screening described above could be used to identify low-expressing factor subclones. isolate. For example, using probes containing fragments of the sequences claimed herein. By pronto analysis, subclones of parental cells were determined for expression of C-type RNA sequences. Evaluate. Until the RNA level becomes undetectable or no longer decreases. Additional low-expressing subclones are screened until various tran The expression level of C-type mRNA, A, can vary markedly in transfected cell lines; It can be expected that changes may occur even in subclones that do not act.

The present invention also includes more effective selection means. Provided herein as a probe Using the obtained cDNA, the gene encoding the coat protein of type C particles was detected. Isolate from cDNA or genomic libraries. Then to the envelope encryption array, Link current vectors directly at the DNA level and with partial overlap at the mRNA level do. Generate protein assemblies after providing sequence from a substantial open reading frame and expressed in a suitable recombinant host such as E. coli according to known techniques. do. Anti-coat protein antibodies are then generated by immunization with the protein. let

Because retroviral coat proteins are expressed on the surface of cells that secrete C-type particles, A number of direct selection methods using type C coated antibodies can be used. 1 implementation In embodiments, the host cells are incubated in the presence of anti-coat protein antibodies and complement. to Antibodies and complement lyse expressing cell lines, leaving only non-expressing cell lines viable. remains. If there are no non-expressing cells in the initial cell population, select low expressing cells. Routine experimentation with multiple antibody concentrations may be necessary to determine.

A second approach, using a fluorescence-activated cell sorter (F, AC5), Gives flexibility in cell selection. Anti-coat-secondary antibody and fluorescein-conjugated secondary Stain the host cells using the following antibodies: High expressing cells show high fluorescence, low expressing cells Cells show low fluorescence. F　A　CS shows individual cells according to fluorescence density. By performing physical sorting, a collection of cells that express C-type coat protein at low levels can be obtained. Enrich the group. Selection operation is improved by repeating FAC5 cell sorting many times. do.

Once a low-expressing cell population is obtained, subclones are expanded and screened as described above. More rigorous analysis of C-type expression is performed as outlined in the protocol.

The nucleic acid sequence encoding the retrovirus-like particle of the invention is preferably one copy. One or more cells are integrated into the host cell.

With reference to the teachings contained herein, the teachings of the present invention can be applied to a specific problem or situation. It is within the ability of those of ordinary skill in the art to apply the invention to Below is the actual Representative Examples of Products of the Invention and Their Isolation, Use, and Manufacture A specific example of this method is shown below. The examples are for illustrating the invention and are intended to explain the invention. It is not intended to limit the scope.

~212 pages (1986) Ko. This subclone is derived from Fuzumi dhfr and human Recombinant coat glycoprotein (gpl, 2) of human immunodeficiency virus type 1 (HIV-1) 0) After transfection with the expression vector containing the gene, dihydrofolate [Rubini] derived from CHO-DUXB11 cells lacking reductase (dhfr) Ecke, A., S. et al., Developments in Biological Standards. Heine, Vol. 70, pp. 187-191 (1989) , U, I, et al., Journal of General High Surgery, Vol. 44, 45- 55 (1979)], CHO-Kl cell line (CHO-DUXBl 1 line) progenitor cells) from Chinese hamsters [Cricellus griseus (Cricellus griseus)]. tulu griseus)] was first induced by the ovarian bioboom of adult animals. [Liber 1M, M, et al., Science, vol. 182, 56-5] 8 pages (1973)], American Type Culture ° Collection (Am Obtained from erican Type Curture Co11ection) (ATCCCCCL61). Similarly, mouse myeloma cell line X63-Ag3.653 [Rubiniecki, A.S., and May, L.H., Development Manager Twin Biological Standards/Yon, Vol. 60, pp. 141-146. (1985) Co, and the human diploid fibroblast cell line MRC5 [Hodgman, F., et al. , Developments in Biological Standardization, Volume 70, Pages 195-202 (1989), ATCCCL171 also included from ATCC. I got it.

