CN1041005A - Post-transfusion non-A non-B hepatitis virus and antigen - Google Patents

Abstract

The present invention provides purified viral particles, antigens, antibodies reactive with viral antigens, and viral genetic material associated with non-A non-B hepatitis. Also disclosed are genetic material useful for the identification of whole virus particles of the invention and clones for use in diagnostic techniques and/or antigen production.

Description

The invention relates to viruses related to post-transfusion (PT) non-A, non-B (NANB) hepatitis, PT-NANB antigens produced by recombinant methods, and products and methods related to PT-NANB hepatitis diagnosis, prevention and vaccination .

Acute viral hepatitis is a systemic infectious disease mainly characterized by liver lesions. Five types of viral agents are known to cause hepatitis: hepatitis A virus (HAV), hepatitis B virus (HBV), posttransfusion (PT) and enterically transmitted (ET) non-A non-B (NANB) hepatitis virus and HBV-related delta viruses. The association of specific viral factors with HAV, HBV and delta viruses is known. However, although many publications have reported factors associated with the hepatic form of PT-NANB, no causative factor appears to have been identified. Harrison (Principals of Internal Medicine, 11th ed., 1987) reported at least two different causative agents of hematologic NANB hepatitis, but mentioned that no virus or viral antigen had been definitively identified.

Routine screening of blood donors for anti-HBV antibodies and HBV antigen (HBsAg) has reduced the incidence of posttransfusion hepatitis B, but posttransfusion PT hepatitis caused by NANB hepatitis factor infection remains a serious problem because currently There is no effective serological screening test for the identification of PT-NANB hepatitis virus. The identification of new viruses and the use of genetic information derived from the viruses to produce recombinant proteins that are safe for use in vaccines and diagnostics are major goals in the development of a safe blood supply.

The PT-NANB virus and antigen have been reported, eg, see U.S. Patent Nos. 4,464,474 and 4,542,016. It has been reported that PT-NANB virus is a coat virus, see, for example, U.S. Patent No. 4,464,474. European patent application 88310922.5 (publication number 0 318 216Al) reports the genetic engineering of the hepatitis virus gene identified as hepatitis C virus.

The present invention provides isolates containing virus particles related to PT-NANB hepatitis and genome materials obtained therefrom, as well as their preparation methods and applications. The characteristics of the virus particle are: it can be obtained from cells sensitive to NANB hepatitis infection in a host infected with NANB hepatitis; it can induce NANB hepatitis in a sensitive host; and it can induce the expression of NANB virus-specific antigens in cells sensitive to virus infection. Express. Viral particles can be used as a source of genomic material required for the preparation of polynucleotide probes for diagnostics, and as a source of antigens and vaccines for therapeutic and diagnostic use. Propagation of viral particles in vitro can be used to identify virus-specific cell surface antigens and serve as a source of these antigens. Attenuated or inactivated virus particles can be used as vaccines.

Figure 1 shows a fragment of PT-NANB virus from clone #30. The upper row represents the amino acid sequence encoded by the nucleotide sequence shown in the lower row.

Figure 2 shows a fragment of PT-NANB virus derived from lambda gt-11 clone PT-2. The abbreviations are the same as those shown in Figure 1.

Figure 3 shows a fragment of PT-NANB virus derived from lambda gt-11 clone PT-8. The abbreviations are the same as those shown in Figure 1.

Figure 4 shows a fragment of PT-NANB virus derived from lambda gt-11 clone PT-19. The abbreviations are the same as those shown in Figure 1.

Figure 5 shows a set of 7 fragments of PT-NANB virus genetic material. Only the cDNA sequence is given in this figure.

The present invention provides a source of well-identified viral genetic material and viral particles associated with post-transfusion non-A, non-B (NANB) hepatitis. The virus particles can be obtained by buoyancy density separation from samples that may contain virus particles, such as serum from infected humans or other great apes, or from cells that are susceptible to NANB virus infection, such as hepatocytes. Viral particle isolates can be used directly as a source of genomic material for the preparation of diagnostic probes, therapeutic and diagnostic antigens, and vaccines, and can also be propagated in sensitive cell lines such as triomas containing human hepatocytes. Infected cells can be used as a source of virus particles, or to identify NANB virus-specific antibodies or antigens, and as a source of such antigens.

Virus particles can be obtained from infected humans or other infected animal sources such as chimpanzees, from blood plasma, or other cells susceptible to NANB virus infection, such as hepatocytes. To obtain viral genomic material, biological samples can be centrifuged and viral RNA extracted from viral particles within the sample. Alternatively, a sample may be fractionated by a density gradient, such as a sucrose density gradient, to obtain a purified fraction containing viral particles. Portions having a buoyant density of about 1.07 to 1.13 g/cm ³ , preferably 1.09 to 1.11 g/cm ³ are collected. Fractions containing virus particles can then be extracted and cDNA clones made from viral RNA. The virus may be further characterized in that its genome contains RNA sequences which can be reverse transcribed to obtain at least one cDNA sequence as shown in Figures 1-5. All of these sequences were obtained from viral genetic material isolated from humans or chimpanzees infected with PT-NANB. For example, the first five sequences shown in Figure 5 were obtained from viruses obtained from humans. These sequences were obtained from different viral genome segments and may not be related to each other. The latter two sequences shown in Figure 5 were obtained from viruses obtained from infected chimpanzees. All these fragments may differ from previously known NANB sequences, such as that disclosed in published European application 0 318 215 A1 (identified as a hepatitis C virus fragment as mentioned above).

Any nucleotide sequence from the sequences described above can be used as a probe or primer for detection or modulation of viral nucleic acid. These probes can be much shorter than the full sequence, but should be at least 16 nucleotides long. Moderate oligonucleotides of 20 to 500, especially 30 to 200 nucleotides in length serve as very specific and fast-acting probes. Longer oligonucleotides, even reaching the full length of the gene, can also be used. Both RNA and DNA probes can be used. In addition, sequences of at least 8, generally at least 12, and often at least 20 amino acids can be used as epitopic sites, immunodominant sequences, haptens, etc. for the production of diagnostic reagents and vaccines, production of antibodies, isolation of serum antibodies, etc. Isolated peptides are generally less than about 125 amino acids, and most are less than about 100 amino acids. An amphipathic sequence or a sequence satisfying Rothbard's rules such as G-V-V-Y-D-N-D-D or E-P-V-N-P-K-D-P can be used.

Sequences homologous to viral sequences can be detected and hybridized to RNA under conditions described by Maniatis et al. . See also pages 324-328, especially page 325, paragraph 6, for DNA hybridization conditions.

Since the meaningful sequence of the genetic material (cDNA) has been well characterized, it is possible to chemically synthesize a variety of DNA and RNA sequences based on this natural sequence, which can then be recombined using known recombinant DNA techniques. Insert into any of a number of suitable DNA vectors. The present invention can thus be practiced using the relevant reagents, plasmids, and microorganisms that were readily available to the public at the time of filing this patent application.

For example, nucleotide sequences greater than 100 nucleotides in length can be readily synthesized using the Applied Biosystems Model 380A DNA Synthesizer as recommended in commercial advertisements (see e.g. Genetic Engineering News, November/December 1984, p. 3). Many techniques for preparing overlapping complementary sequences (eg, coding strand of 1-100 nucleotides, complementary strands of 0-50 and 51-150 nucleotides, coding strand of 101-200 nucleotides, etc.) are available. The oligonucleotides are readily cut and then hybridized and the strands ligated.

In addition, robotics can also be used to directly synthesize any of the peptides disclosed in the present invention. Commercially available automated peptide synthesizers with coupling efficiencies exceeding 99% were advertised in the same issue of the above-mentioned publication (Genetic Engineering News) (p. 34). This device provides the peptides of the invention using direct synthesis or the synthesis of a series of smaller fragments which are then coupled using other known techniques.

