JP2006068004A - env GENE OF NEW HUMAN ENDOGENOUS RETROVIRUS HC2 - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an env gene of a new human endogenous retrovirus related to onset of IgA nephropathy. <P>SOLUTION: An HC2env gene (env gene of the human endogenous retrovirus HC2) is disclosed. One or several bases therein may be deleted, substituted or added. The onset of the IgA nephropathy is a mechanism in which the HC2env gene is expressed to produce an HC2ENV protein that is an expression product thereof. When a human has an antibody reactive with the HC2ENV protein by an autoimmune mechanism, an immunoreactive conjugate of the antibody deposits in a glomerular mesangium region to cause chronic inflammatory reactions. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a novel human endogenous retrovirus HC2 env gene.

  IgA nephropathy, a type of chronic glomerulonephritis, is a disease characterized by cellular and substrate proliferative responses in the glomerular mesandium region and immunoglobulin A (IgA) deposition in the region. IgA nephropathy accounts for 30% or more in adults and 20% or more in children among chronic glomerulonephritis in Japan (see Non-Patent Document 1). This disease occurs frequently in countries in the Asia-Pacific region including Japan and southern European countries such as France, but is rare in Northern Europe and North America. In this regard, research on this disease should be especially emphasized in countries with a high incidence of this disease including Japan. In addition, the prognosis of this disease is much worse than previously assumed, and about 38% will fall into end stage renal failure in 20 years after renal biopsy (see the same document).

  The cause of IgA nephropathy remains unclear. As a cause of this disease, the theory that blood immune complexes including IgA are deposited in the glomerular mesandium region and cause a chronic inflammatory response is probable, but the identification of the antigen that forms the IgA immune complex It has not been successful yet (see Non-Patent Documents 2 and 3). In addition, treatment of IgA nephropathy is only symptomatic treatment, and if the patient's renal function eventually decreases, there is no other way than relying on dialysis therapy.

On the other hand, diagnosis of IgA nephropathy is performed by confirming IgA deposition by kidney biopsy using a fluorescent antibody method. However, kidney biopsy is extremely invasive to patients and has a life-threatening risk. There is also a problem that iterative implementation is difficult.
Intractable Disease Information Center, Foundation for Intractable Diseases Research, “Diagnosis and Treatment Guidelines IgA Nephropathy” [online], May 12, 2004 search, Internet <URL: http://www.nanbyou.or.jp/sikkan/ 001_i.htm> Rantala, I., J. Mustonen, M. Hurme, J. Syrjanen, & H. Helin. Pathogenetic aspects of IgA nephropathy. Nephron 88: 193-198, 2001. Smith, AC, & J. Feehally. New insights into the pathogenesis of IgA nephropathy. “New insights into the pathogenesis of IgA nephropathy” Springer Semin. Immunopathol. 24: 477-493, 2003.

  The present invention has been made in view of such circumstances, and an object thereof is to provide a gene that is related to the development of IgA nephropathy and a protein that is an expression product thereof.

In order to achieve the above object, the present invention relates to an env gene of human endogenous retrovirus HC2 (hereinafter also referred to as HC2 env gene), comprising any one of the following polynucleotides (1) to (6): Provide a gene.
(1) A polynucleotide comprising the base sequence of SEQ ID NO: 1 in the sequence listing.
(2) A polynucleotide comprising a nucleotide sequence in which one or several bases are deleted, substituted or added in SEQ ID NO: 1 in the sequence listing, and a nucleotide sequence complementary to the polynucleotide of (1) above A polynucleotide that hybridizes under stringent conditions.
(3) A polynucleotide comprising a nucleotide sequence in which one or several bases are deleted, substituted or added in SEQ ID NO: 1 in the sequence listing, wherein the polynucleotide has homology with the polynucleotide of (1) above. A polynucleotide which is 80% or more.
(4) A polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2 in the sequence listing.
(5) Anti-gp70 antibody 12H5.1, which is a mouse monoclonal antibody, or an anti-Rauscher consisting of an amino acid sequence in which one or several amino acid residues are deleted, substituted or added in SEQ ID NO: 2 in the sequence listing A polynucleotide encoding a protein capable of antigen-antibody reaction with leukemia virus gp70 antibody.
(6) A polynucleotide comprising a base sequence complementary to the polynucleotide of any one of (1) to (5).

In order to achieve the above object, the present invention provides a protein (hereinafter, also referred to as HC2ENV protein) that is an expression product of the env gene of human endogenous retrovirus HC2, which is described in (7) or (8) below. Provide protein.
(7) A protein comprising the amino acid sequence of SEQ ID NO: 2 in the sequence listing.
(8) The anti-gp70 antibody 12H5.1, which is a mouse monoclonal antibody, or an anti-Rauscher consisting of an amino acid sequence in which one or several amino acid residues are deleted, substituted or added in SEQ ID NO: 2 in the sequence listing Protein that reacts with leukemia virus gp70 antibody by antigen-antibody reaction.

  The present inventors have conducted extensive research on primary glomerular diseases such as IgA nephropathy, and in the process, IgA immune complexes are chronically present in the blood of patients with this disease, so IgA reacts. The idea was that the antigenic molecule might be a kind of autoantigen rather than a foreign antigen. As a result of further research based on this idea, the present inventors discovered a novel human endogenous retrovirus HC2 env gene and a protein that is a product of the HC2 env gene, and this protein is related to IgA nephropathy. As a result, the present invention was reached. Therefore, IgA nephropathy can be diagnosed by detecting or measuring the HC2 env gene of the present invention or its HC2 env gene product protein, or by detecting or measuring an antibody immunoreactive with the HC2 env gene product protein of the present invention. It becomes. In addition, the present inventors have also found that a single nucleotide polymorphism exists in the HC2env gene of the present invention. By this polymorphism analysis, for example, the onset diagnosis of IgA nephropathy, the type of onset, etc. Diagnosis is possible. Then, by suppressing the transcription and expression of the gene of the present invention or inhibiting the immune reaction between the protein of the present invention and human IgA antibody, the onset prevention or treatment of IgA nephropathy can be achieved. Furthermore, since the HC2env gene product protein of the present invention is derived from a human endogenous virus, it is expected that no immune response (neutralizing antibody production) will occur even if it is repeatedly administered to humans. It can also be used for retroviral vectors.

  In addition, as will be described later, the present invention has been made against the conventional common sense, and can only be accomplished by the present inventors. That is, over the past 20 years, research on mice (mouse) that spontaneously develop renal glomerular lesions and vascular lesions similar to so-called autoimmune diseases, including human IgA nephropathy, has been conducted. Discuss whether mouse endogenous retrovirus gene products deposited on glomeruli and blood vessel walls are involved in lesion formation, or whether antibodies against nuclear components detected in serum (antinuclear antibodies) are pathogenic It has been. However, no one has directly demonstrated that autoantibodies to endogenous retroviral gene products have the ability to form lesions. The present inventors have succeeded for the first time in the world in establishing a number of monoclonal antibodies that react with endogenous retrovirus env gene products from mouse strains that spontaneously develop autoimmune glomerulonephritis. Most have demonstrated that glomerular lesions can be induced by intravenous transfer into normal mice that do not spontaneously develop lesions (Tabata, N., M. Miyazawa, R. Fujisawa, YA Takei, H Abe, and K. Hashimoto. Establishment of monoclonal anti-retroviral gp70 autoantibodies from MRL / lpr lupus mice and induction of glomerular gp70 deposition and pathology by transfer into non-autoimmune mice. Induction of glomerular gp70 deposition and lesions by establishment of monoclonal autoantibodies and introduction into non-autoimmune mice ”J. Virol. 74: 4116-4126, 2000.]).

  The present invention originates from the discovery that one of the antibodies established at this time reacts with an unknown antigen present in the lesion of human IgA nephropathy. Conventionally, as for the possibility of human endogenous retroviruses being involved in the formation of human lesions, the more negative the more a retrovirus specialist is, the more dominant the negative idea. For example, Jonathan P. of the National Institute for Medical Research, a leader in mouse endogenous retrovirus research and an authority on human endogenous retrovirus gene analysis. Stony and John L. of the National Institutes of Health, National Institute of Allergy and Infectious Diseases, renowned for many years of mouse retrovirus research. Portis has expressed a strong negative view on the involvement of endogenous retroviruses in human autoimmune diseases (see [Stoye, JP The pathogenic potential of endogenous retroviruses: a skeptical view. Trends Microbiol. 7: 430, 1999., Portis, JL Perspectives on the role of human endogenous retroviruses in autoimmune diseases. Views on the role of human endogenous retroviruses in autoimmune diseases ] Virology 296: 1-5, 2002.]).