The cell culture medium used to prepare centrifuged cell culture concentrates/particles was manufactured under serum-free manufacturing conditions. obtained from cells grown in Typically 20-30 liters of clarified cell culture medium A high-performance continuous flow ultracentrifuge rotor (Beckman CF 3) Type 2) and centrifuged at 1,100,000x. Pelletized particles were treated with 150mM Na In 10mM Tris (pH 7,5) containing Cl, 0.1% BSA (TN-BSA) It was recovered by scraping it from the rotor wall. The collected particles are then transferred to Beckma. Pellet by centrifuging for 90 minutes at 1100000x in a Ti450-tar, and - Redissolved in BSA in a final volume of 4-5 ml and stored at -80°C. This operation As a result, particles present in the starting cell culture solution could be concentrated 4,000 to 6,000 times.

Reverse transcriptase (RT') assay Reverse transcriptase assay requires 0.05ml of sample per assay. The test was carried out using a total volume Q of 1 ml. Immediately before incubation, sample RT activity was solubilized by treatment with 02% Triton-X100 for 30 minutes on ice, and 37 The assay was performed by incubation at °C for 1 hour. In the presence of 02ff1M manganese Insertion of 13H Co-TTP into DNA Using Re(rA) Oligo(dT) Template , monitored by counting the radioactivity bound to DE81 filters. . As a control for the presence of nonspecific polymerase activity, poly(dA)・oligo(d T) Polymerase activity was monitored in the presence of template. Regarding Wakasen's samples, please refer to Ma. RT activity was compared in the presence of 51DM magnesium. Also sample 1 Using 5:1 with a total volume of 65:l and using:3:p3~TTP as the label. A modification test was conducted in Wakachi's experiment. Sucrose interchamber gradient fractionation to purify particles To do this, we used the double sucrose gradient method. ,j! Pour the compressed culture solution through a solid sieve. X[L, 40%, 30 %, 20% sucrose in TN buffer (101nii Tris-HCl (pH 7, 5) Add an equal volume of 150 ml of NaCl solution to 5 W, 270-liter After centrifugation at 25K rpm overnight, the R-containing peak fractions were pooled and added to TN buffer. 10-60% (w/’w) sucrose dialyzed against and prepared in TN buffer 39Krp from c5W410-ter layered at the top of the linear continuous density gradient of Centrifugation of the sucrose-rich gradient fraction for 2 hours at m to hand the particles at a suitable density. Sample (10-5 (7 L) was dialyzed against TN buffer and placed on the lid. . After the particles have settled for at least 30 minutes, they are immobilized using uranyl acetate. The cells were stained and visualized using a transmission electron microscope.

Type C immunoreactive soil seven core polypeptides using protein plot analysis A protein W sample was separated using a reduced base 5DS-polyacrylamide gel, and then electrolyzed. Dynamically to Inmobilon filter (Phar++acig) I moved. The primary antiserum (Yagami anti-feline leukemia virus p27 serum) was diluted 1400. used.

Secondary detection antibody (Donkey anti-Yaki IgG conjugated to horseradish Belox/dase) was used at a dilution of 1:1000. 4-chloro-1-naphthol/hydrogen peroxide was used as a substrate to generate a signal.

Characterization of nucleic acids present in retrovirus-like particles and phenol-chloroform extraction Nucleic acids were extracted from the sucrose density gradient fraction by extraction. slot plot ma Schleich er and Schuell m1ni-fold)). Run (Genescreen plus), NEN Research Products (■1~Re5search Products)) Exhibition I made him wear it. 0.5M NaC], O, IM HEPES (PH7,2), 5 m1iEDTA, 0.1% Atrium Pyrotnonate, 5X Denhardt's Solution, 10 0 μg/ml denatured salmon sperm DNA, and 50% formamide (high stringency 4 in a buffer containing either 25% formamide (low stringency) or 25% formamide (low stringency) Hybridization was performed at 2°C. Endogenous polytrophic murine leukemia cormorant Irus (MLV) Arashi 11tI body Mx27I Stoy! (Stoye), JP4, Nyanar of High Crossy, vol. 61, pp. 2659-2669 (1987 ) cloned probe for J, M Coffin and Provided by J, P Stoini. Endogenous CHO intracapsular one-molecule family I pCHIAP, SW2 for family II, pcHIAP, Y L9) probe is described in Ike (Andermen, 1990, supra), The probe for mouse β-actin was prepared using the published sequence [Tokugawa ), K. New York Un's Research, Vol. 14, pp. 28-29 (19 (1986) synthesized based on 2 and containing a complementary sequence at its 3” end. It consisted of a 45-mer oligonucleotide. All probes are Klenow fragment extension or plasmid probing to duplicate cleotide probes α-[32P3-dCTP was radioactively labeled by insertion of

Isolation and characterization of type C cDNA clones. Type C-related sequences were isolated from a randomly primed cDNA library. ML~ ' - Using a low stringency hybridization tube using the MX27 probe, A plaque was identified. DNA sequencing was performed on subcloned single-stranded templates. 7 methods of dideoxy chain termination ethod), Sanger, F. Bronodings of the ・National Academy of Sciences of the USA, Volume 74, 3463-3467 (1977)].