In addition to the specific polypeptide sequences shown in Figures 1 to 5, peptide fragments based on these sequences and fragments representing minor variations thereof will also possess various peptide biological activities. For example, fragments of known peptide sequences recognized by NANB hepatitis-specific immunoglobulins can be readily prepared and screened. Small peptide fragments (such as less than 100 amino acids) can be prepared using a peptide synthesizer, or larger fragments can be prepared using genetic engineering techniques. A simple screening method for identifying appropriate polypeptide fragments involves preparing a monoclonal antibody directed against the entire encoded antigen, attaching the antibody to an affinity column, and eluting the peptide fragment retained by the bound antibody, using if necessary Monoclonal antibodies were replaced by antiserum from clones. The applicability of this technique has been demonstrated experimentally.

The ability to make and select appropriate immunologically active fragments from larger proteins is known in the art and has been described in numerous publications, including the patent literature. As described in U.S. Patent No. 4,629,783 for the preparation of immunologically active fragments of viral proteins, a common variation is to prepare the polypeptides of the present invention in the form of fusion polypeptides. Such peptides are generally prepared using the promoter region of a gene known to be expressible in the host, whereby nucleotides encoding all or most of the amino acids of the present invention are inserted into the gene sequence encoding the host protein. Examples of such fusion proteins include the proteins discussed below fused to beta-galactosidase.

Another technique for preparing immunoreactive peptide fragments is to synthesize a series of 5-100 amino acids in length (or any intermediate length, such as 10, 15 amino acids, or any other multiple of 2, 3, or 5 in length within this range Amino acid sequence) and use antiserum (or monoclonal antibody) to screen for immunologically active fragments. Fragments exhibiting the best cross-reactivity are selected along the entire length of the peptide (eg, a series of peptides 20 amino acids in length and containing AA ₁ -AA ₂₀ , AA ₅ -AA ₂₅ , AA ₂₀ -AA ₃₀ , etc.). In this way, the selected fragments correspond to particularly suitable corresponding nucleotide sequences, so that these nucleotide sequences can be used to prepare large quantities of peptides recombinantly for use in the methods described herein.

In addition, minor variations of the above-mentioned peptides and DNA molecules should also be considered equivalent to the peptides and DNA molecules detailed herein, as will be recognized by those skilled in the art. For example, it is contemplated to substitute isoleucine or valine for leucine, glutamic acid for aspartic acid, serine for threonine, serine or alanine for cysteine, or Structurally related amino acids are substituted for similar amino acids (i.e., conservative substitutions) without significantly affecting the biological activity of the resulting molecule, especially when substituting amino acids that do not include binding or other biologically active sites. In an immunologic or diagnostic assay that relies on immunogen specificity, it is easy to determine whether a variant results in a functional peptide by direct functional analysis. An example of this method is described in detail below. Peptides with more than one substitution are readily detected in the same way. Preferred peptides differ by no more than 12, most preferably by no more than 5, in any contiguous group of 20 amino acids. The standard conservative groups of amino acids are given by the one-letter amino acid symbols within the following brackets: nonpolar (A, V, L, I, P, M); aromatic (F, T, W); uncharged polar Sexual (G, S, T, C, N, Q); Acidic (D, E); Basic (K, R, H). Aromatic groups are sometimes considered to belong to the broad sense of nonpolar (F, W) or uncharged polar (T) groups.

Based on the codons listed in Table 1, other DNA molecules encoding such peptides can be easily identified, which are also considered equivalent to the DNA sequences in Figures 1 to 5. Because DNA codons and amino acids in a peptide have a fixed relationship, any discussion in this application of substitutions or other changes in a peptide applies equally to the corresponding DNA sequence or DNA molecule, recombinant vector, or sequence Transformed microorganisms that have been localized therein (and vice versa).

Table 1

genetic code ¹

The first position (5' end) The second position The third position (3' end)

U C A G

PHE SER TYR CYS U

PHE SER TYR CYS C

U LEU SER TERMINATION TERMINATION A

LEU SER Termination TRP G

LEU PRO PRO HIS ARG U

LEU PRO PRO HIS ARG C

C LEU PRO GLN ARG A

LEU PRO GLN ARG G

ILE THR ASN SER U

ILE THR ASN SER C

A ILE THR LYS ARG A

MET THR LYS ARG G

VAL ALA ASP ASP GLY U

VAL ALA ASP ASP GLY C

G VAL ALA GLU GLY GLY A

VAL ALA GLU GLY G

1. Given the position of the base in the codon, the corresponding amino acid can be found. For example, the codon (5')AUG(3') on mRNA specifies methionine, while CAU specifies histidine. UUA, UAG, and UGA are termination signals. AUG is part of the initiation signal, and it also encodes an intrachain methionine.

In addition to the specific nucleotide sequences listed in Figures 1 to 5, the DNA (or corresponding RNA) molecules of the invention may have additional nucleotides preceding or following the specifically listed sequence. For example, poly A can be added to the 3' end of the cDNA and a short sequence (e.g., less than 20 nucleotides) can be added to both ends to provide a terminal sequence equivalent to an endonuclease restriction site , a stop codon may be added after the peptide sequence to terminate translation, etc. In addition, DNA molecules can be prepared that contain promoter regions or other control regions upstream of the gene. All DNA molecules containing the sequences of the present invention can be used for at least one purpose, because all of these DNA molecules can be cut into small fragments to generate oligonucleotide probes and used to isolate or detect PT of biological origin - NANB-specific nucleic acid sequences.

Homogeneous preparations of the peptides of the invention can be first prepared by direct synthetic methods or using cloned genes or fragments thereof as described herein. By "uniform" is meant that, with respect to peptide or DNA sequence, the primary molecular structure (ie, amino acid or nucleotide sequence) of substantially all molecules present in the composition in question are identical. The term "substantially" as used in the preceding sentence means at least 95%, preferably at least 99%, most preferably at least 99.8% by weight. When there are fragments derived from the complete molecule of the homogeneous peptide or DNA sequence, if they do not exceed 5%, preferably not more than 1%, and most preferably not more than 0.2% by weight, they are not included in the determination of homogeneity. Considered, because the term "homogeneous" refers to the presence of a single intact molecule (and its fragment).

As used herein, the term "isolated" refers to pure peptide, DNA or RNA, respectively, separated from other peptides, DNA or RNA, and present only, if any, in solvents, buffers, ions, or their usual biochemical solutions. of the other ingredients. "Isolated" does not include a natural substance in its native state, nor a natural substance that has been separated into its components (eg, in an acrylamide gel), but not obtained as a pure substance or a solution. The term "pure" as used herein preferably has the same quantitative limitations as "substantially" above. The phrases "substituted" or "substituted" as used herein do not necessarily refer to any effect that necessarily occurs, but rather refer to the peptide present when there is a specified amino acid at the same position as the specified amino acid but different in structure.

Salts of any of the biomolecules described herein are naturally formed when these molecules are present in (or isolated from) aqueous solutions of different pH. All salts of molecules exhibiting the indicated biological activity are considered to be within the scope of the present invention. Examples of salts that can be formed with peptides include alkali metal, alkaline earth metal or other metal salts of their carboxylic acid residues, acid addition salts (such as hydrochloride) of amino residues, and carboxylic acid and amino residues in the same molecule. Zwitterions formed by the reaction between them.

The present invention specifically contemplates the various possible variations of polynucleotides that can be produced by selecting various combinations of the possible codon usages listed in Figures 1-5 and Table 1, all of which are considered has been specifically disclosed. To avoid repetition, these variations are not described here.

