JP2005053924A - Enterically transmitted non-a/non-b viral hepatitis factor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new composition comprising a viral protein derived from an enterically transmitted non-A/non-B viral hepatitis factor (ET-NANB) and fragments thereof, to provide a method for preparing the composition, and to provide a method for using the composition. <P>SOLUTION: The protein is such as to be derived from ET-NANB viral hepatitis factor having a genome containing a region homologous to the EcoRI insertional segment of 1.33 kb DNA existing in plasmid pTZ-KF1 (ET 1.1) borne on an Escherichia coli BB4 strain (ATCC 67717). The invention relates to the method for preparing the ET-NANB virus protein comprising the identification of an ET-NANB genomic sequence followed by cloning and expression in host cells. The recombinant virus protein is used as a diagnostic or a vaccine, and the genomic sequence and fragments thereof is used for preparing the ET-NANB virus protein and as probes for detecting virus. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

(Cross-reference for related applications)
This application is a continuation-in-part of U.S. Patent Application No. 336,672, filed April 11, 1989, and the latter is U.S. Patent Application No. 208,997, filed June 17, 1988. Is a continuation-in-part application. Both of these continuation applications are hereby incorporated by reference.

(Introduction)
(Field of Invention)
The present invention relates to recombinant proteins, genes and gene probes, and more particularly to proteins and probes derived from intestinal transmissible non-A non-B hepatitis virus factors. The present invention also relates to a diagnostic method using the protein and probe and application to a vaccine.

(background)
Intestinal tract non-A non-B (ET-NANB) hepatitis virus factor has been reported to cause some epidemic and diffuse hepatitis found in Asia, Africa and the Indian subcontinent. Infection is generally caused by contaminated water in the stool, but the virus can also be transmitted by close contact with the body. However, the virus is not expected to cause long-term infection. Etiology in ET-NANB has been demonstrated by volunteer infection using pooled stool isolates. In addition, studies by immunoelectron microscopy (IEM) showed the presence of virus particles with a diameter of 27-34 nm in the stool of infected persons. Because this viral particle reacts with serum antibodies from infected individuals in geographically separated areas, a single virus, one type of virus, is responsible for the majority of ET-NANB hepatitis found worldwide. It was suggested. It was pointed out that the two non-A non-B types have different specificities since antibody responses are not found in the serum of infected people with blood-borne non-A non-B viruses.

  In addition to serological differences, two types of non-A non-B infection have different clinical features. ET-NANB has a characteristic acute infection, often accompanied by fever and joint pain, and portal vein inflammation and bile congestion in liver biopsy specimens (Alankare). Symptoms usually disappear within 6 weeks. In contrast, blood-borne non-A non-B results in a chronic infection in about 50% of cases. In this case, the development of fever and joint pain is rare and inflammation is prominent in the parenchyma (Couleau, 1980). The two viruses can also be distinguished on the basis of primate host sensitivity. Unlike blood-borne viruses, ET-NANB can be transmitted to cynomolgus monkeys. Bloodborne viruses infect chimpanzees more easily than ET-NANB (Bradley, 1987).

  Great efforts have been made around the world to identify and clone the viral genomic sequences associated with ET-NANB hepatitis. One goal of this effort is to identify and characterize the nature of the virus and its production proteins with the aim of elucidating virus-specific genomic sequences. Another goal is to produce recombinant viral proteins that can be used in antibody diagnostics and vaccines. Contrary to such efforts, the nucleotide sequence of the virus related to ET-NANB hepatitis has not been identified and cloned so far, and no virus-specific protein has been identified and produced.

(Related literature)
V. A. Alancare et al., The Lancet, 550 (March 12, 1988)
D. W. Bradley et al., J Gen. Virol. 69: 1 (1988)
D. W. Bradley et al., Proc. Natl. Acad. Sci. USA, 84: 6277 (1987).
C. R. Gravera et al. Infect. Diseases, 131: 167 (1975)
M.M. A. Carne et al., JAMA, 252: 3140 (1984)
M.M. S. Coulomb Am. J. et al. Med, 48: 818 (1980)
M.M. S. Kuoguchi et al., Am. J. et al. Med. 68: 818 (1983)
T.A. Maniatis et al., Molecular Cloning: A Laboratory Manua1, Cold Spring Harbor Laboratory (1982)
B. Seto et al., Lancet, 11: 941 (1984).
M.M. A. m. Srinivasan et al. Gen. Viro1. , 65: 1005 (1984)
E. Tabor et al. Infect. Dis. 140: 789 (1979)

(Summary of the Invention)
The present invention provides a novel composition comprising a viral protein derived from the ET-NANB viral factor and fragments thereof, and methods for preparing and using the composition. Preparation of ET-NANB viral protein involves identification of the ET-NANB genomic sequence, followed by cloning and expression in a host cell injection. The resulting recombinant viral protein is used as a diagnostic and vaccine. Genomic sequences and fragments thereof are used for the preparation of ET-NANB viral proteins and as detection probes for viruses.

  The present invention relates to intestinal transmissibility having a genome containing a region homologous to an EcoRI insert of 1.33 kb DNA present in plasmid pTZ-KF1 (ET1.1) carried in E. coli BB4 strain of ATCC Deposit No. 67717. Proteins derived from non-A non-B hepatitis virus factors are provided.

  In a preferred embodiment, the protein is encoded by a coding region in the 1.33 kb EcoRI insert.

The present invention also provides a non-intestinal transmissible non-intestinal tract having a genome comprising a region homologous to double-stranded DNA having the following first sequence, second sequence, third sequence or fourth sequence, or a sequence complementary thereto. Recombinant protein derived from A non-B hepatitis virus factor:
First array

Second array

3rd array

4th array

  The present invention also includes plasmid pTZ-KF1 (ET1.) Which is (a) immunoreactive with antibodies present in intestinal transmissible non-A non-B infected individuals and (b) carried in E. coli BB4 strain of ATCC Deposit No. 67717. Provided is a protein derived from intestinal transmissible non-A non-B hepatitis virus factor having a genome containing a region homologous to the EcoRI insert of 1.33 kb DNA present in 1).

  In a preferred embodiment, the protein is encoded by a coding region in the EcoRI insert of the 1.33 kb DNA.

  The present invention also provides a method for detecting intestinal transmissible non-A non-B virus infection in a test individual, comprising: (a) immunoreactive with an antibody present in an intestinal infectious non-A non-B infected person; ) Intestinal transmissible non-A non-B having a genome containing a region homologous to the EcoRI insert of 1.33 kb DNA present in plasmid pTZ-KF1 (ET1.1) carried in E. coli BB4 strain of ATCC Deposit No. 67717 A detection method comprising preparing a peptide antigen derived from a hepatitis virus factor, reacting serum obtained from a test individual with the antigen, and detecting the antibody to examine the presence of the bound antibody. I will provide a.

  In a preferred embodiment, in the detection method described above, the serum antibody is an IgM or IgG antibody or a mixture of both, the prepared antigen is bound to a support, and the reaction is performed between the antibody and the support. And the detection comprises a reaction of the support and bound serum antibody with a reporter labeled anti-human antibody.

  The present invention also provides a kit for confirming the presence of a serum antibody capable of diagnosing intestinal transmissible non-A non-B hepatitis infection, comprising: Reactive and the genome of (b) contains a region homologous to the EcoRI insert of 1.33 kb DNA present in the plasmid pTZ-KF1 (ET1.1) carried in the E. coli BB4 strain of ATCC Deposit 67717 Provided is a kit comprising a support having a surface-bound recombinant peptide antigen derived from intestinal transmissible non-A non-B hepatitis virus factor having a genome, and a reporter-labeled anti-human antibody.