Chinese hamster I) NA genome DNA pro, throat standard method Genomic DNA was prepared from CHO cells using [T Maniathes et al., 198 9th year, Molecular Cloning, A Laboratory Manual, Cold ・Suji 1 Lulu Harbor Lahoratory, Cold Sbringle/)-t<- 1 or Y, )]. High molecular weight Chichinese hamster liver DN, K, Luda Kindly provided by Lueders et al. Restriction enzyme HindIII DN A was completely digested, and 2 μg of the sample was electrolyzed on a 07% agarose scale It was fractionated by pneumophoresis. Nylon membrane charged with DNA (Scenesc) Lean Plus, NEN Research Products) using a capillary tube, and then Hybridize using the conditions and probes shown in the slot plot. We conducted a training session.

Spirit! : Purification of RT-containing particles from CHO cell culture: extracellular retrovirus removal from CHO cells. We devised a protocol to purify Rus-like particles. The method involves using a large volume of culture solution. The heart concentrates and consists of two consecutive fractionations on a sucrose density gradient (methods and (Refer to Materials section) Cell culture medium concentrated 4000 times by C ultracentrifugation was First fractionated by centrifugation into a sucrose step gradient (equilibrated with Fractions containing peak reverse transcriptase (RT) at 1.12-1.14 g/ml pool and dialyze the top of a sequential 10-60% pre-sucrose density gradient. It was layered on the edge and centrifuged to reach an equilibrium. Standard purification protocol The RT patterns of the first and second gradients are shown in FIG. Background in adjacent fractions A single peak of RT activity was observed in the second sucrose gradient with only 2nd activity. Ru. Fractions containing the RT peak of the second density gradient are collected for subsequent characterization. I did a rule. The RT activity of the purified Launoyer Fuzumi leukemia virus was expressed on the same gradient. and were banded with similar density (results not shown).

Polymerase activity is higher in the presence of poly(rA) template compared to poly(dA) template. The polymerase activity of the peak fraction represents reverse transcriptase. It was confirmed that there was. Also, it is more active in the presence of manganese than magnesium. This superiority indicates that this particle is related to mammalian type C retroviruses. (The results are not shown).

Electron microscopy (EM) evaluation of particles purified from CH03-3000-44 cells: A sample of the fraction containing RT was examined by electron microscopy to detect retrovirus-like particles. I investigated its existence. Visible using this technique after negative staining with uranyl acetate Pleomorphic particles comparable in size and morphology to other retroviruses were observed. (Figure 2).

Protein and nucleic acid content of purified retrovirus-like particles Second step of three particle purification Pools of four fractions, each obtained from a glucose density gradient (Figure IB), were combined and retro Tested for the presence of viral structural antigens and nucleic acid sequences. feline leukemia virus (FeLV, mammalian type C retrovirus) purified p27 core protein. Western blot analysis of density gradient pools using antisera against 31k d-core protein (p31) and 54kdgag precursor (p64) peak was found to be present in a density gradient pool containing fractions with RT activity ( Figure 3A).

Isolate nucleic acids from each pool and membrane them using a slot plot manifold. and hybridize with the retroviral probe and control probe. (Figure 3B). Fuzumi leukemia virus (type C) probe (MLV-MX27 (original) Nucleic acids that hybridize specifically to (see Materials and Methods)) have RT activity. All the samples containing detectable p31 and p64 polypeptides consisted of fractions with observed only in swimming pools. Also, the nucleic acids of fraction 1 containing RT and p31 Also, particles (IAP) are distributed in family I and family II CHO cell capsules 4. Column (pCHI AP, SW2 and pcHIAP, YL9 (raw materials and procedures) A pooled probe hybrid was formed (see Methods section). Experimental ability after that 1 et al., Fami+)-II (1) CHIAP, YL9) was revealed. However, it is not homologous to the family 1 (pcHI, heP, 5W2) I to P sequence. (Results not shown.) What nucleic acid is homologous to king cell mRNA (β-actin)? No force was detected in this density gradient fraction.