Although the genes and corresponding proteins can be prepared using total synthetic techniques as described above, in preferred embodiments of the invention, the genetic information is obtained from natural sources and identified as described herein. The genetic material is first obtained in the form of a gene library using any of a number of known techniques. The first step is to randomly shear genomic DNA, and insert the sheared material into an expression vector such as λgtl1. If enough recombinants are produced, there is a good chance that at least one recombinant in the population will express a fusion protein equivalent to the antigen of interest. Indeed, for the size of the viral genome (approximately 10Kbp DNA), at least approximately 6 x ¹⁰³ independent recombinants are required. Only in this way can the recombinant have a complete genome with inserts with an average size of 100 bp, wherein at least one end of one insert falls within any 10 base pair region. Assuming only 1 of 6 such insertions is in the correct orientation and reading frame, functional recombinants should exist in a library approximately one fusion per 10 base pairs.

A second option for generating a gene library is to make complementary DNA (cDNA) copies of total viral RNA and clone these copies in expression vectors as recombinant molecules. Preferably, cDNA libraries are used to obtain genetic information for use in the present invention. Such a library has been generated from NANB-infected human plasma and screened with NANB-infected human sera. Recombinants expressing serum-reactive determinants include those shown in FIGS. 1 to 4 .

cDNA libraries can be screened using anti-NANB polyclonal antiserum to locate the desired genetic material. The cDNA fragment is inserted into the expression vector, and after being transformed into a suitable host, the host cell can be screened according to whether it produces a protein that can bind to the polyclonal antiserum. Initially identified recombinants can be isolated in this way. The resulting clones can then be used as probes to further examine libraries containing larger fragments or partially overlapping fragments until the complete cDNA is identified.

NANB genetic material can be used to produce intact fragments or modified peptides using standard techniques for manipulating and culturing unicellular microorganisms. Antigens that may be used as vaccines and/or diagnostic reagents include antigens recognized by the sera of infected patients. In addition, any gene sequence can be used as a probe for hybridization detection.

The above techniques, if combined with the knowledge of professionals in the field of genetic engineering and the points mentioned above, will be able to isolate the desired genetic material and use it in a recombinant DNA vector with the disclosed sequence, but other methods that produce the same result It is also known and can also be used to prepare the DNA recombinant vector of the present invention.

Multiple copies of the gene in the transformed host can be used to enhance the protein ( Such as the expression of proteins used as vaccines, whereby large quantities of proteins are produced by the insertion of exogenous DNA. Any other known method can also be used to enhance peptide expression. In all cases, when the DNA sequence is functionally inserted into the vector, it can be expressed Viral protein. The so-called "functional insertion" refers to insertion with correct reading frame and orientation, which is clear to those skilled in the art. Generally speaking, the gene will be inserted into the downstream of the promoter, followed by a stop codon subs, but preparations like hybrid proteins (possibly followed by cleavage) can also be used if desired.

In addition to the general methods described above that can be used to prepare recombinant DNA molecules and transform unicellular microorganisms in accordance with the practice of the present invention, other known techniques or modifications thereof can also be used to practice the present invention. Especially those technologies related to genetic engineering developed in recent years. There are a number of recent US patents disclosing plasmids, genetically engineered microorganisms, and various genetic engineering methods useful in the practice of the present invention. For example, U.S. Patent No. 4,273,875 discloses a plasmid and its isolation method. U.S. Patent No. 4,304,863 discloses a method for producing bacteria using genetic engineering techniques, which includes constructing hybrid plasmids and transforming bacterial hosts. U.S. Patent No. 4,419,450 discloses a plasmid useful as a cloning vector in recombinant DNA manipulations. U.S. Patent No. 4,362,867 discloses methods for the construction of recombinant cDNAs and the resulting hybrid nucleotides which can be used in cloning procedures. U.S. Patent No. 4,403,036 discloses genetic reagents for producing plasmids containing multiple copies of DNA fragments. U.S. Patent No. 4,363,877 discloses recombinant DNA transfer vectors. U.S. Patent 4,356, No. 270 discloses a recombinant DNA cloning vector, and its disclosed content is particularly useful for those with little experience in the field of genetic engineering, because many terms used in genetic engineering are defined and many terms used therein are defined. The basic method used. U.S. Patent No. 4,336,336 discloses a fusion gene and its preparation method. U.S. Patent No. 4,349,629 discloses plasmid vectors and methods for their production and use. U.S. Patent No. 4,332,901 discloses a cloning vector that can be used for DNA recombination. Although some of these patents do not relate to the production methods of specific gene products within the scope of the present invention, those skilled in the field of genetic engineering can completely modify the methods described in the above-mentioned patents by replacing the existing open reading frame sequence with the target sequence for the practice of the invention described in this specification.

The significance of the present invention is that the NANB virus protein and the genetic material of the strain of interest can be provided without limitation for the establishment of a hybridization assay or any other type of assay using these materials as reagents for diagnosis, immunization, treatment and research work middle. The method for using the genetic material in the hybridization assay and the equipment for amplifying the genetic material may be a commercially available PCR system (Perkin-Elmer Cetus).

It is particularly desirable to isolate genes and viral genomes expressing viral proteins of interest using oligonucleotide probes based on the principles disclosed herein and varying nucleotide sequences. When used, probes are typically detectably labeled (e.g., with radionuclides such as ³² P, ³ H, or biotin) and combined with single-stranded DNA or RNA was incubated together. After separation (usually using nitrocellulose filters) of single- and double-stranded (hybridized) DNA (or DNA/RNA), hybridization is detected with the aid of a marker. Hybridization techniques suitable for oligonucleotides are known. The identity of viruses or genetic material from any source to the viruses or genetic material of the invention can be further confirmed by hybridization assays using probes prepared from the gene sequences described herein.

While probes are generally used with detectable labels for ease of identification, unlabeled oligonucleotides can also be used, both of which can serve as precursors for labeled probes and for detection of double-stranded DNA (or DNA/RNA) for direct detection (e.g. adsorption on nitrocellulose filters). Thus, the term "oligonucleotide probe" refers to both labeled and unlabeled forms.

Additionally, it is possible to purify viral particles from any source and reduce fractionation of potentially virus-containing biological samples (such as viral particles obtained from serum or other body fluids of apes infected with NANB hepatitis) based on buoyant density to identify those associated with NANB hepatitis The amount of viral particles and genetic material necessary for screening. Parts are selected to have the appropriate buoyant density. That is, the obtained sample enriched in the specific virus of the present invention. A titer of 100% of the inoculum was in fact recovered from the above buoyant density fraction.

Some clones have been produced using these techniques. They were characterized in terms of their immunoreactive protein (a β-gal fusion product) recognized by NANB antiserum. This genetic material is exogenous to both human and chimpanzee genomes (infected and uninfected), and after amplification of genetic material extracted from buoyant density fractionated serum, compared with samples obtained from NANB-infected chimpanzees The hybridization was positive but negative when the same analysis was performed on amplified genetic material obtained from HBV-infected control chimpanzees.

As a method for proliferating viral genetic material in large quantities, the virus of the present invention can be cultured in vitro using a hybrid cell line that is sensitive to virus infection. U.S. Patent Application No. 846,757 (filed April 1, 1986) specifically describes immortalized virus-specific tissue cells that can be used to grow the NANB virus of the present invention. The aforementioned patent application and U.S. Patent No. 4,464,474, the disclosure of which is hereby incorporated by reference, describe techniques for producing viral particles from cell culture.