  The present invention further relates to intestinal transmissibility having a genome comprising a region homologous to an EcoRI insert of 1.33 kb DNA present in plasmid pTZ-KF1 (ET1.1) carried in E. coli BB4 strain of ATCC Deposit No. 67717. DNA fragments derived from non-A non-B hepatitis virus factors are provided.

  In a preferred embodiment, the fragment is derived from the 1.33 kb EcoRI insert.

The present invention further includes the following first sequence, second sequence, third sequence or fourth sequence, or an intestinal transmissible non-intestinal tract having a genome containing a region homologous to double-stranded DNA having a sequence complementary thereto. DNA fragment derived from A non-B hepatitis virus factor:
First array

Second array

3rd array

を提供する。 4th array

I will provide a.

  In a preferred embodiment, the DNA fragment comprises a coding sequence homologous to a sequence complementary to the second to 101st nucleotides of the first sequence, the 594th to 643th nucleotides of the second sequence, or the coding sequence. Including.

  The present invention also provides a region homologous to an EcoRI insert of 1.33 kb DNA present in plasmid pTZ-KF1 (ET1.1) carried in E. coli BB4 strain of ATCC Deposit No. 67717 in one or more containers. A kit comprising a pair of single-stranded primers derived from homologous regions of double-stranded DNA alleles derived from intestinal transmissible non-A non-B hepatitis virus factor having a genome comprising:

  In a preferred embodiment, the kit primers are derived from the opposite strand of the EcoRI duplex insert in the plasmid.

  The present invention is also a method for detecting the presence or absence of intestinal transmissible non-A non-B hepatitis virus factor in a biological sample, comprising preparing a mixture of double-stranded DNA fragments derived from a sample and denaturing the double-stranded fragments The denatured DNA fragment has a genome containing a region homologous to the EcoRI insert of 1.33 kb DNA present in the plasmid pTZ-KF1 (ET1.1) carried in the E. coli BB4 strain of ATCC No. 67717 A pair of single-stranded primers derived from the homologous regions of double-stranded DNA alleles derived from intestinal tract non-A non-B hepatitis virus factor are added, and two from intestinal tract non-A non-B hepatitis virus factor The primer is hybridized in the presence of DNA nucleotides until the primer is hybridized to the homologous region of the allele of the strand DNA fragment and the amplification of the sequence is reached. Is reacted with NA polymerase, to form DNA2 stranded new comprising said primer sequence, and the modified, added, hybrid, and sequence repeated the steps of, providing a detection method comprising the.

  In a preferred embodiment, in the above method, the primer is derived from the opposite strand of the EcoRI insert double strand in the primer.

  In another preferred embodiment, the method is used to detect the presence or absence of a viral factor in a cultured cell sample infected with the viral factor.

  The present invention further relates to a vaccine for immunizing an individual against intestinal transmissible non-A non-B hepatitis virus factor, a physiologically acceptable adjuvant, and a plasmid carried by E. coli BB4 strain of ATCC No. 67717 Allele homology of double-stranded DNA derived from intestinal transmissible non-A non-B hepatitis virus factor having a genome containing a region homologous to the EcoRI insert of 1.33 kb DNA present in pTZ-KF1 (ET1.1) A vaccine comprising a recombinant protein derived from the region is provided.

  In a preferred embodiment, the vaccine is such that the protein is derived from an EcoRI insert in the plasmid.

  The present invention also provides a method for isolating intestinal tract non-A non-B hepatitis virus factor or a nucleic acid fragment produced by the viral factor, wherein the virus factor source is infected with intestinal tract non-A non-B hepatitis. Provided is a separation method characterized by utilizing bile obtained from a human or cynomolgus monkey.

  In a preferred embodiment, in the above method, bile is obtained from an infected cynomolgus monkey.

  The present invention further relates to intestinal transmissibility having a genome comprising a region homologous to an EcoRI insert of 1.33 kb DNA present in plasmid pTZ-KF1 (ET1.1) carried in E. coli BB4 strain of ATCC Deposit No. 67717. Provided is a human polyclonal antiserum obtained from a human immunized with a protein derived from a homologous region of an allele of double-stranded DNA derived from non-A non-B hepatitis virus factor.

  The present invention provides a novel composition comprising a viral protein derived from the ET-NANB viral factor and fragments thereof, and methods for preparing and using the composition. Preparation of ET-NANB viral protein involves identification of the ET-NANB genomic sequence, followed by cloning and expression in a host cell injection. The resulting recombinant viral protein is used as a diagnostic and vaccine. Genomic sequences and fragments thereof are used for the preparation of ET-NANB viral proteins and as detection probes for viruses.

(Description of special aspects)
Compositions comprising gene sequences derived from ET-NANB virus and fragments thereof, as well as recombinant viral proteins produced using the genomic sequences and methods of using the compositions are provided.

  The genome of the ET-NANB virus factor has been confirmed to contain a region homologous to the EcoRI insert of 1.33 kb DNA carried in E. coli strain BB4 of ATCC Deposit No. 67717. Early studies determined the sequence of both terminal and intermediate regions of this insert. The 5 'terminal region of this insert contains the following sequence:

The middle region has the following sequence:

The 3′-terminal region has the following sequence:

  As discussed below, additional studies provided full sequences in both directions. Since it is unclear which DNA strand the gene is located on, the sequences of both DNA strands are provided. However, due to the statistical similarity of known proteins, unidirectional sequences are called “forward” sequences. This sequence is described below along with three possible translation sequences. There is one long open reading frame that begins at nucleotide 145 for isoleucine and extends to the end of the sequence. There are many stop codons in the other two open reading frames. In this specification, standard abbreviations for nucleotides and amino acids are used.

  The complementary strand referred to herein as the “reverse sequence” is described below in the same manner as the forward sequence shown above. Some open reading frames are shorter than the long open reading frames found in the forward sequence, and this reverse method sequence is also found.

  Identification of the above sequence as a sequence in a virulence factor was confirmed by determining a sequence corresponding to a virus strain isolated in Burma. This Burma isolate contains the following nucleotide sequence:

Non-A non-BET: Burma stock

  The ability of the methods described herein to isolate and identify genetic material from other NANB hepatitis strains was confirmed by identifying genetic material from isolates obtained in Mexico. The sequence of the above isolate showed 75% identity with the ET1.1 sequence. The sequence is shown in II. It was identified by hybridization under the conditions described in Section B. A partial sequence of cDNA consisting of 1611 nucleotides is shown below.

Non-A non-BET: Mexican stock

  When the above Burma strain and the Mexican strain are compared, duplication is recognized at the 1372th nucleotide starting from the 13th nucleotide of the Mexican strain and the 331st nucleotide of the Burma strain.

  It will be apparent to those skilled in molecular genetics that both of the above two sequences disclose opposite complementary DNA sequences and RNA sequences for both the main and complementary DNA. In addition, there is an open reading frame encoding a peptide, and an expressible peptide does not specify the amino acid sequence and is disclosed by the above nucleotide sequence. It is done in the same way as when the amino acid is known, as in the case of the ET1.1 sequence.

(Detailed description of the invention)
(Section I Definition)
The terms defined below have the following meanings:

  1. “Intestinal tract non-A non-B (ET-NANB) hepatitis virus factor” means (1) causes water-borne infectious hepatitis, (2) can be transmitted in cynomolgus monkeys, (3) hepatitis A virus (HAV) (4) a genomic region homologous to the 1.33 kb cDNA in plasmid pTZ-KF1 (ET1.1) carried in E. coli BB4 strain identified as ATCC deposit 67717 A virus, a virus type or a virus species.