When nucleic acids are treated with Rλ8 enzyme or alkali before plotting, retro Virus probes (MLV-1X27 and CHIAP-YL9) Although the brining signal decreased significantly, it was Even if the sample is treated with DNase that does not contain RNAse, no No decrease in the intensity of the detected nuclei was observed. It can be seen that acid +: RN, weak and not DN A. Presence of RNA within the particle The morphological and immunological characteristics of these particles associated with retroviruses are consistent. I will.

~Characterization of particle cDNA sequences homologous to ILV Several cDNA clones were isolated and characterized. made a decision. Three of these clones were studied in detail. These (ma) All showed homology with the endonuclease domain of ML~'. largest A Clone (pcHoCNiL 10 (949bp)) t:, Mooney MLV ( 7) Publication tL)': Sequence [men-y　'), T2, supra, Nature (19 (81) showed 73% nucleotide identity with the two (Fig. 4, Table 1). evolutionarily divided Reduced homology in endonuclease domains of divergent species of type C retroviruses type C retrovirus of pcHOc~1L10 clonal fungi It was strongly suggested that this species is most closely related to S. (Table 1). pcHoc, ~IL1 The 0 sequence contains possible multiple breaks in the coding sequence in all three open reading frames. and thus could not encrypt the intact endonuclease (FIG. 5).

[Table IF Mammalian type C retrovirus genome of pcHOc, MLloc DNA Nucleotide homology to Nucleotide homology Type C Retrovirus Identity (%) Score 1 Moloney Fuzumi Hemorrhagic Disease Virus 74 200g (Mennick et al., 1981) (cited above) Feline leukemia virus 7 0 1910 (Donahieu et al., 1988) (Journal of Hierarchy, Volume 62, pages 722-73176) Crack endogenous virus 64 1714 (Cado et al., 1987) (Japanese Journal of Genetics (Japn, J, Genetics), 62, 127 ~137 pages) Actual performance reported by Fitch and Smith Based on parameters (1983) (81).

Presence of homologous NA sequences in the Chinese hamster genome pcHOc.

Chinese hamster DNA preparation using MLIO clones as probes DNA of parental and recombinant CHO cell lines and Chinese The conserved homology in NA (isolated from hamster liver) (Figure 6A) It has been found that there are 100 to 300 copies of this sequence in each chemome in DNA. won. Using these same high stringency hybridization conditions, human MR Homologous sequences were not detected in DINA isolated from C5 cells or murine myeloma cells. (Results not shown)

CHOIAP family 11 RNA sequences were also detected in density gradient purified particles. (see above). Related DNA sequences are present in the genomes of recombinant and parental CHO cell lines. It was previously demonstrated that they exist as a medium-frequency repeat family [Anderson et al. , supra (1990). DNA from several CHO cell lines and Chinese A CHOIAP family II probe of DNA from hamster liver was used. Comparison by Southern blot analysis (Figure 6B) reveals that this repetitive sequence has a conserved family of It turns out that Millie is also present in the germline of Chinese hamsters. .

Consideration Particles morphologically similar to retroviruses were previously observed in CH○ cells; , detailed characterization was not reported. Large using high-performance fluidized ultracentrifugation Concentrating extracellular retrovirus-like particles from the culture medium of a large number of recombinant CH○ cell lines. This facilitated the purification and biochemical characterization of these particles.

The extracellular particles of CHO cells are mammalian type C particles, which have been characterized based on a number of criteria. Similar to retrovirus particles. They have a favorable density in the sucrose density gradient. It shows RT activity with selectivity for manganese. particles The repeptide is immunologically similar to the polypeptide of mammalian type C retrovirus particles. RNA from purified particles was cloned under low stringency conditions into murine type C Hybridizes specifically to the genome.