The general procedure for infecting and culturing hybrid cells with a human infectious virus of choice is as follows: Infect hybrid cells with plasma from NANB-infected humans or other sources (e.g., chimpanzee) and periodically detect virus-associated virus in cultures after virus infection. Cell changes. NANB virus infection is characterized by the appearance of virus-specific antigens, so immunological methods should be used to detect antigens appropriately after viral infection. After infection and propagation of the virus, the virus can be isolated if desired using conventional methods for releasing and purifying virus particles from cells. For example, cells can be lysed and the lysate fractionated according to buoyant density as described below. The virus particles are thus isolated without the need for additional purification techniques that would destroy the virus particles. When uninfected hybrid cells and virus particles are contacted, the isolated particles will recapitulate the cellular changes associated with the virus.

Further purification of the particles present in a sample containing the virus particles of the invention may be required for a number of reasons. For example, if viral particles are to be processed for use as vaccines or for immunoassays, there should generally be as little contamination as possible with foreign proteins. Accordingly, the particles should be substantially free of primate proteins.

NANB virus antigens are available from a variety of sources. The antigen may be present on intact virions, on partially degraded virions, on protein or carbohydrate-containing molecules in solution, or in any other physical state, including antigens chemically or physically bound to particles or solid surfaces, Examples include attaching antigen to the surface of a test tube or to suspended particles such as red blood cells or latex particles. An antigen according to the invention is defined as a substance containing at least one epitopic site of a virus particle.

In order to obtain NANB virus antigens, soluble or other antigens are generally separated from water-insoluble pollutants (such as animal cells or cell fragments and unicellular microorganisms such as bacteria) that are larger than intact particles or have different densities. This coarse separation step is generally accomplished by low speed centrifugation or using standard filtration techniques. In order to retain crude pollutants and allow antigens to pass through, ordinary filter membranes with an average pore size of 0.45 microns can be used.

Alternatively, the antigens of the present invention can be separated from unwanted water-soluble substances after removal of gross contaminants. To recover intact virus particles or their water-insoluble fragments, it is straightforward to remove all water-soluble components from the sample. Appropriate techniques include ultrafiltration with membranes, use of selective flocculants or protein precipitants (such as polyethylene glycol and ammonium sulfate), and chromatography. Chromatography is the most versatile method because it is easily scaled up for commercial production of antigens. Gel chromatography systems utilizing cross-linked dextran beads are most commonly used. A suitable gel column can be selected that allows diffusion of proteins and entry of low molecular weight species into the external water volume of the gel beads, thereby preventing these contaminants from passing through the column and allowing virtually unimpeded passage of all virus particles. If a specific antigen is required, other specifications of the gel can be used to separate antigens of any specific size. The choice of gel should be a matter of routine experimental knowledge.

The various techniques described herein can be used in combination as desired. For example, cesium chloride or sucrose density gradients can be used to separate the particles, followed by disruption of the particles by either technique and gel electrophoresis to separate the viral antigens and select for proteins that bind to antibodies such as antisera specific for NANB antigens.

A particularly suitable technique for isolating soluble protein antigens or particle fragments is affinity chromatography. An antibody capable of binding an antigen of the present invention is covalently linked or adsorbed to an insoluble carrier using conventional methods. Add the conjugated antibody to the column. Passing the antigen-containing sample through the column allows the antigen to bind to the conjugated antibody. Immunologically bound antigens are washed with a buffer, and the antigens can then be released by, for example, changing the ionic strength or pH of the wash buffer. In general, an acidic pH is effective for releasing bound antigen. This technique is very effective in isolating closely related proteins from the antigens of the present invention.

The antigens of the invention are useful as vaccines. A preferred starting material for the preparation of vaccines is particulate antigen produced from tissue cultures of infectious virus. It is best to initially recover the antigen as intact particles as described above. However, suitable vaccines can also be prepared from particulate or non-particulate recombinant antigens isolated from other sources. When using nonparticulate antigens (typically soluble antigens), it is best to use proteins inherent to the viral capsid or viral envelope to prepare the vaccine. These proteins can also be purified by affinity chromatography as described above.

If the purified protein itself is non-immunogenic, it can be rendered immunogenic by conjugating it to a carrier. Carriers include bovine serum albumin, keyhole Hemocyanin etc. Preferably (but not necessarily) the antigen is purified to be essentially free of human protein. But what is more important is to make the antigen free of proteins, viruses and other substances of non-human origin. These substances may be derived from the nutrient medium, cell lines, tissues or pathological fluids used for culturing or preparing viruses, or the contents of these materials. pollution introduced.

Vaccination can be carried out by conventional methods. For example, antigens (whether viral particles or proteins) may be added in a suitable diluent such as water, saline, buffered saline, complete or incomplete adjuvant, and the like. Immunogens can be vaccinated using standard techniques for inducing antibody production, such as subcutaneous administration of inactivated or attenuated viral particles or antigens in physiologically compatible sterile solutions. Virus particles in an amount capable of generating an immune response are generally given at each vaccination injection, and the volume is generally 1 ml or less.

In addition to their use as vaccines, these compositions can also be used to raise antibodies against NANB virus particles. Antibodies can be used directly as antiviral agents. To prepare antibodies, host animals may be immunized with viral particles or, where appropriate, non-particulate antigens inherent to the viral particles may be linked to carriers as described above for vaccines. After a certain period of time, host serum or plasma is collected to obtain a composition containing virus particle reactive antibodies. Gamma globulin fractions or IgG antibodies can be prepared using saturated ammonium sulfate or DEAE Sephadex, or other techniques known to those skilled in the art. These antibodies are substantially free of the various deleterious side effects that may be associated with other antiviral agents such as drugs.

Antibody compositions can be made more compatible with the host system by minimizing potentially deleterious immune responses. This can be achieved by removing all or part of the Fc fragment of the foreign antibody, or by using antibodies of the same species as the host animal (eg, using antibodies produced by human/human hybridomas) (see below).

Because antibody-virus complexes are recognized by macrophages, antibodies can also be used as a means to enhance the immune response. Antibodies can be administered in amounts required for treatment with other antibodies. For example, pooled gamma globulin can be administered at 0.02-0.1 ml per pound of body weight during the early incubation period of other viral diseases such as rabies, measles and hepatitis B to interfere with viral entry into cells. Therefore, NANB virus particle-reactive antibodies can be passively administered alone or in combination with other antiviral agents to hosts infected with NANB virus to enhance the immune response and/or the efficacy of antiviral drugs.

Alternatively, anti-idiotypic antibodies can be administered as immunogens to induce anti-NANB virus antibodies. Generally, anti-idiotypic antibodies are induced in host animals using purified anti-NANB virus antibody preparations prepared as described above. The composition is administered to a host animal in an appropriate diluent. Following administration (usually repeated administration), the host develops anti-idiotypic antibodies. In order to eliminate the immunogenic reaction to the Fc region, an antibody produced by the same species of the host animal can be used, or the Fc region of the administered antibody can be removed. After the anti-idiotypic antibody is induced in the host animal, serum or plasma is collected to prepare the antibody composition. The composition can be purified as described above for the purification of anti-NANB virus antibodies, or by affinity chromatography using anti-NANB virus antibodies bound to an affinity matrix. The generated anti-idiotypic antibodies are conformationally similar to real NANB antigens and can be used to prepare NANB vaccines without using NANB particle antigens.

When used as a means of inducing anti-NANB virus antibodies in patients, the method of injecting antibodies is the same as that of vaccination, that is, adding them to a physiologically applicable diluent and injecting them through intramuscular, intraperitoneal, subcutaneous, etc. at an effective concentration. With or without adjuvants. One or more booster injections may be needed. An anti-idiotypic approach to induce anti-NANB virus antibodies could alleviate problems caused by passive administration of anti-NANB virus antibodies, such as deleterious immune responses, as well as problems associated with administration of purified blood components, such as infection with other as-yet-uncharacterized agents .