  2. If two nucleic acid fragments are capable of hybridizing to each other under hybridization conditions that contain no more than about 25-30% base pair mismatches in the hybrid strand, then these fragments are “homologous”. In general, two short nucleic acid species are washed under the conditions described in Maniatis et al. (Op. Cit., Pp. 320-323) [2 × SCC, 0.1% SDS, twice at room temperature for 30 minutes each. 2 × SCC, 0.1% SDS, 50 ° C. once for 30 minutes; 2 × SCC, 0.1% SDS, twice at room temperature for 10 minutes each], these nucleic acids are said to be homologous. Like. More preferably, the homologous nucleic acid strand contains 15-25% base pair mismatch, and more preferably 5-15%. These degrees of homology can be selected by changing the washing conditions of the gene library (or other source of genetic material) as is well known in the art.

  3. A DNA fragment is “derived from” a viral factor if it has the same or substantially the same base pair as the ET-NANB viral factor genome.

  4). A protein is “derived from” a viral factor if it is encoded in the open reading frame of DNA or RNA from the ET-NANB viral factor.

(Section II Collection of Cloned ET-NANB Fragments)
According to one aspect of the present invention, virus-specific DNA clones can be obtained by: (a) isolating RNA from bile of cynomolgus monkeys with known ET-NANB infections; (b) cloning cDNA fragments to live fragments And (C) screening the library by discriminating hybridization against radiolabeled cDNA obtained from infected or non-infected bile sources.

(A) cDNA fragment mixture ET-NANB infection in cynomolgus monkeys was performed using a 10% by volume suspension obtained from the stool of an infected human (27-34 nm ET-NANB particles-average particle size 32 nm-positive). Begin by inoculating intravenously. Infected animals are monitored for elevated levels of alkaline aminotransferase and examined for hepatitis infection. ET-NANB infection is confirmed by examining the immunospecific binding of serologically positive antibodies according to the existing method (Grabber). That is, a stool (or bile) specimen obtained from an infected animal 3 to 4 weeks after infection was diluted 10-fold with a phosphate buffer, and this 10% suspension was clarified by low-speed centrifugation. And filter through a 0.45 micron filter. The material can be further purified by pelleting through a 30% sucrose layer (Bradley). After overnight incubation, the mixture is centrifuged overnight to pellet immunoaggregates, they are stained, and the VLP-conjugated antibody is examined with an electron microscope.

  ET-NANB infection can also be confirmed by seroconversion of VLP positive serum. Here, the sera of infected animals are mixed as described above with 27-34 nm VLPs separated from human stool specimens with infectious diseases, and antibodies that bind to VLPs are examined by immunoelectron microscopy.

  Bile can be collected by intubation into the bile duct and collection of bile, or bile duct drainage during autopsy. Total RNA is extracted from bile by hot phenol extraction as outlined in Example 1A. The RNA fragment is used to synthesize considerable double-stranded cDNA by random priming, also as outlined in Example 1A. The cDNA can be fractionated by gel electrophoresis or density gradient centrifugation to obtain fragments of the desired size (ie, 500-4, OOO base pair fragments).

  Other viral materials such as VLPs obtained from stool specimens (described in Example 4) can also be used to produce cDNA, but bile sources are preferred. According to one aspect of the invention, bile obtained from monkeys infected with ET-NANB has a greater number of complete virus particles than material obtained from stool samples, as revealed by immunoelectron microscopy. I understood that. Bile obtained from ET-NANB infected humans or cynomolgus monkeys (for use as a source of ET-NANB viral protein or genomic material), or complete virus, forms part of this invention.

(B) cDNA library and screening The cDNA fragment obtained above is cloned into an appropriate cloning vector to form a cDNA library. This can be done by connecting an appropriate end linker, such as an EcoRI sequence, to the blunt end of the fragment and inserting the fragment into the appropriate insertion site of the cloning vector as at the unique EcoRI site. After initial cloning, the percentage of the vector containing the fragment insert can be increased if necessary. The creation of the library described in Example 1B is an example. Here, the cDNA fragment is blunt-ended, connected to the EcoRI site, and inserted into the EcoRI site of the λ phage vector. Library phage are less than 5% of the insert, and when this is isolated and the fragment insert is recloned into the λgt10 vector, more than 95% insert-containing phage is produced.

  The cDNA library is screened against ET-NANB specific sequences by differential hybridization with cDNA probes from infected or non-infected sources. CDNA fragments obtained from bile or stool isolates of infected or non-infected sources can be prepared as described above. Fragment radiolabeling is by random labeling, nick translation or end labeling according to conventional methods (Maniatis, p. 109). The cDNA library is screened by transfer to two sets of nitrocellulose filters and hybridization with infectious and non-infectious (control) radiolabeled probes as described in Example 2. In order to hybridize at a preferable upper limit of 25 to 30% of base pair mismatch, the conditions described in Maniatis et al. (Op. Cit., Pp. 320-323) and washing conditions [2 × SCC, O.D. 1% SDS, 2 times at room temperature for 30 minutes each; 2 × SCC, 0.1% SDS, once at 50 ° C. for 30 minutes; 2 × SCC, O.D. The clones are selected by examining whether or not to hybridize according to 1% SDS, twice at room temperature for 10 minutes each]. Under these conditions, the above Mexican isolate can be identified by using ET1.1 as the probe. Plaques that show selective hybridization to an infectious source probe are preferably re-plated at a low plate concentration and isolate single clones that are spectral in the ET-NANB sequence. As shown in Example 2, 16 clones that specifically hybridize with the source probe were identified through a series of procedures. One of these clones is designated λgt101.1 and contains a 1.33 kb insert.

(C) ET-NANB sequence The base sequence of the cloning region of the ET-NANB fragment from (B) is determined by standard sequencing methods. In one example described in Example 3, an insert fragment from a selected cloning vector is cut out and inserted into a cloning vector whose base sequence on one side of the insertion site is unknown. The specific vector used in Example 3 is the pTZ-KF1 vector shown on the left side of FIG. The ET-NANB fragment from λgt101.1 phage was inserted into the unique EcoRI site of the pTZ-KF1 plasmid. Recombinants with the desired insert were identified by hybridization with the isolated 1.33k fragment as described in Example 3. One selected plasmid is identified as pTZ-KF1 (ET1.1) and after digestion of the vector with EcoRI yields a 1.33 kb fragment as expected.

  E. coli BB4 strain infected with pTZ-KF1 (ET1.1) plasmid was deposited with the American Standard Culture Collection (Rockville, MD) and identified as ATCC Deposit No. 67717.

  The pTZ-KF1 (ET1.1) plasmid is illustrated at the bottom of FIG. This insert has a 5 'and 3' end region designated A and B, respectively, and an intermediate region designated B. These regions are sequenced by standard dioxy methods. The three short sequences (A, B and C) are of the same insert strand. As seen in Example 3, since the sequence of the B region was actually determined from the allelic strand, the above B region sequence is the complementary sequence of the sequenced strand. The number of bases in the partial sequence is an approximate value.

  In our subsequent work, the complete sequence was identified as described above. Each fragment of this entire sequence can be easily prepared using restriction endonucleases. A number of cleavage sites were identified by computer analysis of both the forward and reverse method sequences. Specific cleavage sites (in the forward direction) are shown in the table below.

(Section III ET-NANB fragment)
According to another aspect, the invention includes an ET-NANB genomic sequence or a cDNA fragment derived therefrom. These fragments may include full-length cDNA fragments as described in Section II, or may be derived from short sequence regions in the cloned cDNA fragment. Short fragments can be prepared by enzymatic digestion of full length fragments under conditions that yield fragments of the desired size. This is described in Section IV. Fragments can also be prepared by oligonucleotide synthesis using sequences derived from cDNA fragments. Methods or institutions that prepare oligonucleotide fragments of a specific sequence can also be used.