In addition, the purified particles contained two sequences related to the intracapsular A particle (CHIAP) of CH○ cells. A clone representing one of the characteristic families [Anderson et al., supra, 1990]. Contains RNA that specifically forms a hyperprint on the cloned probe. this One possible explanation for the results is that a similar buoyant density during the sucrose density gradient There are two types of particles with . these at least one of the particles contains a structural antigen that cross-reacts with the type C antiserum. No difference. Or, if not, C enveloping two different types of RNA. There may be a single type of particle containing type structural proteins. different types No examples of retrovirus particles containing torovirus RNA species have been reported. ``Scolnick, E. No. 129, pp. 964-972 (1990), Chervi n), S, A., Nyarnal of Pyrology, vol. 26, pp. 257-264. (1978)]. Another possibility to consider is that the particle RNAs are both unrelated. A recombinant proviral gene composed of sequences from two related retrovirus species. It represents the product of genes. Availability to distinguish between these alternatives No possible data.

Sequence analysis of cDNA clones representing purified particle RNA revealed that several mammalian species A significant linkage with the endonuclease encoding the type C retrovirus gene of Creotide homology was revealed (Table 1). However, the cloned cDNA sequence does not contain an open reading frame capable of encoding an intact endonuclease. functional retroviral endonucleases are essential for the retroviral replication cycle. These findings suggest that CH○ details require proviral integration. This provides one possible explanation for the non-infectious nature of the particles observed within the cells.

Nucleotide homology of the particle cDNA with the murine C virus genome indicates that larger than that observed in type C retrovirus genomes of more distant mammalian species. This suggests that these particles are of rodent superthickness. moreover Intracellular DNA and CHO cells of several species were analyzed using Southern blot analysis. Comparing DNA derived from Neese hamster liver, it was found that within purified extracellular particles, Two types of retrovirus-like sequences were identified (CHOIAP family 1 and It was found that all types C) are present in the germ line of Chinese hamsters.

Therefore, extracellular retrovirus-like particles in CHO cells are similar to those in Chinese hamsters. It is likely a product of endogenous proviral elements present in the germline.

Sequence list (1) General information (1) Name of applicant Noe Funtech 3 Incorporate, Do(ii) R Ming: Number of retrovirus-like particle (iii) sequences in Chinese hamster ovary cells 2 (1v) Letter address (A) Recipient: Nenentech, Inc. (B) Street, Point To San Bruno Bourno h-do No. 460 (C) State/Suf(D) California (E) Country, United States of America (F) Postal code: 94080 (V) Computer reader form (with) medium type 5 25 inch, 360 Kb floppy disk (B) computer IB M PC compatible (C) operating system PC-DO3/MS -DO3(D) Software patin (Noe Futech) (■1) Current output request data (A) Application number (B) Application 09-\01'-1990 (C) Classification.

(vii) Previous application data.

(^) Application number: (B) Application date (viii) Patent attorney/agent information (A) Name, Adler, Cash Line R (B) Registration number 32.324 (C) Reference/Docket Number: 678 (1x) Telecommunication Information.