In addition to therapeutic applications, the particles and antigens of the invention, as well as genetic material, can also be used in diagnostic methods. Methods for detecting NANB hepatitis include analyzing biological samples such as blood or liver biopsies for the presence of analytes associated with NANB hepatitis virus. The analyte may be a nucleotide sequence that hybridizes to a probe comprising at least 16 contiguous nucleotides, typically 30 to 200 nucleotides, up to substantially the cDNA shown in Figures 1 to 4 all in sequence. The analyte can be RNA or cDNA.

The analyte may be a viral particle having at least one of the following characteristics: it can be prepared from a cell susceptible to NANB hepatitis infection; it can induce expression of a virus-specific surface antigen in a cell susceptible to particle infection, which can be detected by NANB is recognized by sera from infected hosts but not by sera from non-infected hosts; buoyant density is approximately 1.09 to 1.11 g/cm ³ . The virus particle may be further characterized as having an RNA viral genome comprising a sequence that is at least about 80% homologous to at least 12 contiguous nucleotide sequences of the sequences shown in Figures 1 to 5, and typically at least 90% homologous. % homologous to at least 60 consecutive nucleotides within the sequence. The viral particle may comprise sequences substantially homologous to the sequences shown in Figures 1-5. The analyte may contain an antibody that recognizes an antigen on the NANB virus particle, such as a cell surface antigen. The analyte may also be a NANB virus antigen.

For detection of an analyte, if the analyte hybridizes to a probe, the probe may contain a detectable label. Likewise, when the analyte is an antibody or antigen, labeled antigen and antibody, respectively, can be used to bind the analyte to form immune complexes that can then be detected by the label.

Typically immunoassays are based on the detection of analytes such as surface antigens and/or whole particles. Immunoassays can be performed by detecting antibodies produced in the host due to infection with NANB hepatitis virus, or by directly detecting the presence of viral particles or antigens. These techniques are known and need not be described in detail here. Examples include both heterogeneous and homogeneous immunoassay techniques. Both techniques are based on the formation of an immune complex between viral particles or their antigens and corresponding specific antibodies. Heterogeneous assays for viral antigens typically use a specific monoclonal or polyclonal antibody bound to a solid surface. The sandwich method is becoming more and more common. Homogeneous assays performed in solution without a solid phase can also be used, for example to measure differences in enzyme activity due to binding of free antibody to the enzyme-antigen conjugate. U.S. Patent Nos. 3,817,837, 4,006,360 and 3,996,345 disclose a variety of suitable assays.

When detecting antibodies induced by NANB virus, the viruses and antigens of the present invention can be used as specific binding agents to detect IgG or IgM antibodies. Because IgM antibodies are generally the first antibodies to arise during an infection when IgG synthesis may not have begun, specifically distinguishing between IgM and IgG antibodies present in the host's bloodstream allows a doctor or other researcher to determine whether the infection is recent or chronic.

The genetic material of the invention itself can be used in a number of assays as probes for the detection of genetic material present in naturally occurring infection processes. One method for amplifying target nucleic acids for subsequent hybridization analysis is known as the polymerase chain reaction or PCR technique. The PCR technique using oligonucleotide primers spaced apart from each other and based on the gene sequence shown in FIGS. These primers are complementary to opposite strands of a double-stranded DNA molecule, generally separated by about 50 to 450 or more nucleotides. The method involves preparing specific oligonucleotide primers, followed by repeated cycles of target DNA denaturation, primer binding, and extension with a DNA polymerase to obtain probes of the desired length. The extension product generated from one primer is used as an additional target sequence for the other primer. The degree of amplification of the target sequence is controlled by the number of cycles performed and can be calculated theoretically using a simple formula 2 ⁿ , where n is the number of cycles. Assuming an average efficiency of approximately 65% to 85% per cycle, 25 cycles yield 0.3-4.8 x ¹⁰⁶ copies of the target sequence. There are many publications describing the PCR method, including Saiki et al., Science (1985) 230:1350-1354; Saiki et al., Nature (1986) 324:163-166; Scharf et al., Science (1986) 233:1076-1078, See also U.S. Patent Nos. 4,683,194, 4,683,195 and 4,683,202.

For in vivo use of antibodies against NANB virus particles and proteins and anti-idiotypic antibodies, and for diagnostic applications, monoclonal antibodies are preferred. Monoclonal anti-virion antibodies or anti-idiotypic antibodies can be prepared as follows. Spleen or lymphocytes are taken from the immunized animal and immortalized, or used to prepare hybridomas by methods known to those skilled in the art. To prepare human/human hybridomas, human lymphocyte donors are selected. People who are known to be infected with NANB virus (for example, those who have been infected by the presence of anti-viral antibodies in their blood or virus culture) can be suitable lymphocyte donors. Lymphocytes can be isolated from a peripheral blood sample or, if the donor has had a splenectomy, from the spleen. Epstein-Barr virus (EBV) can be used to immortalize human lymphocytes, and human cell fusion partners can also be used to generate human/human hybridomas. Peptides can also be used for primary immunization in vitro when generating human monoclonal antibodies.

Antibodies secreted by immortalized cells are screened for clones secreting antibodies of the desired specificity. For monoclonal anti-virion antibodies, these antibodies must bind NANB virions. For monoclonal anti-idiotype antibodies, these antibodies must bind anti-viral particle antibodies. Cells are selected that produce antibodies with the desired specificity.

The present invention has been generally described above, and the present invention will be understood more deeply with reference to the following examples. The purpose of these examples is for illustration only, and should not be regarded as a limitation of the present invention, unless otherwise specified.

Two hybrid hepatocyte cultures infected with NANB virus particles were deposited with the American Type Culture Collection (ATCC) on June 24, 1988 (12301 Parklawn Drive, Rockville, Maryland 20882). The two hybrid cultures were GLH03 and GLH04 with ATCC accession numbers CRL9754 and CRL9755, respectively. The uninfected hybrid hepatocyte culture GLH02 was deposited with the ATCC on March 26, 1986, and its ATCC accession number is HB9027.

Example 1

Preparation of cDNA clones from NANB particles isolated from human serum

Sera from patients diagnosed with NANB hepatitis were centrifuged at 30,000 rpm for 2-1/2 hours at 5°C using a SW40 rotor (Beckman). The supernatant was discarded and the pellet was dissolved in 50 mM sodium acetate buffer (pH 4.8) containing 1% sodium dodecyl sulfate (SDS). RNA was selectively extracted using phenol equilibrated in the same buffer without SDS. Nucleic acids in the aqueous phase were then precipitated with two volumes of absolute ethanol. RNA was reverse transcribed into double-stranded cDNA using a DNA synthesis kit following the manufacturer's (Boehringer-Mannheim Biochemioals, Indianapolis, Indiana) protocol, except that random primers were used in place of the oligo-dT primers provided in the kit. The resulting double-stranded DNA was ligated to an EcoRI linker to create a cohesive EcoRI site and inserted into λgtll as described by the supplier (ProMega Biotech, Madison, Wisconsin). The plaques were screened with human antiserum against NANB infection and positive clones were isolated. Clones were then screened again with human antiserum against NANB infection and subjected to plaque purification.

Analysis of the sequences shown in Figures 1 to 4 using existing sequence homology search programs showed that the sequences of the clones were not similar to any sequences entered into the database (GenBank version 54). No homology to the polypeptide sequence contained in GenBank version 52 was found.