  In addition, it was conventionally thought that the env gene is not present in human endogenous retrovirus HC2, but the present inventors suspected this common sense and reacted with the mouse endogenous retrovirus env gene product prepared by the present inventors. As a result of a detailed examination of the mouse endogenous retrovirus-like gene present on the human genome, the env gene of the human endogenous retrovirus HC2 of the present invention was found. It was. Therefore, even if the mouse endogenous retrovirus and its env gene have been publicly known, the preconception that the human endogenous retrovirus HC2 has no env gene is generally prevalent. Contrary to common sense, it is difficult to predict the presence of an env gene similar to that of a mouse endogenous retrovirus in humans, and it can be said that it is difficult to obtain the HC2 env gene of the present invention.

  The HC2env gene of the present invention has the 69th position (T → A), the 524th position (T → C), the 586th position (C → G), and the 592nd position (G) in the nucleotide sequence of SEQ ID NO: 1 in the sequence listing. → T), 887th (C → T), 970th (C → T), 1000th (C → T), 1140th (A → G), 1143th (T → C) and It may be a gene containing at least one single nucleotide polymorphism selected from the group consisting of the 1212th (C → A). In the HC2env gene of the present invention, the polynucleotides (1) to (6) may be DNA or RNA. However, in RNA, in the base sequence in the sequence listing, t (thymine) base is read as u (uracil) base.

  The polynucleotide of the present invention is a polynucleotide comprising all or part of the polynucleotide of any one of (1) to (6) above. The polynucleotide of the present invention can be used as a primer or probe for the HC2env gene of the present invention.

  In another aspect, the polynucleotide of the present invention is a polynucleotide used as a primer or probe for amplifying or detecting all or a part of the HC2env gene of the present invention, which is from 3 to 24 and 30 in the sequence listing. A polynucleotide comprising any one of 36 nucleotide sequences.

  The probe of the present invention is a probe for detecting the single nucleotide polymorphism, which is complementary to all or part of the polynucleotide of any one of (1) to (6), and It is a probe consisting of a base sequence containing the single nucleotide polymorphic base in SEQ ID NO: 1 in the sequence table.

  The mRNA of the present invention is a transcript mRNA of the HC2env gene of the present invention.

  The IgA nephropathy marker of the present invention is at least selected from the group consisting of the HC2env gene of the present invention, the mRNA of the present invention, the HC2ENV protein of the present invention, and a human antibody that undergoes an antigen-antibody reaction using the HC2ENV protein of the present invention as an antigen. It is a single IgA nephropathy marker.

  The gene expression measurement method of the present invention is a method for measuring gene expression of the HC2env gene of the present invention, and is a measurement method for measuring the amount of mRNA of the present invention or the HC2ENV protein of the present invention. The measurement of the amount of mRNA of the present invention is preferably performed using the polynucleotide of the present invention used as a primer or probe of the HC2env gene of the present invention. The measurement of the HC2ENV protein of the present invention is preferably performed using an antibody having the HC2ENV protein of the present invention as an antigen.

  The method for measuring a human antibody of the present invention is a method for measuring a human antibody using the HC2ENV protein of the present invention as an antigen, wherein the HC2ENV protein of the present invention and the human antibody are reacted with each other, and further, the human antibody, This is a method for measuring a complex of the HC2ENV protein of the present invention, the human antibody and the labeled antibody by subjecting the labeled antibody using the human antibody as an antigen to an antigen-antibody reaction. In the method for measuring a human antibody of the present invention, the HC2ENV protein of the present invention is preferably immobilized on a solid phase. In the human antibody measurement method of the present invention, the human antibody is preferably a human antibody in plasma.

  The IgA nephropathy test kit of the present invention is an IgA nephropathy test kit in which the gene expression of the HC2env gene of the present invention is measured by the gene expression measurement method of the present invention, and is used as a primer or probe. It is a test kit for IgA nephropathy containing an antibody having a nucleotide or HC2ENV protein of the present invention as an antigen.

  In another aspect, the IgA nephropathy test kit of the present invention is an IgA nephropathy test kit for measuring a human antibody having the HC2ENV protein of the present invention as an antigen by the method for measuring a human antibody of the present invention. A test kit for IgA nephropathy comprising the HC2ENV protein of the invention and a labeled antibody using the human antibody as an antigen. In the IgA nephropathy test kit of the present invention, the HC2ENV protein of the present invention is preferably immobilized on a solid phase.

  The pharmaceutical product of the present invention is a pharmaceutical product for at least one of treatment and prevention of IgA nephropathy, comprising a base sequence complementary to a gene containing all or part of the HC2env gene of the present invention, and comprising the HC2env of the present invention. A pharmaceutical comprising a polynucleotide capable of suppressing gene transcription or protein expression.

  In another aspect, the pharmaceutical agent of the present invention is a pharmaceutical agent for at least one of treatment and prevention of IgA nephropathy, and the expression of the env gene of human endogenous retrovirus HC2 in the amino acid sequence of the HC2ENV protein of the present invention A pharmaceutical product comprising a polypeptide comprising an amino acid sequence of an antigenic determinant (epitope) for a human antibody whose product is an antigen.

  The expression vector of the present invention is an expression vector for introducing a gene containing all or part of the HC2env gene of the present invention into a target cell, the gene containing all or part of the HC2env gene of the present invention, An expression vector comprising a regulatory sequence necessary for expression of the gene, wherein the gene is operably linked to the regulatory sequence.

  The retrovirus vector for gene transfer of the present invention is a retrovirus vector for gene transfer in which the coat protein of the virus particle contains all or part of the HC2ENV protein of the present invention.

  The gene introduction method of the present invention is a gene introduction method using a retrovirus vector for gene introduction, and the gene introduction retrovirus vector is the retrovirus vector for gene introduction of the present invention.

  The method for producing a gene-transferred cell according to the present invention is a method for producing a gene-introduced cell comprising a step of introducing a target gene into a target cell by the gene-transfer method of the present invention.

  The gene introduction kit of the present invention is a gene introduction kit comprising a packaging cell transformed with the expression vector of the present invention and a gene introduction plasmid having a ψ sequence.

  As another aspect, the gene introduction kit of the present invention is a gene introduction kit comprising the expression vector of the present invention and a gene introduction plasmid having a ψ sequence. The gene introduction kit of the present invention preferably further comprises a plasmid that expresses at least one of the gag and pol genes.

  Next, the present invention will be described in detail.

First, the HC2env gene of the present invention will be described. As described above, the HC2env gene of the present invention is a gene comprising the polynucleotide described in any one of (1) to (6) below.
(1) A polynucleotide comprising the base sequence of SEQ ID NO: 1 in the sequence listing.
(2) A polynucleotide comprising a nucleotide sequence in which one or several bases are deleted, substituted or added in SEQ ID NO: 1 in the sequence listing, and a nucleotide sequence complementary to the polynucleotide of (1) above A polynucleotide that hybridizes under stringent conditions.
(3) A polynucleotide comprising a nucleotide sequence in which one or several bases are deleted, substituted or added in SEQ ID NO: 1 in the sequence listing, wherein the polynucleotide has homology with the polynucleotide of (1) above. A polynucleotide which is 80% or more.
(4) A polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2 in the sequence listing.
(5) Anti-gp70 antibody 12H5.1, which is a mouse monoclonal antibody, or an anti-Rauscher consisting of an amino acid sequence in which one or several amino acid residues are deleted, substituted or added in SEQ ID NO: 2 in the sequence listing A polynucleotide encoding a protein capable of antigen-antibody reaction with leukemia virus gp70 antibody.
(6) A polynucleotide comprising a base sequence complementary to the polynucleotide of any one of (1) to (5).

  The base sequence of SEQ ID NO: 1 in the sequence listing is an example of the base sequence of the env gene of human endogenous retrovirus HC2 found by the present inventors. The base sequence of SEQ ID NO: 1 in the sequence listing exists in the human genome database, but there has been no report that this base sequence is the HC2env gene of the present invention, and the present inventors have found it for the first time. As shown in FIG. 4, the present inventors have identified that the HC2env gene of the present invention is present in the vicinity of 19p13.1 of the short arm of human chromosome 19.