(A) Telephone: 415/266-2614 (B) Fax: 415/952- 9881 (C) Telex 910/371-7168 (2) Sequence number 1゜ (i) Characteristics of array (A) Array length 949 (B) Sequence type: Nucleic acid (C) Number of chains - terminal chain (D) Topology, linear (xi) Sequence description Sequence number 1. G^ATTCAATG TCCTTGTGGG GAAGAACGAT ATT CTA, AAACAGGTAACTGA 50GCA＾Ding GTGAT GCGT GCGCCCGAGTCCAACGCATCCAGACTG ^^GCTrCCT C100CCGGGAACCG GTCAGAGGCT ACCGGCCCG ^^CACATTGG GAGATAGATT 150-cho CAC-cho GAGA-cho TAAACCAGGA AAAATATGGAT ACAAGTATCT ATT AATTTn 200 GTAGACACCT TTTCAGGATG GGTT GAAGCCTTCCCCTACTA^^CATGA^^C250AGCCAGA TCG TTACTAAGAA ATTGCTTG^^GAAATCTTTCC CCGTTATCG 300GATGCCTCAG GTATTGGGAA C AGACAATGG GCCCGCCTTCGTCTCCAGGT 350^^ GTCAGTCA GTGGCCACCT TATTGGGGAT TGATT GGAAA TTACATTGTG 400CTTATAGACCCCAAAG TTCA　GGACAGGTAG　AAAGGATGAA　TAGAACAAT C450AAGGAGACTT TAACAAAATT GTCGCTTGCA ACTGGCACTA GAGAGCTGGG 500TCC Ding CC Ding ACT CCCCCTAGCA CTCTACCGCG CTCGTAATAACCCC TGGACCA 550CATGGGCTCA CACCCTTTGA GAT CCTGTAT GGAGTACCTA GCTCCTATCA 600TTA ACTTTCT　TGATCAAGAT　GTCTCAGm　TGCTAACT CCCCTTCTCTCC650AAGCTCATTT ACAGGCCCTC CAACTAGTACAACGGGAGGTCTGGAAACCC700Cn GCTCAAG CTTATAAAAGA CCAGAGGGACCATCCCA CCA TCCCCCATTC750CTACCAGATCGGGGACACT G　mGGGTccG　GCGTCACCAG　GCCAAGAACC800T TGAACCCCCG CTGGAAGGGA CCCTACATCG TTTT GCTTACCACTCCCACC850GCACTCAAGG TAGACG GCAT T CAGCTTGG ATACATGCTT CACATGTAA A 900GCCAGCCCAA CCCACCGAn CAGCCACTGC ATCAGAATGG AGCCACACC949 (2) Sequence number 2゜ (1) Characteristics of array: (A) Array length 949 (B) Sequence type Nucleic acid (C) Number of strands Knee end strand (D) Topology linear (xi) Sequence description, Sequence number 2: GGTGTGGCTCCATTCTGATG CAGTGGCTGA ATCG GTGGGT　TGGGCGCGGT　50TTACATGTGA　AGCAT GTATCCAAGCTGCAA TGCCGTCTACCTTGAGTGCG 100GTGGGAGTGG TAAGC, 4AAACGATGTAGGT CCCTTCC, 4GCGGGGTTCAAG IJGTTCTTGGCCT GGTGACGCCGGACCCAAACAGTGTCCCCG ATCTGG TAGG 200AATGGGGGAT GGTGGGATGG TCCCTC TGGT C77, 4TA, 4GCTTGAGC, 4,4GG 250GGT TTCCAGA CCTCCCGTTG TACTAGTTGG AGGGCC TGTA AATGAGCTTG 300GAGAGAAGGG GAGTTA GCAA AACT AGACA TCTTGATCAA GAAAGTTAA Ding 350 GATAGGAGCT AGGTACTCCA TACAGGATC Ding CAAAGGGTGT GAGCCCATGT 400GGTCCAGGG G TATTACGAGCGCGGT, ^GAG Ding GC Ding, GGGGGA G-cho AGGAGGAC450CCAGCTCTCT, 4GTGCCAGTT GCAAGCGACA A Ding Ding Ding TGTT, AA AGTCTCCTTG 50 0ATTG Ding TCTAT TCATCCTTTCTACCTGTCCT GAA CTnGGG GτCTATAAGC550ACAATGTAAT ncc^^ TCAA　TCCCCAATAA　GGTGGCCACT　GACTGAC code TA　600CCTGGAGACG　AAGGCGGGGCCCATTGTCTG TTCCCAATAACCTGAGGCATCC650GATAACGGGG AAAGAnTCT TCAAGCAATT TCTTAGTAACGATCT GGCTG 700TTTCATGTTT AGTAGGG^^G GCTTC 8^ To CC ATCCTGA^^^ GGTGTCTAC^ 750A^^AT T^^TA　GATACTTGTA　TCCATATTTT　CCTGGTTT ^^TCTCAGTG^^800^TCTATCTCCCCAATGTGTTCC GGGCCGGTA GCCTCTGACCGGTTCCCGGG 850AG GAAGCTTCAGTCTGGATG CGTTGACTCG GGCGCA CGCA TCACATTGCT 900CAGTTACCTG TTTTAG AATA TCGTTCnCCCCACAAGGACATTGAATTC949 FIG, IA Granuloid fraction FIG, IB grand end fraction pcHOc, MLt.