Example 2

Purification of NANB virus particles from infected chimpanzee plasma

NANB virus particles derived from human plasma were isolated as follows. Chimpanzees were inoculated with infected plasma containing NANB virus particles derived from patients with confirmed NANB hepatitis but carried in chimpanzees. Plasma inoculum from vaccinated chimpanzees was plated on a 20-55% linear sucrose gradient in Tris-HCl (0.01M, pH 8.0) containing 0.001M EDTA, 0.1M NaCl. The gradient was prepared using a Hoefer gradient preparation apparatus. The chimpanzee inoculum contained ¹⁰⁶ times the chimpanzee infectious dose (CID) of NANB virus particles. The gradient was centrifuged at 30,000 rpm at 5°C for 18 hours using a SW40 rotor. Following gradient fractionation, fractions were assayed for infectivity by reinjection into chimpanzees. Fractions with buoyant densities ranging from 1.09 to 1.11 g/ ^cm3 were infectious at a 1: ¹⁰⁶ dilution based on elevated alanine aminotransferase (ALT) approximately 30 days after inoculation into chimpanzees.

Example 3

Preparation of cDNA from purified virus particles

cDNA was prepared by the method described in Example 1 using the virus particles having a buoyant density of 1.09 to 1.11 g/cm ³ prepared as described in Example 2. The resulting cDNA was then amplified using the techniques described in co-owned patent application 208,512, filed June 17, 1988, the disclosure of which is hereby incorporated by reference. As a control, amplified cDNA was prepared from buoyant density fractionated chimpanzee plasma chronically infected with HBV by the same method as the preparation of NANB cDNA. The cDNA amplified from the infected and control groups was separated by agarose gel (2%) electrophoresis, and then transferred to a nitrocellulose filter by the method of Southern (J.Mol.Biol. (1975) 98:503).

According to the instructions provided by the kit manufacturer (Boehringer-Mannheim Biochemicals, Indiannapolis, Indiana), clone #30 was radiolabeled with ³² P nucleotides and random primer kits (the preparation method was described in Example 1, and the sequence was shown in Figure 1 Show). Filters containing amplified cDNA (prepared from fractionated virus particles) were then probed with radiolabeled clone #30 as a hybridization probe. As detected by autoradiography, it only hybridized specifically to cDNA prepared from NANB-infected chimpanzees. This suggests that molecular clone #30 isolated from NANB-infected humans (identified using sera from different NANB-infected humans and determined to be exogenous to human and chimpanzee genomes) detected some homologous sequences present in cDNA , the cDNA was prepared from material enriched in the infectious NANB particles passaged in chimpanzees but derived from infected humans. Clones PT'2, PT'8 and PT'9 also hybridized specifically to cDNA prepared from NANB-infected chimpanzees.

Example 4

Infection of immortalized hepatocytes with NANB virus

Hybrid hepatocytes were plated in 24-well titer plates at 1× ¹⁰⁶ cells/well and covered with 100 μl of (a) chimpanzee plasma known to contain NANB virus factors after passage in a second chimpanzee; or (b) Human plasma from patients with acute post-transfusion NANB hepatitis. After pre-incubation of chimpanzee serum and cells, 0.5 ml of growth medium containing IMDM and 20% FCS was added to each well, and cells were incubated at 37°C in a humidified 7% CO ₂ incubator. The growth medium was changed every 3 to 4 days, and liver hybrid cells were collected weekly to detect the presence of NANB.

Cells were analyzed for expression of NANB virus-specific surface antigens to detect viral particles. The method used is as follows: Take a portion of the culture medium containing about 1×10 ⁷ cells from the culture well, and centrifuge at 200×g for 10 minutes to pellet the cells. After the cells were washed 3 times with PBS, the cells were resuspended at a concentration of 2.5×10 ⁶ cells/ml, and 10 μl of the cell suspension was dropped onto a microscope slide and air-dried. Acetone was then added to fix the dried cells on the slide for 1 min. To minimize non-specific binding, slides were pre-incubated with normal goat serum (diluted 1:10) for 30 minutes at room temperature in a humid incubator. The slides were washed 3 times with PBS and once with distilled water, and then 70 μl of test serum from one of a group of chimpanzees (shown in the left column in Table 2 below) was added to the slide. Each serum sample was preabsorbed with uninfected liver hybrid cells ( ¹⁰⁷ cells/ml serum) to remove serum factors that might bind non-specifically to the cells. The slides with the added serum were incubated at room temperature for 90 minutes in a humid incubator, and then washed three times with PBS and once with distilled water.

Goat anti-human IgG and IgM (FITC-conjugated antibodies) conjugated to fluorescein isothiocyanate were obtained from a commercial source (Zymed Labs). They were all diluted with PBS to a final concentration of approximately 1 μg antibody/ml. Anti-IgM or anti-IgG FITC-conjugated antibody (70 μl) was added to the washed cells and slides were incubated at room temperature for 30 minutes. After washing with PBS and distilled water as above, add 1 drop of 50% glycerol dissolved in PBS and observe under a fluorescent microscope. Cells were recorded as weak (+), moderate (++), and strong (+++) fluorescence, respectively.

The first immunofluorescent indications appeared approximately 6 to 8 weeks after the cells were initially infected with each virus source. Table 2 shows the results obtained after 6 weeks of infection with chimpanzee plasma known to contain NANB virus.

Table 2

Expression of NANB virus cell surface antigen in immortalized hepatocytes infected by NANB virus

Reactivity with liver hybridomas

Chimpanzee Disease Infected Uninfected

A recovery period HAV - -

B ¹ normal - -

B Acute NANB + + -

C Normal - -

D ¹ normal - -

D Acute NANB ++   -

E Normal - -

F HBV in recovery period - -

G Chronic NANB +++ -

H Chronic NANB - -

¹ These animals were prevaccinated with NANB.

As can be seen from the left-hand column of Table 2, specific immunofluorescence was observed only with sera from NANB-infected animals, but not with sera from uninfected or HBV-infected animals. The results showed that: (a) liver hybrid cells could be infected by NANB virus; (b) the infected hybrid cells expressed a virus-specific surface antigen recognized by NANB serum antibodies obtained from NANB-infected chimpanzees; (c) The incubation time required for surface antigen expression is about 4 to 6 weeks.

The results shown in Figure 2 were obtained with anti-IgG antibodies. No immunofluorescence was observed for FITC-conjugated anti-IgM antibodies, which is what would be expected when chimpanzee anti-NANB antibodies were IgG antibodies. Plasma from acutely transfused NANB patients produced results similar to those obtained above with plasma from infected chimpanzees. After 6 weeks, liver hybrid cells infected with patient plasma displayed specific immunofluorescence against sera from NANB-infected chimpanzees but not against sera from control (uninfected) chimpanzees.

Example 5

Recovery of NANB infectious virus particles from hybrid hepatocytes

The presence of infectious virus in NANB-infected hybrid cells was also examined. Infected hybrid cells (prepared as described in Example 4) were harvested 12 weeks after infection by centrifugation and washed 3 times with PBS. Cells were resuspended in PBS to approximately 5 x ¹⁰⁶ cells/ml and clarified by sonication. The supernatant was then inoculated on uninfected hybrid cells (0.5 ml/well) and cultured as described in Example 4 for infecting cells with chimpanzee plasma. Cell-free lysates can also be prepared by hypotonic lysis or freeze-thaw methods. After approximately 6 to 8 weeks of continuous culture, specific immunofluorescence was observed for chimpanzee NANB sera, but not for sera from uninfected animals, suggesting that the thus proliferated cell particles retained their infectivity.