  The human endogenous retrovirus HC2 is derived from the literature [Kabat, P., M. Tristem, R. Opavsky, & J. Pastorek. Human endogenous retrovirus HC2 is a new member of the S71 retrovirus subgroup with a full-length pol gene. “Human endogenous retrovirus HC2 is a subgroup of S71 retroviruses with the full-length pol gene” Virology 226: 83-94, 1996.], but conventionally, HC2 without the env gene It was a provirus belonging to the group. Similarly, in previous reports, all HC2 group proviruses were considered incomplete and the env gene was deleted. However, studies by the present inventors have confirmed that the HC2env gene of the present invention is present in a portion erroneously designated as 3 'LTR (terminal repeat sequence) in the same literature. The position of the HC2env gene of the present invention on the human chromosome differs from the previously reported positions of HC2 and its group of endogenous retroviruses. From these facts, it can be said that the HC2env gene of the present invention is a part of a novel provirus belonging to the HC2 group.

  Furthermore, as shown in Examples described later, the present inventors have a true 3 ′ LTR downstream of the stop codon of the HC2env gene of the present invention on the human chromosome, and 5 upstream of the gag-pol gene region. 'Newly found that LTRs exist, and further confirmed that these new LTRs function (see Example 1, Figs. 5 and 6). These facts also indicate that the portion of the human endogenous retrovirus HC2 previously considered to be the 3 ′ LTR is not a true LTR, but is a part of the HC2env gene of the present invention, and the gene of the present invention is an HC2 provirus. The env gene is clearly shown.

In the HC2env gene of the present invention, the number of bases that can be deleted, substituted or added in the nucleotide sequence of SEQ ID NO: 1 in the polynucleotide sequence (2) is, for example, 1 to 376, 1 The number is preferably 282 to 282, more preferably 1 to 188. Hybridization under stringent conditions means that the two DNA fragments hybridize to each other under standard hybridization conditions as described by Sambrook J et al. (Reference [Expression of cloned genes in E. coli (see Molecular Cloning: A laboratory manual (1989)) Cold Spring harbor Laboratory Press, New York, USA, 9. 47-9.62 and 11.45-11.61]). More specifically, for example, it means performing hybridization and washing (for example, about 2.0 × SSC, 50 ° C.) based on the Tm value obtained by the following formula.
Tm = 81.5-16.6 (log ₁₀ [Na ⁺ ]) + 0.41 (% G + C) − (600 / N)
(Wherein, N is the chain length of the polynucleotide, and% G + C is the content of guanine and cytosine residues in the polynucleotide.)

  In the HC2env gene of the present invention, the homology of the polynucleotide (3) is, for example, 80% or more, preferably 85% or more, and more preferably 90% or more.

  In the HC2env gene of the present invention, the protein encoded by the polynucleotides (4) and (5) is the HC2ENV protein of the present invention described later. Regarding the protein encoded by the polynucleotide of (5) above, the number of residues that can be deleted, substituted or added in the amino acid of SEQ ID NO: 2 in the sequence listing is the anti-gp70 in which the encoded protein is a mouse monoclonal antibody. The antibody 12H5.1 or the anti-Rauscher leukemia virus gp70 antibody is not particularly limited as long as it reacts with an antigen antibody. For example, the number is 1 to 125, preferably 1 to 94, more preferably 1. ~ 62, and even more preferably 1 to 31.

  In the HC2env gene of the present invention, the polynucleotide is not particularly limited, and may be, for example, DNA or RNA. However, in RNA, in the nucleotide sequence of the polynucleotide sequence table, t (thymine) base is read as u (uracil) base.

  The method for producing the HC2env gene of the present invention is not particularly limited, and for example, it may be produced by chemically synthesizing the polynucleotides (1) to (6) by a conventionally known method, and enzymatically in vivo or in vitro. May be manufactured. The HC2env gene of the present invention can be cloned, for example, as follows. First, for example, a bacterial population chromosome (BAC) clone (clone name: CTC-575C13) containing the HC2env gene of the present invention is obtained from an official clone distributor (for example, Invitrogen, Research Genetics) of the public human genome project. Then, the BAC DNA is purified by a conventional method. For example, the full length of the HC2env gene is amplified by the PCR method using the following primers 011295ORF-L and 0121295ORF-R (SEQ ID NOS: 3 and 4 in the sequence listing), and an appropriate plasmid vector (For example, pBluescriptII etc.) It can clone by inserting. It is preferable to determine the nucleotide sequence of the inserted sequence as necessary and confirm that it is completely identical to SEQ ID NO: 1 in the sequence listing.

  In addition, as a primer pair that can be used in the PCR method for more selectively amplifying the HC2env gene of the present invention, a combination of the following forward primer Primer L and reverse primer Primer R (SEQ ID NOs: 5 and 6 in the sequence listing). can give.

  The HC2env gene of the present invention has the 69th position (T → A), the 524th position (T → C), the 586th position (C → G), and the 592nd position (G) in the nucleotide sequence of SEQ ID NO: 1 in the sequence listing. → T), 887th (C → T), 970th (C → T), 1000th (C → T), 1140th (A → G), 1143th (T → C) and At the 1212th (C → A), there are single nucleotide polymorphisms shown in parentheses, respectively. Therefore, the HC2env gene of the present invention may be a gene containing these single nucleotide polymorphisms.

  The method for detecting the single nucleotide polymorphism of the HC2env gene of the present invention is not particularly limited. For example, first, only the gene of the present invention is selectively PCR avoiding many similar genes scattered on human chromosomal DNA. And a method of determining the nucleotide sequence of the PCR product using a primer that is amplified using a method and hybridizing within the gene region of the present invention to detect the single nucleotide polymorphism. In addition, as another method for detecting the single nucleotide polymorphism, it is complementary to all or a part of the polynucleotide of any one of (1) to (6), and the sequence in SEQ ID NO: 1 in the sequence listing is used. There is a method of detecting by hybridization using a probe having a base sequence containing a single nucleotide polymorphic base.

  FIG. 14 shows an example of the single nucleotide polymorphism detection method using Primer L and Primer R (SEQ ID NOs: 5 and 6 in the sequence listing), which are primer pairs that can more selectively amplify the HC2env gene of the present invention. Show. As shown in the figure, Primer L has A at the 5 'end of the gene of the present invention at its 5' end, and Primer R hybridizes to the 3 'flanking region of the gene of the present invention. The region where Primer R hybridizes is a region that does not contain a repetitive sequence, and it has been found by NCBI-BLAST database search that there is no completely identical sequence on the sequence-analyzed human chromosomal DNA. That is, only the gene of the present invention can be selectively amplified by using the primer pair. Next, using the DNA containing the HC2env gene thus amplified as a template, base sequencing is performed using primers that hybridize within the gene region of the present invention, and the obtained base sequences are compared with each other to obtain a single base. Polymorphism can be detected. Examples of the primer used for determining the base sequence include a primer for a coding strand consisting of the base sequence of SEQ ID NO: 9 to 16 in the sequence listing and a primer for a non-coding strand consisting of the base sequence of SEQ ID NO: 17 to 24 of the sequence listing. can give. These primers are shown in Table 1 below.

  Next, the polynucleotide of the present invention will be described. The polynucleotide of the present invention is a polynucleotide comprising all or part of the polynucleotide of any one of (1) to (6) above. The polynucleotide of the present invention is not particularly limited, and may be, for example, DNA or RNA. However, in RNA, in the base sequence in the sequence listing, t (thymine) base is read as u (uracil) base. The polynucleotide of the present invention may be single-stranded or may be two or more multiple-stranded. The polynucleotide of the present invention includes an oligonucleotide comprising a part of the polynucleotide of any one of (1) to (6). Here, the oligonucleotide generally refers to a product in which several to several tens of nucleosides are polymerized by 3 ′, 5′-phosphodiester bonds. In the present invention, the length of the oligonucleotide is particularly limited. For example, it is 10mer-50mer, Preferably, it is 12mer-32mer. These oligonucleotides may be used as a primer or probe for detecting the HC2env gene of the present invention, or as a primer or probe for detecting and measuring the gene transcript mRNA of the HC2env gene of the present invention, as described later. it can.