  To confirm that an ET-NANB fragment is actually derived from ET-NANB viral factor, it is shown that the fragment selectively hybridizes with cDNA from the infectious source. For example, to confirm that the 1.33 kb fragment of the pTZ-KF1 (ET1.1) plasmid originated from ET-NANB, the fragment was excised from pTZ-KF1 (ET1.1), purified, and randomized. Radiolabeled by labeling. Radiolabeled fragments were hybridized with fractionated cDNA from infected or non-infected sources to ensure that the probe reacted only with the infectious source cDNA. This method is shown in Example 4, which examines whether the radiolabeled 1.33 kb fragment from the pTZ-KF1 (ET1.1) plasmid binds to cDNA prepared from infected and non-infected sources. It was. The sources of infection were (1) bile from cynomolgus monkeys infected with a virus strain from a stool sample of a Burma patient with a known ET-NANB infection, and (2) of a person infected with ET-NANB in Mexico. A viral factor derived from a stool sample. The cDNA in each fragment mixture was first amplified by the linker / primer amplification method described in Example 4. Fragment separation was carried out on an agarose gel, followed by the Southern method, and further by hybridization to fractionated cDNA. The column containing cDNA from the infectious source, as expected, showed a smear band of bound probe (cDNA amplified by the linker / probe amplification method was expected to have a wide range of sizes). No probe was found bound to amplified cDNA from a non-infected source. This result shows that the 1.33 kb probe is specific for the cDNA fragment associated with ET-NANB infection. This same type of study showed a tendency for hybridization to ET-NANB samples collected from Tashkent, Somalia, Borneo and Pakistan using ET1.1 as a probe. Second, due to the fact that the probe is specific for ET-NANB related sequences from various continents (Asia, Africa and North America), cloned ET-NANB sequences from Africa are -It can be seen that it is derived from NANB virus (i.e. virus species responsible for worldwide ET-NANB hepatitis infection).

  A related confirmatory study examined the binding of probes to fractional genomic fragments prepared from human or rhesus monkey genomic DNA. Probe binding was not observed for either genomic fragment, indicating that the ET-NANB fragment is not an endogenous fragment of human or rhesus monkey.

  Another means of confirming the ET-NANB specific sequence in the fragment is the ability to express the ET-NANB protein obtained from the coding region in the fragment. Section IV below discusses protein expression methods using fragments.

  One important use of ET-NANB specific fragments is to identify ET-NANB derived cDNAs that contain other information about the sequence. The newly identified cDNA subsequently gives rise to new fragment probes, allowing further reaction until the entire viral genome has been identified and sequenced. The procedure for identifying additional ET-NANB library clones and for generating new probes therefrom is performed after the cloning and selection described in Section II.

  Fragments (and oligonucleotides prepared based on the sequences below) are also useful as polymerase chain reaction probes to detect patient ET-NANB genomic material. This diagnostic method will be described in Section V below.

(Section IV ET-NANB protein)
As described above, ET-NANB protein can be prepared by expressing an open reading frame region with an ET-NANB fragment. In one preferred embodiment, the ET-NANB fragment used for protein expression has undergone a process that produces fragments of the desired size, preferably random fragments with a major size between about 100 and 300 base pairs. It is derived from cDNA. Example 5 describes a method for preparing fragments by DNA digestion. Since it is preferable to obtain a peptide antigen having about 30 to 100 amino acids, it is desirable that a digested fragment having a range of about 100 to 300 base pairs is fractionated by size, for example, by gel electrophoresis.

(A) Expression vector The ET-NANB fragment is inserted into an appropriate expression vector. An exemplary expression vector is λgt11, which contains a unique EcoRI insertion site 53 base pairs upstream of the translation termination codon of the β-galactosidase gene. Thus, the inserted sequence is the N-terminal gene of β-galactosidase, the heteropeptide, and optionally the C-terminus of the β-galactosidase peptide (the C-terminal portion is when the heteropeptide encoding the sequence does not have a translation initiation code. Expressed). This vector also produces a temperature sensitive repressor (c1857) that renders the virus lysogenic at permissive temperatures (eg, 32 ° C.) and lyses the virus at high temperatures (eg, 42 ° C.). Furthermore, since an inactive β-galactosidase containing a heteroinsert is produced, a phage with the insert can be easily identified by a β-galactosidase color-substrate reaction.

  For insertion into an expression vector, the digested fragment of the virus can be modified as necessary to include a specific restriction site linker, such as an EcoRI linker, according to conventional methods. Example 1 exemplifies a method of cloning a digested fragment into λgt11, which includes a step of making the fragment a blunt end, a step of ligating with an EcoRI linker, and introducing the fragment into λgt11 excised with EcoRI. A process is included. The resulting viral genome library can also be examined to confirm that a relatively large (representative) library is produced. In the case of the λgt11 vector, this can be done by infecting a suitable bacterial host, plating the bacteria and finding a loss of β-galactosidase activity in the plaque. Using the method described in Example 1, loss of enzyme activity was observed in about 50% of the plaques.

(B) Expression of peptide antigens The viral genome library created above is screened for the production of peptide antigens (expressed as fusion proteins) that immunoreact with antisera from ET-NANB serotype-positive patients. . In a preferred screening method, the phage library genome is plated onto a plate as described above, the plate is blotted with a nitrocellulose filter, and the recombinant protein antigen produced by the cells is transferred onto the nitrocellulose filter. Subsequently, the filter is reacted with ET-NANB antiserum, washed to remove unbound antibody, and reacted with a reporter-labeled anti-human antibody. In the sandwich method, this antibody is bound to the filter via an anti-ET-NANB antibody.

  In general, phage plaques identified by examining production of the recombinant antigen of interest are re-examined at relatively low concentrations for production of antibody-reactive fusion proteins. Several recombinant phage clones producing immunoreactive recombinant antigens were identified in this way.

  The selected expression vector can also be used for mass production for the purpose of purifying the recombinant protein. Mass production consists of (a) lysogenic a suitable host such as E. coli with λgt11 recombinant, (b) culturing the transfected cells under conditions that produce high levels of heteropeptides, and (c) from the lysed cells This is done using one of a variety of conventional methods for purifying recombinant antigens.

  One preferred method for the λgt11 cloning vector described above involves infecting a highly productive E. coli host BNN103 with a given library and replicating in two plates. One of these plates is cultured at 32 ° C. where lysogenicity of the virus occurs, and the other is cultured at 42 ° C. where the infected phage is in a lysed state and suppresses cell growth. Thus, cells that proliferate at low temperatures but do not proliferate at high temperatures are thought to skillfully lysate.

  Subsequently, the lysogenized host cells are grown under conditions favorable for high production of the fusion protein containing the viral insert, and lysed by rapid freezing to release the desired fusion protein.

(C) Peptide purification Recombinant peptides are purified by standard protein purification methods such as fractional precipitation, molecular sieve chromatography, ion exchange chromatography, isoelectric focusing, gel electrophoresis, affinity chromatography, etc. be able to. In the case of a fusion protein such as the β-galactosidase fusion protein prepared as described above, the protein separation technique used can be applied from the one used for the separation of the original protein. Thus, for the separation of β-galactosidase fusion protein, this protein is easily separated by simple affinity chromatography (a method in which cell lysate is passed over a solid support having a surface-bound anti-β-galactosidase antibody). be able to.

(D) Viral protein The ET-NANB of this invention may be derived directly from the ET-NANB viral factor. As noted above, VLPs separated from infected stool samples are a suitable source of viral proteins. VLPs separated from stool samples can be further purified by chromatography prior to protein separation (see below). Viral factors are also expressed in cultured cells, which may be a convenient source of viral protein enrichment.

  Shared US Pat. No. 846,757, filed Apr. 1, 1986, describes permanently dividing trioma hepatocytes that support NANB infection in cultured cells. Trioma cell lines were selected with a focus on human chromosome stability. Prepared by fusing mouse / human fusion cells and human hepatocytes. Cells containing the desired NANB viral factor can be identified by immunofluorescence using an anti-ET-NANB human antibody.

  Viral factors are disrupted by conventional methods (sonication, high or low salt treatment, or use of detergents) prior to protein separation.