Example 6

Stability of viral infectivity of NANB virus particles propagated in hybrid hepatocytes

Molecular clones were isolated from NANB-infected cells to determine whether in vitro passaging resulted in the production of defective virus particles that weakened virus infectivity. Methods as below:

Infected cells were allowed to grow in exponential phase and then harvested by centrifugation at 3,000 rpm for 10 minutes. Cell-free lysates were prepared from 5 × ¹⁰⁸ cells via three consecutive cycles of freezing on dry ice/ethanol and thawing at room temperature. The lysate was clarified by centrifugation at 10,000g for 15 minutes in a minicentrifuge. The supernatant was then applied to a linear sucrose density gradient as described above (see Example 2). The fractions with a buoyant density of 1.09 to 1.11 g/cm ³ were collected, and the RNA in the particles was extracted according to the method in Example 3 above. After conversion to cDNA and amplification as described in Example 3, the amplified cDNA was analyzed by Southern blot hybridization to confirm the presence of NANB homologous sequences. The probes used were cDNA clones having the sequences shown in Figures 1 to 4 . The material was then cloned into λgt10, and the molecular clones shown in Figures 1 to 4 were used as probes to select molecular clones by hybridization. The primary nucleotide sequence of molecular clones obtained from infected hybrid hepatocytes was then analyzed to determine whether defective virus particles were produced during in vitro passaging.

All publications and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which the invention pertains. All publications and patent applications cited herein are incorporated by reference with the same reference value as if each individual publication or patent application were specifically or individually indicated to be incorporated by reference.

Having now fully described the present invention, it will be apparent that various changes and modifications may be made therein by those skilled in the art without departing from the spirit or scope of the appended claims.

Claims (30)

1, a kind of hybrid cell that contains non-A, non-B (NANB) viral genome material, described cell is prepared by following method:

Infect described hybrid cell with the sample that contains infectious NANB virion.

According to the hybrid cell of claim 1, it is characterized in that 2, the buoyant density of described infectious viral particle is about 1.09 to 1.11g/cm ³

3, the method for a kind of propagation non-a non-b hepatitis (NANB) viral genome material, described method comprises:

Cultivation is reached the enough time by the hybrid cell system that NANB viral genome material infects, so that described viral genome material propagation.

According to the method for claim 3, it is characterized in that 4, described clone is to infect by the hybrid cell that does not infect is contacted with the sample that contains infectious NANB virion.

According to the method for claim 4, it is characterized in that 5, described sample comprises:

Virion is according to buoyant density with the antigen relevant with non-a non-b hepatitis, described antigen, partly collects as non-A non-B and opens with separated from contaminants, and the buoyant density of this material is about 1.07 to 1.13g/cm ³

According to the method for claim 4, it is characterized in that 6, described part contains buoyant density and is about 1.09 to 1.11g/cm ³Material.

7, by isolating virion in the biological sample, described virion is characterised in that, can be by to the non-B(NANB of the non-A of PT-) make in the cell of hepatites virus infections sensitivity, its buoyant density is about 1.09 to 1.11g/cm ³

According to the virion of claim 7, it is characterized in that 8, described biological sample is by the host's blood plasma or the liver cell of PT-NANB hepatites virus infections.

9, virion according to Claim 8 is characterized in that, described host is the people.

According to the virion of claim 7, it is characterized in that 10, the further feature of described particulate is, have a rna virus cdna group, this genome has 80% order at least with the order that comes from least 12 continuous nucleotides in one of following order:

1 ACACAACCAAATTCTTGGTTCCAGCGCCAACCTGAAACCAAA 42

43 AAAGTGGCAAATATTGCCGGGCAAGCGTCGATTGCTTCGACC 84

85 GCCTATGTGAGCCAAGATGCGGCCATTTCGGCCTACAATAAA 126

27 GTAAAAAATGCTGTGGTCACCGTGCAAAACTTGCAAAAGAAT 168

169 GCGGCCCAAACCCCAGATGGTTTTGCGGGGTTGTTTGGTCAA 210

211 TCAGGGCGTCAAAAGCAAGCCGATAATAATGGCCAAGTTGAA 252

253 ACTGCCTCAGAAGGCTCT 270

Or

1 AAAAATAACAAGATCTTACATTTACGGAAATCTGCGACAAAA 42

43 GTTTCCAAATATAAGATCAAAAAGTTAAGTGTTGGTGTCGCC 84

85 TCCGTTCTGGTGGGGGCCACTTTCTTCCTTGGTTCGACAGCG 126

127 AGTGCAAGTGCTTCTGATGAGCAACTCGCTGATAAGCAGGCA 168

169 GGGGTCACACAACAAACTGATCAAAATGCAACAAACACAAAT 210

211 GATCGGGTATTAAAGTTTGACAATGCAACGTCAACGGCCACA 252

253 ACGGATAATGCTGATTCTAGTGCGGCCAAAATGTCAAACGTT 294

295 GCGCAAGCTGACAATTCAGCCAACAATGCAACAGTAGCTAAT 336

337 AATCTTGATAAAAAATCAATTACCGATTCTACATTATCCAAT 378

379 AATAACGATTTAAAATCAACTGAAATGCAATCAACTGTTACT 420

421 GACCAAGCAGCAGCTGACGATGCAAGTACTGCTGATCAAACA 462

463 GCAACTGAAAAGCAAGCAACTGTGACCAATCAAGCCACAGTT 504

505 GATAACACAGTAAATACTGCTGACCAAGCAACTCAAGCAGCA 546

547 GCTGAAAAGACAACAACGCCTGCAAGTACTGCTGCCAACACG 588

589 CAAGCAGCTGCACTAGTTGCAACGCTACGTGCCGCAGCAACT 630

631 GCGGATACAAGTACGACGACAACTGTTAACAACTGGACT 669

Or

1 CCATCGGCTTCCATCCAGGAAGCAATGGATAAGCAGTTAACG 42

43 GCTGATCGTGAACGAGTGGCAACTATTGCAAAAGCCGAAGGG 84

85 GAGGCACGCTCCATCGAACTCACAACCAAGGCTAAAAATGAC 126

127 GCGTTGATGGCGACGGCGAAAGCCGAAGCTGACGCGACGAAA 168

169 ACCCGTGCTGATGCCGAACGTTACCGAATCGATACGGTACAA 210

211 GCTGGTTTGGCTGGGGCGGATGACAAGTACTTCCAAAACCAA 252

253 TCCATTAACGCATTCGCGACGTTACCCAAT 282

ATCGAGAGCAACGCACTGGCAGTGTCCAACCTGGATTTCTGATC

CTGTTTTGACCCGCAGTACCCAAAAAGGCCAACGCTCAGCGTTGGCCTTT

TTTAATGGCTAAAAAATGACTATGGCGCCAACAGCACCGCCCTCTCCTCG

CGGCACAACTCCAGTAAAAAATCCCACACCACCCTCAACCTTACGGATTT

GTGCAGTTCCCGGCGGGTGCTGATCCAGTAACTGCGTTGCACAGACTCGC

CCGGCAATACACGCACCAGGTCGGGGTCGTCAGCGGCCATGTAGTTGGGC

AATACGGCGATACCCAGGCCGGCGCGAGCGGCTTGTTGCTGGGCAATGAC

GCTGGTGCTGCGAAAGGTCACGGTCGGGGTGCGGCAGAAGGTATTGAGGA

ACAGCAGCTCCTGACTGAACAACAGGTCGTCGACGTAGCCGATCCAGTAG

TGGTTGCCA

Or

GTCATCACGCACAACAGGGGTGTTGAGCGGTGC

ACCGAGTTCTTTCCAGTCCGGGAACAATTCGTTCAGCGCACGGGGTTCAA

ACACGGCACCGGACAGGATGTGAGCGCCGACTTCGGAGCCTTTTTCGACC

ACGCAGACGCTGATTTCCTTACCGGCTTCGGCGGCCTTCTGCTTCAATCG

GCAGGCGGCAGACAGGCCTGCCGGGCCAGCGCCGACGATGACCACGTC

Or

GTTGACCACTCCCTGGCCGTCGAAGCCGGTG

GTTCGACCGACGCCTTCGAGAAGAACCGCGCCATCGAAGACCGCCGCAAC

GAAGACTGTTTCCACTTTATCGAGTGGACCAAAAAGGCCTTCAAGAACGT

CGATGTGATCCGCCGGGCAACGGCATCATGCACCAGATCAACCTGGAGAA

AATGTCGCCGGTGATCCAGGTGCGCGACGGCGTAGCTTCCGGATACCTGC

GTCGGCACCGATAGCCACACGCCCACGTGGATGCCTTGGCGTGATCGCAT

CGGCTCTGGCGCGTA

Or

CCCCATAGAGCCCGGACCCATAGACAGCCCTG

CCCCATAGACAGTCTGGCCCTATAGACAGCCCAGCCCCATAGAGCCCGGC

CCTATAGATAGCCCGGCCCCATACAGCCCGGACCCATAGAGAGCACTGCC

CCATAGAGCCCGGACCCATAGAGCCCTGCCCCATAGACAGTC

Or

GCCAAAGAGTGGCGCACCGACCGTTCCCTCAG

CCGCCTCGAAGCCATGCTCGCCGTGGCCAACAAAGACGCCTCCCTGATCA

TCACCGGCAACGGTGACGTGGTAGAGCAGAAAACGGCTTGATCGCCATG

GGCTCCGGTGGCGGCTACGCCAGGCTGCGGCCAGTGCGCTGTTGAAGAAA

ACCGACCTGTCGGCCCGTGAAATCGTCGAGACCGCCTTGGGCATCGCTGG

CGATATCTGCGTGTTCACCAACCACAACCTGACCATTGAGGAGCAGGACC

TCGCCGAGTAAGCCGTAGGCTTATTC

Or

GGCGATGACGGCTGCACCGCAAGCACCAGTA

TCAGTCCAGCCAAGTGAAACAGTGACACCTGCACAACCCGTCAAAGTTGC

ACCACAAGTGGTTGCAGCGCAACCAACGTCAACACCAACACCAACGGTAA

CAGTTGAGACTGTACCATCAACGCCTACGCCAGTGCCACCAACATTGGCA

ACGCCACCAATTGCACAACCAGTGGTAACTGCTGCGCCAACTGAAGAAGC

AGCCGTTGCCAACCAGTTGTGGGCACGTACGGGACAAAATGCGGTCTTTG

CCGTCCTACAACAAGCGAACGGAGACGCTTAGTCGCGTGAAGGCTGCTTG

GTCAGACTTGATTAGTCAATTTGGTGTTGCTGAACAGGCCTTACTGACGA

TTGCCGCCCCAGTAGCTGCAAGTGAGGAAGGGCTTGTTTTAGCGTTTGAT

TTTCCACCTTTATTGGCGCAAGCTTTACAAGATGCCGCCTTGCAAACGCA

ATTACGGACAGCGCTGGCTGCACAACAATTGCCAACAGAAATGGTGTTGA

TTACCCAAGATAGCTGGCAACAAGAACGCTCTGATTATGTCGCGCAGTTA

AAGGCGGGGACGACTCAACCTTTGAATTTGGCGGATATACCGAGAGTGAG

CCAAACAACCACGACCCAGTCGCAAAGTGCACCGACACCAGAGCAAACGG

GGCTTG

Or

TCGGGCCGGTAATGACCACGGCCACCATAGCACCGCGAAGAAGCCTGCGA

TGGCGACGCTGGAGGCCATGGTGACGACGCTCCAATCGATGTCCGCGCGC

GCTCGGCGGCGCGATCTGCCGGATATCATCCGGCGCACCAGTCGGACGCC

GCAGCCGCGCTACCGGCCCGAGAAAGAAGATCGCCGCCGCCCATTCGATG

GGGAACG.

According to the virion of claim 10, it is characterized in that 11, described viral genome has 90% order at least with the order that comes from least 60 continuous nucleotides in the described order.

12, a kind of purified basically polynucleotide, it has a segmental zone that is complementary to or is same as at least 12 continuous nucleotides in the claim 10DNA order basically.

According to the polynucleotide of claim 12, it is characterized in that 13, described polynucleotide comprise the whole order of one of described order basically.

14, determine the non-B virus antigen of the non-A of PT-of at least one epitope, described antigen comprises:

Partly by basically with a kind of protein of the sequence coding of DNA sequence that comes from claim 10 or complementary order.

15, according to the antigen of claim 14, it is characterized in that there are not other protein in this antigen basically.

16, a kind of carrier, it comprises the dubbing system of energy performance function in bacterium, a mark and the order that comes from the order of at least 60 Nucleotide in claim 10 order basically together that the detection transformant is required.

17, a kind of method that detects the non-A, non-B hepatitis virus existence, this method comprises:

Analysis of biological samples is to determine existing of a kind of analyte, and wherein said analyte includes a nucleotide sequence, this Nucleotide have one can with a kind of order of probe hybridization, described probe comprises at least 16 continuous nucleotides in claim 10 order.

18, the vaccine of the Hepatitis virus factor anti-non-B(NANB of non-A of a kind of immune host), described vaccine comprises:

A kind of interior virus antigen of the compatible solution of physiology or virion of deactivation or attenuation of being present in, wherein said antigen or particle are by being made by the cell of NANB hepatites virus infections.

19, according to the vaccine of claim 18, it is characterized in that, described virus antigen or described virion comprise a kind of protein, this protein portion ground by basically with the sequence coding of claim 10 order homologous, and this albumen is determined at least one epitope.

20, according to the vaccine of claim 19, it is characterized in that, described epitope can by the own chimpanzee that is infected by NANB or people's NANB serum antibody identification.

21, the non-B(PT-NANB of non-A after a kind of antiviral agent that contains antibody compositions, wherein said antibody can be attached to and transfuse blood) on the hepatitis virus particles; Described production of antibodies method is: give described host animal a kind of immunogenic composition; said composition contains separation from the PT-NANB of biological sample virion or the non-particulate antigen of PT-NANB virion inherent; described virion is characterised in that it can make by PT-NANB is infected responsive cell.

22, a kind of antiviral agent according to claim 21 is characterized in that, the Fc district of described antibody has at least a part to be removed.

23, a kind of antiviral agent according to claim 21 is characterized in that, described production of antibodies is permanence by a kind of hybridoma of preparation, and this hybridoma contains the cell that synthesizes described antibody of described host animal.

24, a kind of antiviral agent according to claim 23 is characterized in that, described hybridoma is people-people's hybridoma.

25, a kind of antiviral agent according to claim 24 is characterized in that, described antibody is monoclonal antibody.

26, a kind of hybridoma, its produce can with blood transfusion back non-A, non-B (PT-NANB) hepatitis virus particles bonded antibody.

27, a kind of hybridoma according to claim 26 is characterized in that, described antibody is monoclonal antibody.

28, a kind of anti-id AB composition, it contains its conformation at least basically similar in appearance to the antibody of natural non-A, non-B (PT-NANB) hepatitis virus antigen, and wherein said production of antibodies method is: the immunogenic composition that host animal is contained anti-PT-NANB antiviral antibody.

29, a kind of antiviral that strengthens has the method for the intravital effectiveness of host animal of blood transfusion back non-A, non-B (PT-NANB) hepatitis virus in the infection with described pharmacological agent, and described method comprises: with anti-PT-NANB antibody compositions in conjunction with the described host animal of described pharmacological agent.

30, according to the method for claim 29, it is characterized in that, described treatment comprises, give conformation at least basically similar in appearance to the anti-id AB of natural PT-NANB hepatitis virus antigen to described host animal, thereby in described host animal body, induce described anti-PT-NANB antibody compositions.

Images