  Furthermore, the polynucleotide of the present invention is a sequence other than the base sequence of the polynucleotide of any one of (1) to (6) above, in order to amplify or detect all or part of the HC2env gene of the present invention. Polynucleotides that can be used as primers or probes.

  Specific examples of the polynucleotide of the present invention that can be used as a primer or probe for amplifying or detecting all or part of the HC2env gene of the present invention include any of SEQ ID NOs: 3 to 24 and SEQ ID NOs: 30 to 36 in the Sequence Listing. And a polynucleotide comprising such a base sequence. Although the usage method in particular of these polynucleotides as a primer or a probe is not restrict | limited, For example, it is as above-mentioned or below-mentioned.

Examples of the method for detecting the HC2env gene of the present invention using the polynucleotide of the present invention as a primer or a probe include Southern blotting, Northern blotting, PCR, nucleotide sequencing and the like. The polynucleotide of the present invention may be modified with a labeling substance or the like as necessary according to each detection method. When the polynucleotide of the present invention is used as a gene detection probe, for example, it can be prepared by adding a label by the random primer method using the full length of the HC2env gene of the present invention consisting of the base sequence of SEQ ID NO: 1 in the sequence listing as a template. . Examples of the labeling substance for the probe include a radioactive substance such as ³² P and a non-radioactive chemical substance such as digoxigenin.

  Next, the mRNA of the present invention will be described. The mRNA of the present invention is mRNA of the transcript of the HC2env gene of the present invention. Since the mRNA of the present invention serves as an index of the transcriptional activity of the HC2env gene of the present invention, by measuring the expression level of the mRNA of the present invention, for example, as described later, the disease state of IgA nephropathy can be determined, or the prognosis Can be predicted.

Next, the HC2ENV protein of the present invention will be described. The HC2ENV protein of the present invention is a protein of an expression product of the HC2env gene of the present invention, and is a protein of the following (7) or (8).
(7) A protein comprising the amino acid sequence of SEQ ID NO: 2 in the sequence listing.
(8) The anti-gp70 antibody 12H5.1, which is a mouse monoclonal antibody, or an anti-Rauscher consisting of an amino acid sequence in which one or several amino acid residues are deleted, substituted or added in SEQ ID NO: 2 in the sequence listing Protein that reacts with leukemia virus gp70 antibody by antigen-antibody reaction.

  Regarding the protein of (8), the number of residues that can be deleted, substituted or added in the amino acid of SEQ ID NO: 2 in the sequence listing is as follows. The protein of (8) is a mouse monoclonal antibody, anti-gp70 antibody 12H5. 1 or anti-Rauscher leukemia virus gp70 antibody and antigen-antibody reaction is not particularly limited, but is, for example, 1 to 125, preferably 1 to 94, more preferably 1 to 62 And even more preferably 1 to 31.

The HC2ENV protein of the present invention undergoes an antigen-antibody reaction with an anti-gp70 mouse monoclonal antibody 12H5.1 or a known anti-Rauscher leukemia virus gp70 antibody, as shown in Examples below. Therefore, the HC2ENV protein of the present invention can be detected and measured using the 12H5.1 antibody or the anti-Rauscher leukemia virus gp70 antibody. As described later, the 12H5.1 antibody is a monoclonal antibody that specifically reacts with the mouse endogenous xenobiotic retrovirus env gene product gp70, and the anti-Rauscher leukemia virus gp70 antibody contains both endogenous and exogenous antibodies. It is a polyclonal antibody that specifically reacts with the mouse retrovirus env gene product gp70. The method for producing and purifying the protein of the present invention is not particularly limited and can be produced and purified by a conventionally known method. For example, a labeled peptide such as (His) ₆ is fused to the C-terminus of the HC2env gene of the present invention. For example, the expression vector may be introduced into a cultured cell line and expressed, and purified using an affinity column for the labeled peptide.

  Next, the IgA nephropathy marker of the present invention will be described. The IgA nephropathy marker of the present invention is at least selected from the group consisting of the HC2env gene of the present invention, the mRNA of the present invention, the HC2ENV protein of the present invention, and a human antibody that undergoes an antigen-antibody reaction using the HC2ENV protein of the present invention as an antigen. It is a single IgA nephropathy marker.

  The IgA nephropathy marker comprising the HC2env gene of the present invention can be used for, for example, predicting the onset of IgA nephropathy based on polymorphism analysis, diagnosis of the type of onset, etc. using the single nucleotide polymorphism as a genetic marker. it can. In addition, the IgA nephropathy marker comprising the mRNA of the present invention and the HC2ENV protein of the present invention changes the concentration of the HC2ENV protein antigen in the blood depending on the level of expression of the mRNA, and the antibody against this depends on the concentration of the HC2ENV protein. It is thought that the amount of production will change and that the disease will be hated or relieved, so the subject's disease state should be determined, or prognosis should be predicted, for example, whether strong treatment should be performed or only follow-up It can be used to determine the treatment policy.

  Furthermore, an IgA nephropathy marker comprising a human antibody (hereinafter also referred to as an anti-HC2 ENV protein antibody) that undergoes an antigen-antibody reaction using the HC2ENV protein of the present invention as an antigen includes, for example, IgA nephropathy and other primary glomerular diseases, Patients with a disease suspected of being involved in an autoimmune mechanism such as multiple sclerosis and diabetes can be classified into a group in which the anti-HC2ENV protein antibody is present in a body fluid such as serum and a group in which the anti-HC2ENV protein antibody is not present. A highly effective therapeutic drug in each of the patient group having the anti-HC2ENV protein antibody and the patient group not having the anti-HC2ENV protein antibody by comparing the responsiveness and therapeutic effect of the patient to the therapeutic drug / therapy based on the classification・ Enables selection and development of treatment methods. In addition, the IgA nephropathy marker comprising the anti-HC2ENV protein antibody of the present invention is based on the antibody titer or reaction specificity, and is compared with the severity / activity of the subject's pathology as a novel disease state index. Available.

  Next, the gene expression measuring method of the present invention will be described. The gene expression measuring method of the present invention is a method for measuring gene expression of the HC2env gene of the present invention, and is a gene expression measuring method of measuring the mRNA of the present invention or the HC2ENV protein of the present invention.

  The gene expression measurement method of the present invention for measuring the mRNA of the present invention is preferably performed using, for example, the polynucleotide of the present invention used as a primer or probe of the HC2env gene of the present invention. Specific examples of the primer include a pair of the 011295ORF-L and the 011295ORF-R (SEQ ID NOS: 3 and 4 in the sequence listing), a pair of the Primer L and the Primer R (SEQ ID NOS: 5 and 6 in the sequence listing), And the following HC2env011L1 and the following HC2env011R (SEQ ID NOS: 7 and 8 in the sequence listing) are listed. Among these, the primer pair is preferably a pair of HC2env011L1 and HC2env011R. The measurement of mRNA by the RT-PCR method may be a real-time RT-PCR method, a competitive RT-PCR method, or the like, and the method and reagents are not particularly limited and conventionally known ones can be used. In the Northern blotting, for example, the polynucleotide of the present invention comprising the nucleotide sequences of SEQ ID NOs: 17 to 24 in the sequence listing can be used as a probe. Available.

  The mRNA of the HC2env gene to be measured can be recovered from, for example, the peripheral blood mononuclear cells of the subject. The recovery method is not particularly limited. For example, the peripheral blood mononuclear cells can be extracted as a part of total RNA by a normal method using TRIzol reagent (manufactured by Invitrogen), or the peripheral blood mononuclear cells. An example is a method in which spheres are stimulated with a lectin such as phytohemagglutinin and then extracted in the same manner as described above.

  The gene expression measurement method of the present invention for measuring the HC2ENV protein of the present invention can be performed by a conventionally known immunological measurement method using, for example, an antibody having the HC2ENV protein of the present invention as an antigen. The immunological measurement method is not particularly limited, and examples thereof include Western blot, ELISA, and sandwich ELISA. The antibody having the HC2ENV protein of the present invention as an antigen may be prepared by a conventionally known method using the HC2ENV protein of the present invention, but the anti-gp70 mouse monoclonal antibody 12H5.1 or the anti-Rauscher leukemia virus gp70 Antibodies may be used.