  Purification of the ET-NANB viral protein can be performed by affinity chromatography using an anti-ET-NANB antibody conjugated by standard methods. For separation of the anti-ET-NANB antibody from the immune serum source, the antibody itself can be purified by affinity chromatography in which the immunoreactive recombinant ET-NANB protein as described above is bound to a solid support. The bound antibody is released from the support by standard methods.

  In addition, anti-ET-NANB antibody immunizes mice and other animals with recombinant ET-NANB protein, isolates lymphocytes obtained from animals, and allows cells to acquire permanent division by appropriate fusion cells And a monoclonal antibody (Mab) prepared by selecting a fusion protein that reacts with the recombinant protein immunogen. These can then be purified by affinity chromatography as described above to obtain the original ET-NANB antigen.

(Section V Usage)
(A) Diagnostic Method The particles and antigens and genetic material of the present invention can be used for diagnostic methods. A method for examining the morbidity of ET-NANB hepatitis involves analyzing biological samples such as blood samples, stool samples, and liver biopsy specimens for the presence of ET-NANB liver virus related subjects.

  The analyte may be a nucleotide sequence that hybridizes with a probe comprising a sequence from at least about 16 nucleotides (usually 30 to 200 nucleotides) to substantially the entire sequence of the above base sequence (cDNA). The analyte may be RNA or cDNA. In general, the analyte is ET-NANB or a virus particle suspected of having non-classifiable particles. In addition, the virion is characterized by a sequence that is at least about 80% homologous (generally at least about 60 consecutive in the sequence) to at least 12 consecutive nucleotide sequences of the “forward” and “reverse” sequences described above. The virus particle may comprise a sequence that is substantially homologous to the full-length sequence, having an RNA viral genome consisting of a nucleotide sequence that is at least 90% homologous to the nucleotide). To detect an analyte that hybridizes to a probe, the probe may contain a detectable label.

  The analyte may also consist of an antibody that recognizes an antigen, such as a cell surface antigen, on ET-NANB. Further, the subject can be an ET-NANB virus particle. When the analyte is an antibody or antigen, either the labeled antigen or antibody can be bound to the analyte to form an immune complex, which is detected via the label .

  In general, detection methods for analytes (such as surface antigens and / or total particles) are based on immunoassays. Immunoassays can be performed by measuring host antibodies formed by ET-NANB hepatitis virus infection, or by detection methods that directly measure the presence of viral particles or antigens. These techniques are known and need not be described in detail here. For example, both hetero and homoimmunoassay methods are possible. Both approaches are based on the formation of immune complexes between viral particles or viral antigens and the corresponding specific antibodies. Heterodetection methods for viral antigens generally use specific monoclonal or polyclonal antibodies bound to a solid surface. The sandwich method is becoming increasingly popular. Homo detection methods do not require a solid phase and are performed in solution, for example, by measuring the difference in enzyme activity caused by free antibody binding to the enzyme-antigen conjugate. A number of suitable detection methods are disclosed in US Pat. Nos. 3,817,837, 4,006,360, and 3,996,345.

  When assaying for the presence of antibodies induced by ET-NANB virus, the viruses and antigens of this invention can be used as specific binding agents to detect either IgG and IgM antibodies. Since IgM antibodies are the first antibodies that appear during the course of infection, physicians and other researchers can identify infections by distinguishing between IgG and IgM present in the host's blood before IgG synthesis begins. Can determine if is acute or chronic.

  In one diagnostic method, test serum is reacted with a solid phase reagent having a surface-bound protein antigen. Anti-ET-NANB antibody is bound to the reagent, unbound components are removed by washing, and then the reagent is reacted with the reporter-labeled anti-human antibody, proportional to the amount of anti-ET-NANB antibody bound on the solid support. The reporter binds to the reagent. The reagent is washed again to remove unbound labeled antibody and the amount of reporter bound to the reagent is measured. Usually, the reporter is an enzyme that is detected by incubating the solid phase in the presence of a suitable fluorescent or chromogenic substrate.

  The solid surface reagent in the above detection method is prepared by known methods for binding protein substances to solid support materials such as polymer beads, dipsticks, filter materials. These attachment methods usually involve non-specific adsorption of the protein to the support, or generally through a free amino group, to a chemically reactive group on the solid support, such as an activated carboxyl group, hydroxyl group or aldehyde group. The covalent bond of the protein.

  In a second diagnostic method, known as a homodetection method, an antibody that binds to a solid support results in a change in the reaction medium that can be assayed directly in the medium. In the general type of homo-assay method, (a) a spin label reporter detected by a change in mobility (spread splitting peak broadening) in which an antigen-binding antibody has been reported, and (b) binding is fluorescence efficiency. Fluorescent reporters detected by the change in pH, (C) an enzyme reporter in which antibody binding causes an enzyme / substrate interaction, and (d) a liposome-binding reporter in which the binding leads to dissolution of the liposome and release of the encapsulated reporter . Following conventional methods for the preparation of homodetection reagents, a series of methods for the protein antigens of this invention are applied.

  Each of the detection methods described above may involve a step of reacting serum from a subject with a protein antigen and a step of examining the presence of a bound antibody with the antigen. For the test, as in the first method, the step of reacting a labeled anti-human antibody against a test antibody (IgM in the acute phase or IgG in the recovery phase) and the amount of reporter bound to the solid support are determined. A measuring step may be involved. Further, as in the second method, a step of observing the effect of antibody binding on the homoassay reagent may be involved.

  Further, part of the invention is an assay system or kit for carrying out the assay described above. The kit generally comprises a support with a surface-bound recombinant protein antigen, which is (a) immunoreactive with antibodies present in an infected person due to intestinal infectious non-A non-B viral factor. (B) a region where the viral hepatitis factor genome is homologous to the 1.3 kb DNA EcoRI insert present in plasmid KF1 (ET1.1) carried in E. coli BB4 (ATCC Deposit No. 67717). Including the viral hepatitis factor. The reporter-labeled anti-human antibody of the kit is used to detect surface-bound anti-ET-NANB antibody.

(B) Application of viral genome diagnostic method The genetic material of the present invention itself can be used in various assays as a probe for genetic material in a naturally occurring infectious agent. An amplification method with a target nucleic acid is a subsequent analysis by a hybridization method, but is known as a polymerase chain reaction (PCR method). This PCR method can be applied to detection of virus particles of the present invention in a sample suspected of being infected. The detection is based on the gene sequence described above, using oligonucleotide primers separated from each other. These primers are complementary to opposite strands of a double-stranded DNA molecule and are separated by about 50 to 450 or more nucleotides (usually 2000 nucleotides or less). In this method, specific oligonucleotide primers are prepared, repeated cycles of target DNA denaturation and extension with DNA polymerase are performed to obtain the expected fragment relative to primer spacing. Extension products generated from one primer also serve as target sequences for other primers. The degree of amplification of the target sequence is controlled by the number of repetitions of the cycle, and theoretically calculated by the simple formula 2 ⁿ (n is the number of cycles). If the average efficiency per cycle is in the range of about 65% to 85%, there will be 0.3 to 48,000 target sequence copies in 25 cycles. PCR methods have been published in numerous publications (such as Saiki et al., Science (1985) 230: 1350-1354; Saiki et al., Nature (1986) 324-163-166; and Sharf et al., Science (1986) 233: 1076-1078). It is described in. See also U.S. Pat. Nos. 4,683,194, 4,683,195, and 4,683,202.