10, 6 International Search Report OFT/IK O+/nllF ζ Direct PCT/US 911082 54

Claims (34)

[Claims] 1. Under stringent conditions, the following DNA sequence (SEQ ID NO: 1): [Sequence exists] A nucleic acid sequence containing a sequence that hybridizes to. 2. The nucleic acid of paragraph 1 comprising at least about 10 nucleotides. 3. The following sequence (SEQ ID NO: 1): [There is an array] an isolated nucleic acid sequence with or hybridization specificity. A fragment of that. 4. A fragment of paragraph 3 having at least about 10 nucleotides. 5. The following sequence (SEQ ID NO: 2): [There is an array] an isolated nucleic acid sequence with or hybridization specificity. A fragment of that. 6. A fragment of paragraph 5 having at least about 10 nucleotides. 7. Under stringent conditions, the following DNA sequence (SEQ ID NO: 2): [Sequence exists] A nucleic acid sequence containing a sequence that hybridizes to. 8. 8. The nucleic acid of paragraph 7 having at least about 10 nucleotides. 9. as described in paragraph 3, further comprising a promoter operably linked to the nucleic acid sequence. of nucleic acids. 10. Nucleic acid according to paragraph 3, further comprising an origin of replication functional in a unicellular organism. array. 11. The sequence of the first term, which is the genome sequence. 12. Functional control sequences recognized by the host transformed by the vector An expression vector comprising the nucleic acid sequence of item 1 linked to. 13. The vector of term 12 which is a plasmid. 14. A host cell transformed with the vector according to paragraph 12. 15. 15. The host cell of paragraph 14 which is a mammalian cell. 16. a first term operably linked to an exogenous control sequence recognized by the host cell; A host cell containing the described nucleic acid. 17. 17. The host cell according to paragraph 16, wherein one or more copies of the nucleic acid sequence are present. 18. The host cell of paragraph 16 which is a mammalian cell. 19. isolated with reduced or increased expression of endogenous type C retroviral sequences. mammalian cells. 20. For obtaining cells with increased or decreased transcription of the nucleic acid according to paragraph 1. A method, (a) obtaining cells containing the nucleic acid; (b) introducing a transcriptional regulatory element into the cell; (c) increasing or decreasing transcription of the nucleic acid; A method consisting of screening cells that have been 21. Methods for obtaining cells with reduced expression of endogenous type C retroviral sequences The law is (a) obtaining cells with detectable levels of endogenous type C retrovirus sequences; (b) RNA that is complementary to the messenger RNA of the endogenous type C retrovirus sequence nucleic acid. A method comprising introducing into the cell a nucleic acid encoding an A transcript. 22. 22. The method according to paragraph 21, wherein the C retrovirus sequence is a sequence of paragraph 3. 23. 22. The method of paragraph 21, wherein the complementary nucleic acid is the sequence of paragraph 5. 24. Methods for obtaining cells with reduced expression of endogenous type C retroviral sequences The law is (a) obtaining cells with detectable levels of endogenous type C retrovirus sequences; (b) RNA that is complementary to the messenger RNA of the endogenous type C retrovirus sequence nucleic acid. introducing A into the cell, A method involving stages. 25. 25. The method according to paragraph 24, wherein the type C retrovirus sequence is the sequence of paragraph 3. 26. 25. The method of paragraph 24, wherein the complementary nucleic acid is the sequence of paragraph 5. 27. Methods for obtaining cells with reduced expression of endogenous type C retroviral sequences The law is (a) obtaining cells with detectable type C retrovirus RNA sequences; (b) obtaining type C retrovirus RNA sequences; Ribozyme nucleic acid sequences that cleave RNA adjacent to retroviral RNA sequences introduce into cells, A method involving stages. 28. 28. The method according to paragraph 27, wherein the type C retrovirus sequence is the sequence of paragraph 3. 29. Adjacent to the nucleic acid in a proximate position and orientation sufficient to influence transcription of the nucleic acid. Culture cells containing the nucleic acid according to item 3, in which a transcription regulatory element has been inserted into the DNA. A method consisting of nurturing. 30. Hybridization between the test sample and the probe specific for the sequence of the third term. A method for detecting retrovirus-like nucleic acid sequences in a test sample, the method comprising measuring retrovirus-like nucleic acid sequences in a test sample. 31. Hybridization between the test sample and the probe specific for the sequence of paragraph 5. A method for detecting retrovirus-like nucleic acid sequences in a test sample, the method comprising measuring retrovirus-like nucleic acid sequences in a test sample. 32. Retrovirus-like nucleic acids in test samples, including the use of polymerase chain reaction A method for determining the presence of expression products of. 33. in test samples, including the use of immunoassays directed against expression products. A method of determining the presence of a virus-like nucleic acid expression product. 34. The following sequence (SEQ ID NO: 1): [There is an array] at least one retrovirus-like particle encoded by.