  It is considered that the concentration of the HC2ENV protein antigen in the blood and the amount of antibody produced against the HC2ENV protein of the present invention change depending on the expression level of the mRNA of the present invention, thereby causing the disease to be hated or ameliorated. Therefore, according to the gene expression measurement method of the present invention, the disease state of a subject is determined, or the prognosis is predicted, and for example, a strong treatment or a follow-up treatment should be performed. Can be useful.

  Next, the human antibody measurement method of the present invention will be described. The method for measuring a human antibody of the present invention is a method for measuring a human antibody (anti-HC2ENV protein antibody) using the HC2ENV protein of the present invention as an antigen, wherein the HC2ENV protein of the present invention and the human antibody are reacted with each other by an antigen-antibody reaction, This is a method for measuring a complex of the HC2ENV protein of the present invention, the human antibody and the labeled antibody by reacting the human antibody with a labeled antibody using the human antibody as an antigen.

  In the human antibody measurement method of the present invention, a conventionally known method can be used as a specific measurement method, and is not particularly limited. For example, the HC2ENV protein of the present invention is preferably immobilized on a solid phase. . An example of this measurement method is shown in FIG. As shown in the figure, the HC2ENV protein of the present invention is immobilized on a solid phase such as a plastic plate or a filter membrane as an antigen 1 by a general method, and the immobilized antigen 1 and a human antibody that is a measurement target in the sample Then, the labeled antibody (labeled secondary antibody) 3 is immunoreacted with the human antibody 2 to be bound thereto. Then, by measuring the label of the labeled antibody 3, the presence or absence of the human antibody 2 reacting with the antigen 1 in the sample can be determined. As the sample, for example, a body fluid containing human immunoglobulin such as human blood, serum, plasma, saliva, joint fluid, cerebrospinal fluid, and the like can be used. Examples of the labeling of the labeled antibody 3 (measurement method using the labeled antibody) include, for example, radioactive labeling (radioimmunoassay (RIA)), enzyme labeling (enzyme immunoassay (ELISA)), or fluorescent labeling (fluorescence) Immunoassay (FIA)) and the like.

  By the human antibody measurement method of the present invention, for example, the detection of the anti-HC2ENV protein antibody and the quantification of the antibody titer of the anti-HC2ENV protein antibody can be performed. Furthermore, according to the human antibody measurement method of the present invention, for example, patients with diseases suspected to be involved in autoimmune mechanisms such as IgA nephropathy and other primary glomerular diseases, multiple sclerosis, diabetes and the like. And a group in which an anti-HC2ENV protein antibody is present in a body fluid such as serum and a group that is not. Based on this classification, by comparing the patient's responsiveness and therapeutic effect to therapeutic drugs and treatment methods, treatments with high efficacy in each of the patient group having the anti-HC2ENV protein antibody and the other patient group are not. Drugs and treatment methods can be selected and developed. In addition, the measurement method of the present invention can obtain the antibody titer or reaction specificity of an anti-HC2env gene product antibody of a subject, which is compared and contrasted with the severity and activity of the subject's pathological condition. It can be an indicator.

  Next, the IgA nephropathy test kit of the present invention will be described. The IgA nephropathy test kit of the present invention is a test kit for measuring the gene expression of the HC2env gene of the present invention by the gene expression measurement method of the present invention, and the polynucleotide of the present invention used as a primer or probe, or the present invention It is a test kit for IgA nephropathy comprising an antibody having the HC2ENV protein of the invention as an antigen. The IgA nephropathy test kit of the present invention may contain conventionally known reagents necessary for the gene expression measurement method of the present invention, for example, total RNA extraction reagent, polymerase, nucleotides, labeled compounds, buffers, etc., if necessary. preferable.

  In another aspect, the IgA nephropathy test kit of the present invention is an IgA nephropathy test kit for measuring an anti-HC2ENV protein antibody by the human antibody measurement method of the present invention, comprising the HC2ENV protein of the present invention and the human antibody. An IgA nephropathy test kit comprising a labeled antibody as an antigen. The IgA nephropathy test kit of the present invention preferably contains conventionally known reagents necessary for the human antibody measurement method of the present invention, for example, a labeling compound, a coloring reagent, a buffer and the like, if necessary.

  Next, the pharmaceutical product of the present invention will be described. The pharmaceutical product of the present invention is a pharmaceutical product for at least one of treatment and prevention of IgA nephropathy, comprising a base sequence complementary to a gene containing all or part of the HC2env gene of the present invention, and comprising the HC2env of the present invention. A pharmaceutical comprising a polynucleotide capable of suppressing gene transcription or protein expression. That is, this pharmaceutical is a pharmaceutical to which RNAi (RNA interference) such as antisense method, siRNA method, miRNA method, etc. is applied. The pharmaceutical product of the present invention preferably contains a pharmaceutically acceptable adjuvant or excipient.

  In another aspect, the pharmaceutical agent of the present invention is a pharmaceutical agent for at least one of treatment and prevention of IgA nephropathy, and an antigenic determinant (epitope) for an anti-HC2ENV protein antibody in the amino acid sequence of the HC2ENV protein of the present invention. A pharmaceutical comprising a polypeptide comprising the amino acid sequence of In addition, the pharmaceutical of the present invention may contain a low molecular compound that undergoes an antigen-antibody reaction with an anti-HC2ENV protein antibody in addition to or in place of the polypeptide. The pharmaceutical product of the present invention preferably contains a pharmaceutically acceptable adjuvant or excipient.

  Next, the expression vector of the present invention will be described. The expression vector of the present invention is an expression vector for introducing a gene containing all or part of the HC2env gene of the present invention into a target cell, the gene containing all or part of the HC2env gene of the present invention, An expression vector comprising a regulatory sequence necessary for expression of the gene, wherein the gene is operably linked to the regulatory sequence. By using the expression vector of the present invention, target cells can be transformed to express all or part of the HC2ENV protein of the present invention. And the transformed cell manufactured by the said transformation can be utilized as a packaging cell for manufacturing the retrovirus vector for gene transfer of this invention as mentioned later, for example. In this case, in the expression vector of the present invention, a part of the HC2env gene of the present invention is not particularly limited, but preferably contains the receptor binding domain (RBD) of the HC2ENV protein of the present invention.

  The expression vector of the present invention may be, for example, a single-stranded, double-stranded, circular or supercoiled DNA molecule or RNA molecule. The method for producing the expression vector of the present invention is not particularly limited, and for example, a gene containing all or part of the HC2env gene of the present invention can be expressed in the regulatory sequence in an expression vector having a known regulatory sequence. The method of inserting in is mentioned. Examples of the known expression vectors include retrovirus vectors, adenovirus vectors, adenovirus associated virus vectors, vaccinia virus vectors and other non-viral vectors such as pBK-CMV, pcDNA3, and pZeoSV (manufactured by Invitrogen). Viral vectors are examples. The regulatory sequence is a nucleotide sequence necessary for expression of a gene containing all or part of the HC2env gene of the present invention operably linked in a cell, and is suitable for eukaryotic cells, for example. Examples of the sequence include a promoter, a polyadenylation signal, an enhancer and the like. Said operatively connected means that the components are juxtaposed so that they can perform their functions.

  Next, the retrovirus vector for gene transfer of the present invention will be described. The retrovirus vector for gene transfer of the present invention is a retrovirus vector for gene transfer in which the coat protein of the virus particle contains all or part of the HC2ENV protein of the present invention. The part of the HC2ENV protein of the present invention is not particularly limited, but preferably contains the receptor binding domain (RBD) of the HC2ENV protein of the present invention. The retrovirus vector for gene transfer of the present invention can be used for gene therapy, for example, as described later. In addition, it can be used to introduce a gene into a target cell for research purposes or production of pharmaceuticals.

  Retroviruses convert the RNA genome in their particles into double stranded DNA by reverse transcriptase, and the DNA genome is integrated into the infected host cell chromosome as a provirus. Therefore, it is possible to integrate any target gene into the target cell chromosome using a genetically modified retrovirus in which a heterologous gene is incorporated into the viral genome, and obtain a transformed cell that permanently retains this gene. . Retroviruses are widely used for research purposes and therapeutic purposes as a means of stable gene transfer. Retroviruses for gene transfer use a recombinant RNA genome containing the ψ sequence and target gene necessary for RNA incorporation into the virus particle, protein necessary for virus particle formation, and RNA genome reversal in the infected host cell. It can be produced by expressing in a cell producing a protein required for copying and chromosomal integration. The protein required for the formation of virus particles is the gag gene product, and the protein required for reverse transcription and chromosomal integration of the RNA genome is the pol gene product. The env gene product determines what target cells can be adsorbed and invaded by infectious virus particles budding from virus-producing cells.