  This invention includes specific diagnostic methods for determining ET-NANB viral factors based on selective amplification of ET-NANB fragments. This method uses single-stranded paired primers derived from the heterologous regions of the opposite strand of the double-stranded fragment. This double-stranded fragment is derived from intestinal infectious viral hepatitis factor, and the genome of this factor is contained in plasmid pTZ-KF1 (ET1.1) carried in E. coli BB4 strain (ATCC Deposit No. 67717). It contains a region homologous to the EcoRI insert of the existing 1.33 kb DNA. These primer fragments form one aspect of the invention, but are prepared from ET-NANB fragments as described in Section III above. This method is followed by a method of amplifying a specific nucleic acid sequence as described in US Pat. No. 4,683,202 above.

(C) Peptide vaccine Any antigen of this invention can be used in the preparation of a vaccine. A preferred starting material for vaccine preparation is particulate antigen separated from bile. These antigens are preferably initially recovered as complete particles as described above. However, it is also possible to prepare a suitable vaccine with particles separated from other origins or non-particulate recombinant antigens. When using non-particulate antigens (generally soluble antigens), it is preferred to use viral envelopes or capsids for vaccine preparation. These proteins can be purified by affinity chromatography (see above).

  If this purified protein does not have immunity by itself, it can be bound to a carrier to obtain a protein immunogen. These carriers include bovine serum albumin, keyhole limpet hemocyanin, and the like. It is desirable, but not always, to purify antigens that are substantially free of human proteins. More importantly, however, the antigens do not contain proteins, viruses, or other non-human sources. These may be introduced from nutrient media, cell lines, or pathogens from which the virus is cultured and harvested, or by their contamination.

  Vaccination can be performed by conventional methods. For example, the antigen can be used in a suitable dilution such as water, saline, salted buffer, complete or incomplete adjuvant, whether virus particles or proteins. The immunogen is administered using standard methods of antibody induction, such as by subcutaneous injection of a physiologically compatible sterile solution containing inactivated or attenuated viral particles or antigens. The amount of viral particles that cause an immune response is usually administered in a volume of 1 ml or less of vaccine injection.

  Specific examples of vaccine compositions include recombinant proteins or protein mixtures derived from intestinal infectious non-A non-B hepatitis factors mixed in a physiologically acceptable adjuvant. This includes a region homologous to the EcoRI insert of 1.33 kb DNA present in plasmid pTZ-KF1 (ET1.1) carried in E. coli BB4 strain (ATCC Deposit No. 67717). The vaccine is administered periodically until a significant titer of ET-NANB is detected in the serum. This vaccine administration is for preventing ET-NANB infection.

(D) Antibodies and antigens intended for prevention and treatment In addition to use as vaccines, the above compositions can be used to prepare antibodies against viral particles. To prepare the antibodies, the host animal is immunized with the viral particles or, if necessary, the original non-particulate antigen of the viral particles is bound to a vaccine carrier as described above. When the serum or plasma of the host is collected at an appropriate time interval, a composition containing an antibody reactive to virus particles is obtained. IgG fractions or IgG antibodies are obtained, for example, using saturated NH ₄ SO ₄ or DEAE Sephadex or using methods known to those skilled in the art. These antibodies are substantially free of a number of side effects that other antiviral agents such as drugs may be associated with. Antibody compositions can be made more compatible with host systems by minimizing possible side-effect immune system responses. This is accomplished by removing all or part of the Fc region of the heterologous antibody, or by using an antibody that is homologous to the host animal (eg, using a human / human hybridoma).

  In addition, since the antibody / virus complex is recognized by macrophages, the antibody can also be used as a means to enhance the immune response. These antibodies can also be administered in amounts similar to those used for therapeutic administration of antibodies. For example, pooled IgG is administered at 0.02-0.1 ml per pound of body weight during the initial incubation period of other viral diseases such as rabies, measles, and hepatitis B to prevent the virus from entering the cells. Therefore, antibodies reactive to ET-NANB can be passively administered alone or in combination with other antiviral agents to a host infected with ET-NANB virus to enhance the immune response and / or effectiveness of antiviral drugs. Can do.

  Anti-ET-NANB antibodies can be prepared by administering an anti-idiotype antibody as an immunogen. Conveniently, an anti-idiotype antibody is induced in a host animal using an anti-ET-NANB virus antibody preparation prepared as described above. The composition is dissolved in a suitable diluent and administered to the host animal. After this administration (usually repeated administration), the host produces anti-idiotype antibodies. To eliminate an immune response against the Fc region, antibodies produced by animals of the same species as the host can be used, or the Fc region of the administered antibody can be omitted. After induction of the anti-idiotype antibody in the host animal, an antibody composition can be made except serum or plasma. This composition can be purified by anti-idiotype antibody purification as described above or by affinity chromatography using an anti-ET-NANB virus antibody bound to an affinity matrix. The anti-idiotype antibody produced is similar in conformation to the original ET-NANB antigen and is used to prepare an ET-NANB vaccine rather than direct use of ET-NANB particle antigen.

  When used as a means to induce anti-ET-NANB virus antibodies in patients, the method of injecting antibodies for vaccines is the same for intramuscular, intraperitoneal, subcutaneous, etc. At an effective concentration dissolved in a suitable diluent. One or more booster injections are desirable. Anti-ET-NANB virus antibody-induced anti-idiotypic methods are the administration of purified blood components such as problems that can be caused by passive administration of anti-ET-NANB virus antibodies, such as side-effect immune responses and infection with undiscovered viruses. And alleviate related issues.

  Furthermore, the ET-NANB-derived protein of the present invention may be used for the production of prophylactic antisera before or after virus exposure. Here, the ET-NANB protein or mixture of proteins is formulated with a suitable adjuvant and administered to the volunteer according to known production methods of human antisera. The antibody response to the administered protein is monitored for several weeks following immunization, by detecting the presence of anti-ET-NANB virus antibodies by periodic collection of sera, as noted in Section IIA. Done.

  Antisera from patients from immunization can be administered as a prophylactic method prior to exposure to those at risk of contact infection. Antisera are also useful for post-virus exposure treatments, similar to the use of high-titer antisera for prevention of onset after hepatitis B virus exposure.

(E) Monoclonal antibodies For the in vivo use of both antibodies against ET-NANB virus particles and proteins and anti-idiotype antibodies, as well as diagnostic uses, the use of monoclonal antibodies may be preferred. The pancreas or lymphocytes of the immunized animal are removed and established or utilized to generate hybridomas by methods known to those skilled in the art. Donors who have been infected with ET-NANB virus (in which case infection is confirmed, for example, by the presence of antiviral antibodies in the blood or culture of the virus) may be suitable for lymphocyte donors. Lymphocytes can be isolated from peripheral blood samples or pancreatic cells can be used if the donor is a pancreatic biopsy patient. Epstein-Barr virus (EBV) can be used to establish human lymphocytes, and human lymphocytes can also be used to generate human / human hybridomas. In order to produce a human monoclonal antibody, first, in vitro immunization with a peptide is performed.

  Screening for antibodies secreted by the cell line, search for clones that secrete antibodies with the desired specificity. In the case of a monoclonal antiviral particle antibody, the antibody must be bound to the ET-NANB viral particle. Cells that produce the specific antibody of interest are selected.

  The following examples illustrate various aspects of this invention, but are not intended to limit the scope of the claims.

(material)
The materials used in the following examples are as follows.

  Enzymes: DNase and alkaline phosphatase are from Boehringer Mannheim Biochemicals (BMB, Indianapolis, IN); EcoRI, EcoRI methylase, DNA ligase and DNA polymerase I are from New England Biolabs (NEB, N A was obtained from Sigma (St. Louis, MO).

  Other reagents: EcoRI linker from NEB; also nitroblue tetrazolium (NBT), 5-bromo-4-chloro-3-indolyl phosphate (BCIP), 5-bromo-4-chloro-3-indolyl-β- D-galactopyranoside (X-gal) and isopropyl β-D-thiogalactopyranoside (IPTG) were each obtained from Sigma.

  A kit for cDNA synthesis and a kit for random priming labeling are available from Boehriger Mannheim Biochemical (BMB, Indianapolis, IN).