  In the case of a conventional retrovirus vector for gene transfer, when gene transfer is carried out into a mouse cell, an env gene such as a homotrophic murine retrovirus, particularly Moloney leukemia virus, is used for the formation of particle surface proteins. In addition, when introducing a gene into a human cell, the env gene of xenotropic or amtropotropic mouse retrovirus is used, and infectivity to a wider range of cells is further achieved. To ensure this, the viral particle surface protein of bovine vesicular stomatitis virus (VSV) may be used. However, when such a recombinant virus vector is inoculated into humans, an immune reaction occurs against the heterologous retrovirus env gene product or VSV virus particle surface protein, which is the particle surface protein of the inoculated virus vector. It has been known. Moreover, even when the virus particle surface protein is replaced with a human retrovirus such as human T cell leukemia virus (HTLV) or human immunodeficiency virus (HIV), these infectious human retroviruses are endogenous. Since the infection is not from the body but from outside the body, an immune reaction occurs against the env gene product. In fact, it is known that a host immune response including neutralizing antibody production occurs against HTLV and HIV infection.

  In contrast, in the retroviral vector for gene introduction of the present invention, HC2ENV protein is expressed as a particle surface protein. Since the HC2env gene is originally present on the human chromosome, no immune response usually occurs against the HC2ENV protein, except for patients with autoimmune diseases. Therefore, the retroviral vector for gene transfer of the present invention having the HC2ENV protein of the present invention as a particle surface protein does not cause an immune reaction such as neutralizing antibody production even if the same vector is repeatedly administered, and the same vector is used. It becomes possible to introduce genes repeatedly.

  The method for producing the retroviral vector for gene introduction of the present invention is not particularly limited, and can be produced by a conventionally known method except that the surface protein of the virus particle contains all or part of the HC2ENV protein of the present invention. 16 can be produced. First, a gene introduction plasmid 5 containing a gene 4 to be introduced, a packaging signal (Ψ sequence), an LTR and the like is prepared. As the gene transfer plasmid 5, for example, pCMVβ (Clontech) or the like can be used. Next, this gene introduction plasmid 5 is introduced into the packaging cell 6. The packaging cell 6 is a cell that has a gene containing all or part of the HC2env gene of the present invention and expresses as a surface protein 7 a protein containing all or part of the HC2ENV protein that is the gene product. Then, the retrovirus vector 8 for gene introduction of the present invention germinated from the packaging cell 6 is recovered. The packaging cell 6 preferably further expresses gag gene and pol gene. The type of the gene to be introduced 4 is not particularly limited.

  Next, the gene introduction method of the present invention will be described. The gene transfer method of the present invention is a gene transfer method using the retrovirus vector for gene transfer of the present invention. The gene transfer method of the present invention is not particularly limited, and in the gene transfer method using a conventional retrovirus vector, the retrovirus vector for gene transfer of the present invention can be used instead of the retrovirus vector. it can. According to the gene introduction method of the present invention, for example, as described above, gene introduction can be repeated while suppressing immune reactions such as neutralizing antibody production, and is useful for gene therapy, for example.

  Next, a method for producing the gene-transferred cell of the present invention will be described. The method for producing a gene-transferred cell according to the present invention is a method for producing a gene-introduced cell comprising a step of introducing a target gene into a target cell by the gene-transfer method of the present invention. The target gene is not particularly limited. The target cell is not particularly limited, and examples thereof include in vivo or in vitro human cells.

  Next, the gene introduction kit of the present invention will be described. The gene introduction kit of the present invention is a gene introduction kit comprising a packaging cell transformed with the expression vector of the present invention and a gene introduction plasmid having a ψ sequence.

  In addition, the gene introduction kit of the present invention may be a gene introduction kit including the expression vector of the present invention and a gene introduction plasmid having a ψ sequence as another embodiment. The gene introduction kit of the present invention preferably further comprises a plasmid that expresses at least one of the gag and pol genes. For example, a packaging cell for producing the viral vector for gene transfer of the present invention can be produced by transforming appropriate cells with the expression vector of the present invention and a plasmid expressing the gag and pol genes. it can.

  Hereinafter, the present invention will be further described with reference to examples of the present invention. These examples do not limit the invention in any way.

(Detection of IgA nephropathy patient glomerular deposition antigen)
A part of the patient tissue collected by renal biopsy performed for the definite diagnosis of IgA nephropathy was immediately frozen and fixed in isopentane with dry ice and stored frozen. A frozen tissue section prepared with a cryostat was re-fixed with 95% ethanol, and then exposed to 6M urea solution (6M urea, 0.1M glycine-HCl, pH 3.5) to expose the antigen structure in the immune complex. After treatment at 1 ° C. for 1 hour, it was reacted with mouse monoclonal antibodies specific for various known viral antigens. The reaction of each antiviral antibody with renal tissue was detected with a FITC-labeled anti-mouse immunoglobulin secondary antibody. As a negative control, a section was prepared by reacting only with the diluent instead of the primary antibody and then reacting with the fluorescently labeled secondary antibody. As a result, only the monoclonal antibody 12H5.1 against the mouse endogenous xenobiotic retrovirus env gene product gp70 reacted with a part (about 30%) of tissues derived from IgA nephropathy patients. The result is shown in FIG. The three photographs in the figure show three representative examples in which antigen molecules deposited in the glomeruli of IgA nephropathy patients were detected by the 12H5.1 antibody and FITC-labeled anti-mouse IgG secondary antibody. As shown in the figure, strong granular fluorescence was observed in the glomerular mesandium region.

  The anti-gp70 antibody clone 12H5.1 has been described in the literature [Tabata, N. et al Establishment of monoclonal anti-retroviral gp70 autoantibodies from MRL / lpr lupus mice and induction of glomerular gp70 deposition and pathology by transfer into non-autoimmune mice. Of anti-retroviral gp70 monoclonal autoantibodies from / lpr lupus mice and induction of glomerular gp70 deposition and lesions by introduction into non-autoimmune mice ”J. Virol. 74: 4116-4126, 2000.] A large number of hybridoma cells were produced from MRL / MpJ-lpr / lpr mice that spontaneously develop autoimmune lesions including glomerulonephritis and were forced to express using a vaccinia virus vector. Isolated as an anti-gp70 antibody-producing clone by reactivity with retrovirus env gene product (gp70) It is intended.

(Detection of human genomic gene homologous to the mouse env gene (gp70))
In order to confirm a gene encoding an antigen detected by an antibody specifically reacting with a mouse retrovirus env gene product, present in a glomerular lesion of a human IgA nephropathy patient, prepared based on the mouse retrovirus env gene sequence Southern blot analysis was performed on a human genomic DNA library using the prepared probe.

The probe is a plasmid containing the full length of the env gene derived from the mouse endogenous xenobiotic retrovirus cDNA clone (pGP6-8) used for screening the 12H5.1 antibody as a template, and SEQ ID NO: 25 (gga aga aga agg aca aga A ³² P-labeled DNA probe prepared using a synthetic oligonucleotide having a base sequence of ctg) as a primer was used. FIG. 2 shows the amino acid sequence of the mouse endogenous xenobiotic retrovirus env gene product and the base sequence of the gene. The underlined base sequence in the figure is the probe sequence.

  The DNA library was prepared by obtaining about 20 ml of peripheral blood after obtaining written consent from a IgA nephropathy patient and a healthy volunteer after obtaining written consent. Mononuclear cells are separated from the peripheral blood by specific gravity centrifugation, and genomic DNA is prepared by repeated digestion with a proteolytic enzyme and chloroform / phenol extraction by a conventionally known method, and this is subjected to limited digestion of the restriction enzyme Sau3AI. Thereafter, the human genomic DNA library was obtained by incorporating it into a λEMBL3 phage vector.

  The result of Southern blotting performed on the human genomic DNA library is shown in FIG. In the figure, the numbers shown above are phage clone names, and B and E indicate that the clones were completely decomposed with BamHI (B in the figure) and EcoRI (E in the figure), respectively. . As shown in the figure, it was confirmed that a very small number of gene sequences showing extremely high homology with the mouse endogenous heterotrophic retrovirus env gene were present in human genomic DNA.

(Identification of human endogenous retrovirus HC2env gene by database search)
A sequence in human genomic DNA encoding an amino acid sequence that is homologous to the amino acid sequence of the mouse endogenous heterologous retrovirus env gene product was expressed as National Center for Biotechnology Information (NCBI, National Institutes of Health, USA). ) Using a bacterial artificial chromosome (BAC) contig base sequence database of human chromosomes registered in (1)), and searched by TBASTN. The search condition shows 30 to 40% homology as a whole with the amino acid sequence of the mouse env gene product gp70, and among them, the N-terminal region containing the epitope of the 12H5.1 antibody shows higher homology Selected. As a result, the human endogenous heterotrophic retrovirus HC2env gene consisting of the base sequence of SEQ ID NO: 1 in the sequence listing was identified as a gene encoding the protein consisting of the amino acid sequence of SEQ ID NO: 2 in the sequence listing. As a result of searching using the database, the full-length human HC2env gene consisting of the nucleotide sequence of SEQ ID NO: 1 in the sequence listing is present in the vicinity of the 19p13.1 region of the short arm of chromosome 19 as shown in FIG. It was shown that. A series of operations will be described in more detail below.

  Using the amino acid sequence of the mouse endogenous heterologous retrovirus env gene product as Query, the human genome contig sequence database registered in NCBI was subjected to homology search by TBLASTN. At this time, the sequence used as the query is the N-terminal 191 amino acids of the mouse endogenous xenobiotic retrovirus env gene product (Accession No. X59305), which was shown to react with the 12H5.1 antibody, The amino acid sequence is as follows (SEQ ID NO: 26 in the Sequence Listing).

  As a result, 36 nucleotide sequences were listed. Among these nucleotide sequences, a cysteine residue having a characteristic arrangement in the env gene product of gamma retrovirus (mouse endogenous xenogenic retrovirus is also classified as this group), and amino acid motif (FYLCP) Multiple sequences with) were found.

  Next, in order to know whether the found nucleotide sequence encodes a protein similar to any known viral gene product, the GenBank nr database was searched for TBLASTN using the obtained amino acid sequence as a query. As a result, the found nucleotide sequence was found in the literature [Kabat, P., M. Tristem, R. Opavsky, & J. Pastorek.Human inherent retrovirus HC2 is a new member of the S71 retrovirus subgroup with a full-length pol gene. Human endogenous retrovirus HC2 (Accession No. Z70664) described in “Human endogenous retrovirus HC2 is a subgroup of S71 retroviruses with the full-length pol gene” Virology 226: 83-94, 1996.]. ) Was found to be very similar to the “3′-LTR region”. From this result, the portion of HC2 that was reported to have no env gene in the past was actually the env gene region, and the mouse endogenous xenobiotic retrovirus env gene product. The possibility of encoding molecules with similar amino acid sequences was suggested.

  When an open reading frame present in a portion mistakenly designated as “3′-LTR region” in HC2 (Accession No. Z70664) reported by Kabat et al. Was searched with ORF Finder (NCBI), the following amino acids were found: It was found that a protein having a sequence can be encoded (SEQ ID NO: 27 in the sequence listing).

  Therefore, in order to systematically search for similar base sequences on human chromosomal DNA, the NCBI human genome contig base sequence database was again searched for homology by TBLASTN using this sequence as a query. As a result, 96 base sequences were listed. As a result of searching for an open reading frame of a region containing these nucleotide sequences and 3 kbp on the 3 ′ side thereof, it was similar to the murine leukemia virus env gene product consisting of 626 amino acids on the chromosome 19 genomic contig contained in the human genomic contig database. A nucleotide sequence capable of encoding a polypeptide (that is, the HC2env gene of the present invention) was newly identified. In addition, this base sequence can be obtained from NCBI as GenBank Accession NT_011295 (region: 11743043-11744923). However, there has been no report on the amino acid sequence of a protein translated from this base sequence.

(Acquisition of bacterial artificial chromosome (BAC) clone containing HC2env gene)
Since the HC2env gene was found on the inserted human chromosome fragment of the BAC clone CTC-575C13 by the description of the NCBI human genome contig sequence database, this BAC clone was researched as an authorized clone distributor of the public human genome project. Purchased from Genetics (Invitrogen). BAC DNA was purified by Large Construct Kit (QIAGEN K.K., Tokyo). The base sequence at both ends of the inserted sequence of the purified BAC DNA was determined, and it was confirmed that there was no mistake by comparing it with the BAC clone CTC-575C13 insert base sequence (Accession No. AC078899) registered in the GenBank database. The sequencing primers T7 and Sp6 on the vector used at this time are as follows (SEQ ID NOS: 28 and 29 in the Sequence Listing).

(Isolation of HC2env gene)
Using the BAC DNA prepared as described above as a template, the full length of the HC2env gene open reading frame was amplified by the PCR method using the following primer pairs 011295ORF-L and 0121295ORF-R (SEQ ID NOS: 3 and 4 in the sequence listing), and plasmids Subcloned into vector pSC11-MCS / His. Among the plasmid clones thus obtained, the base sequence of the full length of the insert was determined for 5 independent clones. As a result, it was confirmed that these insert sequences were 100% identical with each other and 100% identical with the nucleotide sequence of the HC2env gene described in SEQ ID NO: 1 in the sequence listing.

(Identification and confirmation of authentic 3 ′ LTR and 5 ′ LTR)
As described above, as evidence supporting the existence of the HC2env gene of the present invention in the past reports that the part that was regarded as the 3 ′ LTR is not actually the LTR, the present inventors A structure judged to be an intrinsic LTR from the characteristics of its base sequence, that is, a 3 ′ LTR, is found downstream of the stop codon of the HC2env gene of the invention, and further gag− on the same chromosome as the HC2env gene of the present invention is found. A 5 ′ LTR having 96% homology with the 3 ′ LTR was confirmed upstream of the pol gene region. The sequences of these LTRs and their structures are shown in FIG. As shown in the figure, the newly found 3 ′ LTR and 5 ′ LTR have a CCAAT box, a TATA box, and a polyadenylation signal that are characteristic of the retrovirus LTR.

  Next, the newly found 3 ′ LTR and 5 ′ LTR are actually functioning LTRs, and the 3 ′ RACE (rapid amplification of cDNA ends) method and 5 ′ RACE method of the HC2env gene are used. , And cDNA full length amplification. A summary of the results is shown in FIG. As shown in the figure, the structure of the 3 ′ end of mRNA expressed from the HC2 provirus coincides with the newly found polyadenylation signal site in the 3 ′ LTR, and the structure of the 5 ′ end is also newly seen. It coincided with the transcription start site structure in the 5 ′ LTR. Furthermore, when the HC2env gene of the present invention is expressed, the mRNA precursor transcribed from the newly identified 5 ′ LTR is spliced upstream of the HC2env gene, and the mature mRNA containing the entire HC2env gene sequence of the present invention is It was also confirmed that it was formed (see FIG. 6).

  Here, cDNA used for each RACE reaction was prepared from human thyroid total RNA using a SMART RACE cDNA amplification kit (Becton Dickinson Bioscience). PCR reaction is performed according to the manual of SMART RACE method, and each fragment obtained is subcloned into the plasmid vector of the kit using TOPO cloning kit (manufactured by Invitrogen), and the primer whose base sequence of each inserted fragment is set on the vector Was used for analysis. The cDNA used for amplification of the full length expression product of the HC2env gene of the present invention was prepared from human thyroid total RNA using Superscript III (manufactured by Invitrogen). The primer used in the 3′RACE method of the env gene is HC2env3RACE1, the primer used in the 3′RACE method of the pol gene is HC2pol3RACE1, and the primer used in the 5′RACE method is 5RACEpol-env. The primer pair used for the full length amplification of cDNA is HC2PV-L and HC2PV-R, and the base sequences of the primers are as follows (SEQ ID NOs: 30 to 34).

  Thus, the part conventionally thought to be the 3 ′ LTR of the human endogenous retrovirus HC2 is not a true LTR but a part of the HC2env gene of the present invention, and includes the entire HC2env gene of the present invention. mRNA expression was observed. Therefore, there is no doubt that the gene of the present invention is the env gene of the HC2 provirus.