(Example 1)
Preparation of cDNA library (A) Source of ET-NANB virus It is clear that 27-34 nm virus-like particles (VLPs) in stool bind to immune sera obtained from ET-NANB patients. 10% suspension of stool pool from a second passage cynomolgus monkey (cyno # 37) infected with an ET-NANB virus strain isolated from a case in Burma, positive for NANB. Noted. In this animal, alkaline phosphatase (ALT) levels increased between 24 and 36 days after immunization, and one excreted 27-34 nm VLPs in bile in the pre-acute phase of infection.

  The bile duct of each infected animal was cannulated and 1-3 cc bile was collected daily. RNA was extracted from one bile (cyno # 121) by hot phenol extraction using standard RNA separation methods. Double-stranded cDNA was generated from the isolated RNA by single-stranded random priming using a cDNA synthesis kit obtained from Boehringer-Mannheim (Indianapolis, IN).

(B) Cloning of double-stranded fragment The double-stranded cDNA fragment was blunt-ended with T4 DAN polymerase under standard conditions (Maniatis, p. 118), extracted with phenol / chloroform, and precipitated with ethanol. The blunt ended cDNA was ligated with EcoRI linkers under standard conditions (Maniatis, pp. 396-397) and digested with EcoRI to remove the extra ends of the linker. Unlinked linker was removed by repeated isopropanol precipitation. λgt10 phage vector (Huynh) was obtained from Promega Biotec (Madison, W1). This cloning vector has a unique EcoRI cloning site in the phage cI repressor gene. The cDNA fragment is 0.5-1.0 ug EcoRI digested gtl0, 0.5-3 μl of the double-stranded fragment, 0.5 μl of 10X ligation buffer, 0.5. μl ligase (200 units) and distilled water were mixed (5 μl) and introduced into the EcoRI site. The mixture was incubated overnight at 14 ° C. and then packaged in vitro according to standard methods (Maniatis, pp. 256-268).

  The package layer was used to infect E. coli hf1 strains such as HG415 strain. In addition, the C600hf1 strain of E. coli available from Promega Biotec (Madison, Wis.) Could be used. The percentage of recombinant plaques obtained by insertion of EcoRI-end digested fragments was less than 5% when 20 random plaques were analyzed.

  The resulting cDNA library was spread on a plate, an elution buffer was added, and the phage was eluted from the selection plate. After extracting the DNA from the phage, the DNA was digested with EcoRI to release the heteroinsert and the DNA fragment was fractionated with agarose to remove the phage fragment. 500-4000 base pair inserts were isolated, recloned into the above λgt10 and packaged phage were used for infection of E. coli strain HG415. The percentage of successful reclassifications exceeded 95%. A total of 8 plates were used at approximately 5000 plaques / plate and the phage library was plated with E. coli strain HG415.

(Example 2)
Selection of ET-NANB cloned fragment (A) cDNA probe A double-stranded cDNA fragment derived from a non-infected and ET-NANB infected cynomolgus monkey was prepared in the same manner as in Example 1. The cDNA fragment was radiolabeled by random priming using a random priming labeling kit manufactured by Boehringer Mannheim (Indianapolis, IN).

(B) Selection of clones The cDNA library plated in Example 1 was transferred to each of two nitrocellulose filters, and phage DNA was filtered by heating according to standard methods (Maniatis, pp. 320-323). Fixed. The two filters hybridized with either the source of infection or the control cDNA probe obtained as described above. A test to identify library clones that hybridize with radiolabeled cDNA probes that are derived from infectious sources only (ie, did not hybridize with cDNA probes from non-infected sources) on the filter autoradiograph Went. Such a reduction selection method identified 16 such clones out of a total of about 40000 tested clones.

  All 16 clones were selected at low concentration on agar plates and re-cultured on the plates. Clones on each plate were transferred to nitrocellulose in duplicate and subjected to hybridization studies with infected and non-infected source radiolabeled cDNA probes as described above. The clones were chosen to selectively bind to the infectious source probe (ie, bind to the infectious source probe and not substantially bind to the non-infectious source probe). One of the clones that selectively binds to the source probe was isolated for further study. The selected vector was identified as λgt10-1.1 shown in FIG.

(Example 3)
ET-NANB sequence The clone of Example 2, λgt10-1.1, was digested with EcoRI in order to remove the hetero-insert, and separated from the Becu fragment by gel electrophoresis. The electrophoretic mobility of the fragments was consistent with the 1.33 kb fragment. This fragment contained the EcoRI end and incorporated into the EcoRI site of the pTZ-KF1 vector, but its structure and properties are described in the co-owned US Patent Application No. 125650 filed November 25, 1987 “Cloning Vector System”. And identification of unique clones ". In summary, as illustrated in FIG. 1, this plasmid contains a unique EcoRI site, next to the T7 polymerase promoter site and plasmid and origin of phage replication. The flanking sequences on both sides of the EcoRI site are known. E. coli BB4 obtained from Stratagene (La Jolla, CA) was transformed with the plasmid.

  The radiolabeled ET-NANB probe was prepared by excising a 1.33 kb insert from the λgt10-1.1 phage in Example 2 and separating the fragments by gel electrophoresis and labeling them randomly as described above. did. Transfected with the above pTZ-KF1. Bacteria with the desired ET-NANB insert were selected by the replica method and hybridization with radiolabeled ET-NANB probe according to the method outlined in Example 2.

  One bacterial colony that was successfully recombined was used to sequence a portion of the 1.33 kb insert. This isolate is named pTZ-KF1 (ET1.1) and has been deposited with the American Standard Culture Collection, ATCC Deposit No. 67717. Approximately 200-250 base pairs were obtained from the 5 'and 3' end regions of the insert using primers with sequences on the sides of the EcoRI site by standard dideoxy methods. The sequence is shown in Section II. By determining the later sequence using the same technique, the sequence in both directions was completely known.

Example 4
Determination of ET-NANB Sequence A mixture of cDNA fragments obtained from bile of uninfected and ET-NANB infected cynomolgus monkeys was prepared as described above. A cDNA fragment obtained from a human stool specimen was prepared as follows. As a result of the sudden outbreak of ET-NANB, 30 ml of 10% stool suspension obtained from a Mexican who was diagnosed with ET-NANB infection and the same volume of stool obtained from a healthy non-infected person were obtained with a density of 30% sucrose. A gradient method was applied and the mixture was centrifuged at 25000 × g for 6 hours at 15 ° C. with a SW27 rotor. The stool precipitate obtained from the infectious body contained 27-34 nm VLP particles characteristic of ET-NANB infection in infected stool specimens. RNA was isolated from the sucrose gradient pellets of both infected and non-infected samples, and the separated RNA was used for the preparation of the cDNA fragment described in Example 1.

  A mixture of cDNA fragments obtained from infected and non-infected bile and from infected and non-infected human stool, respectively, is a common patent application entitled “Method for DNA Deployment and Reduction” filed June 17, 1988. It was developed by a new linker / primer replication method described in 07/208512. In summary, the fragments in each specimen were blunt ended with DNA polymerase I, then extracted with phenol / chloroform and precipitated with ethanol. Substances with blunt ends were linked with a linker having the following sequence.

5'-GGAATTCGCGGCCGCTCG-3 '
3′-TTCCCTTAAGCGCCGGCGAGC-5 ′.

  To remove the dimeric linker, the double-stranded fragment was digested with NruI, mixed with a primer with 5'-GGAATTCGCGCCGCCTCG-3 ', and then thermally denatured at room temperature to form a single-stranded DNA / primer complex. Until cooled. The complex was replicated and a double stranded fragment was formed by adding Thermus aquaticus (Taq) polymerase and four total deoxynucleotides. The replication process, including continuous strand modification, strand / primer complex formation and replication was repeated 25 times.

The developed cDNA sequence was fractionated by agarose gel electrophoresis using a 2% agarose substrate. After transferring the DNA fragment from the agarose gel to nitrocellulose paper, (i) treating the pTZ-KF1 (ET1.1) plasmid obtained above with EcoRI, (ii) the released 1.33 kb ET-NANB Filters were hybridized to randomly labeled ³² P probes by separating the fragments and (iii) randomly labeling the separated fragments. Hybridization of the probe was performed by a simple Southern method (Maniatis, pp. 382-389). FIG. 2 shows hybridization patterns obtained with cDNAs derived from bile (2A) of infected (I) and non-infected (N), and human stool (2B) of infected (N) and non-infected (N). Show. Apparently, the ET-NANB probe hybridized with fragments obtained from both infectious agents, but none of the sequences obtained from non-infectious agents were found to be equivalent, so the specificity of the derived sequences Was confirmed.

  Southern blots of radiolabeled 1.33 kb fragment and genomic DNA fragments from both human and cynomolgus monkey DNA were prepared. Since probe hybridization was not seen in either genomic fragment mixture, it was confirmed that the ET-NANB sequence was exogenous in either the human or cynomolgus monkey genome.

(Example 5)
Expression of ET-NANB protein (A) Preparation of ET-NANB coding sequence The pTZ-KF1 (ET1.1) plasmid obtained in Example 2 was purified from a linearized gel electrophoresis, 1.33 kb ET- To remove the NANB insert, it was digested with EcoRI. The purified fragment is suspended in standard digestion buffer (0.5 M Tris HCl, pH 7.5; 1 mg / ml BSA; 10 mM MnCl ₂ ) to a concentration of about 1 mg / ml, and DNase I is used. Digested for about 5 minutes at room temperature. These reaction conditions were determined from previously performed calibration tests, where the incubation time required to generate a fragment consisting of 100-300 base pairs was determined. The material was extracted with phenol / chloroform prior to precipitation with ethanol.

  Fragments in the digestion mixture had blunt ends as in Example 1 and formed ligands with EcoRI. Other fragments were analyzed by electrophoresis (5-10 V / cm) on a 1.2% agarose gel using PhiX174 / HaeIII and lambda / HindIII size markers. A 100-300 bp fraction eluted in the NA45 strip (Schleicher and Schuell), which was then transferred into a 1.5 ml tubule containing the elution solution (1M NaC1, 50 mM arginine, pH 9.0) and 67 ° C. Incubated for 30-60 minutes. The eluted DNA was extracted with phenol / chloroform and then precipitated with twice the amount of ethanol. The precipitate was resuspended in 20 μl TE (0.01 M Tris-HCl, pH 7.5, 0.001 M EDTA).

(B) Cloning in Expression Vector The λgt11 phage vector (Huyny) was obtained from Promega Biotec (Madison, W1). This cloning vector has a unique EcoRI cloning site 53 base pairs above the β-galactosidase translation terminal codon. The obtained genomic fragment was divided into 0.5-1.0 μg EcoRI-cleaved gt11, 0.3-3 μl of the above-mentioned fragment, 0.5 μl of 10X ligation buffer (above), 0.5 μl ligase. (200 units), and introduced to the EcoRI site by mixing distilled water (5 ml). The mixture was incubated overnight at 14 ° C. and then packaged in vitro according to standard methods (Maniatis, pp. 256-268).

  The packaging phage was used to infect E. coli strain KM392 assigned by Dr. Kevin Moore of DNAX (Palo Alto, CA). In addition, E. coli available from the American Standard Culture Collection (ATCC # 37197). Coli strain Yl090 could be used. Infectious bacteria are plated and the resulting colonies are depleted of β-galactosidase activity in the presence of X-gal (clear plaque) using standard X-gal substrate plaque assay (Maniatis). Checked. In about 50% of the phage plaques, the loss of β-galactosidase enzyme activity was observed (recombinant type).

(C) Screening for recombinant ET-NANB protein ET-NANB convalescent antisera were obtained from patients infected with ET-NANB suddenly developed in Mexico, Borneo, Pakistan, Somalia, and Burma. The serum showed immunoreactivity with VLPs in stool specimens obtained from several ET-NANB hepatitis patients.

A clone of E. coli KM392 cells infected with about 10 ⁴ pfu of the phage stock was prepared on a 150 mm plate, inverted, and incubated at 37 ° C. for 5-8 hours. The expressed ET-NANB recombinant protein was transferred from plaque to paper by covering the clones with a nitrocellulose sheet. The plate and filter were indexed to align the corresponding plate and filter. The filter was washed twice with TBST buffer (10 mM Tris, pH 8.0; 105 mM NaCl, 0.05% Tween 20), inhibited with AIB (TBST buffer containing 1% gelatin), rewashed with TBST, and antiserum. (Diluted 1:50 with AIB, 12-15 ml plate) was added and incubated overnight. The sheet was washed twice with TBST and then contacted with an enzyme-labeled anti-human antibody in order to attach the labeled antibody to the site containing the antigen recognized by the antiserum in the filter. After the final wash, 33 μl NTB (mixed in a 50 mg / ml stock solution kept at 5 ° C.) in 5 ml alkaline phosphatase buffer (100 mM Tris, pH 9.5, 100 mM NaCl, 5 mM MgCl ₂ ), mixed with 16 μl BCIP Filters were developed in substrate medium containing 50 mg / ml stock solution kept at 5 ° C. When the antiserum was confirmed, the antigen production site was purple.

(D) Plate culture for screening The antigen-producing region determined in the previous stage was again subjected to plate culture at about 100-200 pfu on a plate having a diameter of 82 mm. In order to purify phages that secrete antigens capable of reacting with ET-NANB antibodies from plaques, the above steps were repeated, beginning with 5-8 hours incubation and undergoing NBT-BCIP development. The identified plaques were selectively eluted with phage buffer (Maniatis, p. 443).

(E) Epitope Identification In Example 2, a series of subclones derived from the native pTZ-KF1 (ET1.1) plasmid were isolated using the same method as described above. Each of these 5 subclones had immunoreactivity with the anti-ET antiserum pool of (C). The subclone contained a short sequence from the previously described “reverse” sequence. The starting and ending points of the sequences in the subclones (relative to the complete reverse sequence) were identified as in the table below.

  Since the entire gene sequence identified in Table 2 should contain the sequence encoding the epitope, the coding sequence for the epitope is between the 594th nucleotide (5 ′ end) and the 643rd nucleotide (3 ′ end). It is clear that they are in the area. Thus, gene sequences that are equal in length and complementary to this relatively short sequence, as well as peptides generated using this coding region, are particularly preferred for various aspects of the invention. A second series of clones, identifying completely different epitopes, were isolated with a Mexican serum.

The coding system for this epitope is between the second nucleotide (5'-end) and the 101st nucleotide (3'-end). Therefore, the gene sequence for this short sequence is also preferred, as is the peptide generated using this coding region.
Although the invention has been described with reference to specific embodiments, methods, constructs and uses, those skilled in the art will be able to make various changes and modifications without departing from the invention.

It is a figure which shows the vector structure and operation which are used when obtaining a cloning ET-NANB fragment and performing sequencing. 2A-2B show infected (I) bile source (2A) and non-infected (N) bile source (2A), and infected (I) stool sample source (2B) and uninfected (N) stool sample It is a development view by the Southern method when an amplified cDNA fragment prepared from RNA isolated from the source (2B) and a radiolabeled ET-NANB are hybridized.

Claims (1)

Intestinal transmissible non-A non-B having a genome containing a region homologous to the EcoRI insert of 1.33 kb DNA present in plasmid pTZ-KF1 (ET1.1) carried in E. coli BB4 strain of ATCC Deposit No. 67717 Protein derived from hepatitis virus factor.

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