(Detection of HC2env gene expression product mRNA)
The HC2env gene expression product mRNA was detected by RT-PCR to confirm that the HC2env gene of the present invention was expressed in human tissues. CDNA was prepared by Super script III (manufactured by Invitrogen) using total RNA extracted from various human tissues by a normal method as a template. Using the obtained cDNA as a template, the HC2env gene cDNA was amplified by the usual RT-PCR method using the following primer pairs HC2env011L1 and HC2env011R (SEQ ID NOs: 7 and 8 in the sequence listing), and the amplified product was obtained by agarose gel electrophoresis. After separation, detection was performed by ethidium bromide staining.

  The result is shown in FIG. In the figure, + or-in the RTase row indicates whether or not Superscript III reverse transcriptase is added to the total RNA of each tissue as a sample, and genomic DNA contamination. Is a negative control to negate the detection of PCR products. As shown in the figure, mRNA, which is an HC2env gene expression product, could be detected specifically in the kidney, prostate, lung and the like.

(Expression of HC2ENV protein which is HC2env gene product protein)
The HC2env gene cloned in Example 1 was inserted into the vaccinia virus transfer vector pSC11-MCS / His shown in FIG. 8 to produce a recombinant vaccinia virus expressing the full length of the HC2env gene. The pSC11-MCS / His is described in the literature [Sugahara, D., S. Tsuji-Kawahara, and M. Miyazawa. Identification of a protective CD4 ⁺ T-cell epitope in p15 ^gag of Friend murine leukemia virus and role of the MA protein. “Identification of protective CD4 ⁺ T cell epitopes in p15 ^gag of Friend mouse leukemia virus and the role of MA protein localization in the plasma membrane plays a role in its immunogenicity” J. Virol. 78 : press, 2004.], which is a vaccinia virus expression vector capable of fusing a (His) ₆ tag to the C-terminus. The HC2env gene was inserted so that the 3 ′ end of the HC2env gene was in-frame with the polyhistidine [(His) ₆ ] residue to obtain a recombinant vaccinia virus vector expressing the HC2env gene.

The expression of the recombinant HC2ENV protein was confirmed by an indirect fluorescent antibody method in which the monkey CV-1 cell line infected with the above recombinant vaccinia virus was stained with a commercially available antibody against (His) ₆ . That is, CV-1 cells infected with the above recombinant vaccinia virus are fixed with 3.7% formaldehyde and subjected to cell membrane removal treatment with 0.4% Triton X-100. An antibody against (His) ₆ (trade name His-probe, manufactured by Santa Cruz Biotechnology) was reacted, washed, and further subjected to FITC-labeled anti-rabbit immunoglobulin antibody (Goat Anti-Rabbit IgG, H + L chain specific Biotechnology, FITC Conjugative Biotech Made). After washing, it was observed with a fluorescence microscope. The result is shown in FIG. As shown in the figure, strong fluorescence was detected in the virus-infected cells in the upper right half region from the center of the figure, confirming the expression of the recombinant HC2ENV protein. On the other hand, no localization of fluorescence due to antibody binding was observed in the non-virus-infected cells in the lower left of the figure.

Purification of recombinant HC2ENV protein expressed in CV-1 cells infected with the recombinant vaccinia virus was performed as follows. First, the infected cells were collected and then treated with a cell solubilization buffer (for example, 6M guanidine-HCl, 20 mM NaPO ₄ pH 7.8, 500 mM NaCl) to solubilize the cells. Next, the cellular genomic DNA was shredded through an injection needle and further centrifuged to collect the supernatant. Then, (His) _6- labeled HC2ENV protein was purified from the collected supernatant using a column packed with a commercially available nickel chelate resin.

(Expression and detection of chimeric gene product of HC2env gene)
Using a plasmid containing the full length of the HC2env gene as a template and using the following primer pair HC-start-RI and HC2-Sma-3 (SEQ ID NOs: 35 and 36 in the Sequence Listing), the receptor binding domain (Receptor- A region including the binding domain (RBD) (the 1st to 651st base sequences of SEQ ID NO: 1 encoding the 1st to 217th amino acid sequences of SEQ ID NO: 2) was amplified by the PCR method.

  When the amino acid sequence of the HC2 env gene product shown in SEQ ID NO: 2 in the sequence listing is compared with the amino acid sequences of the known heterologous murine leukemia virus and ambigenic murine leukemia virus env gene products, the 75th cysteine (Cys) is The sequence of Asp-Leu-Cys-Val (or Asp) -Leu containing is the same as the sequence conserved in mouse leukemia virus, Asp-Phe-Tyr-Leu (or Val containing the 132nd Cys) ) -Cys-Pro-Gly is the same as the conserved sequence of murine leukemia virus, and the Asn-Pro-Leu sequence downstream of the 186th Cys is also murine leukemia including Cys. Ser-Trp-Gly-Leu-Arg-Leu- starting from the 206th Ser The sequence of Tyr and the sequence of Thr-Met-Phe starting from the 220th Thr downstream of the Tyr are completely identical to the murine leukemia virus, and the Pro-Lys (or Arg) -Pro starting from the 235th Pro. The sequence of -Ile-Gly-Pro, the sequence of Asn-Leu-Thr starting from the 292nd Asn, and the sequence of the Cys-Trp-Leu-Cys-Leu starting from the 302nd Cys immediately downstream thereof are also murine leukemia virus It turns out that it is common. From these characteristic amino acid sequences, the region surrounded by the 75th Cys and 114th Cys of the HC2env gene product is important for determining the target receptor specificity in the receptor binding domain (RBD) of murine leukemia virus. The 186th Cys corresponds to the Cys corresponding to the carboxyl terminal boundary of RBD in murine leukemia virus, and the 235th Pro is a proline rich region (PRR). It can be easily guessed that there is. For the definition of the conserved Cys residues of these characteristic amino acid sequences, the VRA region and the PRR region, refer to the literature [Battini JL, Heard JM, Danos O. Receptor choice determinants in the envelope glycoproteins of amphotropic, xenotropic, and polytropic murine leukemia viruses. "Determinants of receptor selection in capsular glycoproteins of bi-, hetero-, and multi-directional murine leukemia viruses" J Virol. 66 (3): 1468-75, 1992].

  As shown in Example 6 described later, the recombinant protein containing up to the 217th glycine in the HC2env gene product of SEQ ID NO: 2 in the sequence listing, that is, the region presumed to be RBD, Bind to the surface. It is also clear from the amino acid sequence of SEQ ID NO: 2 in the sequence listing that the amino acid sequence of the portion presumed to be the PRR region is actually rich in proline residues.

  The amplified product obtained by the PCR method was digested with restriction enzymes EcoRI and SmaI, and then incorporated into a cloning vector pBSII-KS (+) (Stratagene). On the other hand, a plasmid containing the env gene of friend mouse leukemia virus (F-MuLV) (references [Iwashiro, M., T. Kondo, T. Shimizu, H. Yamagishi, K. Takahashi, Y. Matsubayashi, T. Masuda, A Otaka, N. Fujii, A. Ishimoto, M. Miyazawa, MN Robertson, B. Chesebro, and K. Kuribayashi. Multiplicity of virus-encoded helper T-cell epitopes expressed on FBL-3 tumor cells. J. Virol. 67 : 4533-4542, 1993]], the region from the downstream of RBD to the stop codon was cut out using restriction enzyme SmaI and incorporated downstream of the above HC2envRBD. A DNA fragment containing the full length of the HC2-RBD / F-MuLVenv chimeric gene was excised from the thus prepared plasmid (pBS / HC2 / F-Mulv) using the restriction enzyme NotI, and incorporated into an expression vector pCMVβ (Becton Dickinson Bioscience). pC. HC2. Ver1. This expression plasmid expresses the chimeric env gene product protein shown below.

  In the chimeric env gene product protein, M represents a methionine residue corresponding to the start codon, PRR represents a proline-rich region characteristic of a retroviral env gene product, TM indicates a trans-membrane region, and STOP indicates a stop codon.

  Similarly, using the plasmid p20-8 containing the full length of the Friend murine leukemia virus F-MuLVenv gene as a template, using the following primer pair clone57-5kpn and clone57-3kpn (SEQ ID NOs: 37 and 38 in the sequence listing), F- A region including the MuLVenv gene from the start codon to immediately before the RBD was amplified by the PCR method.

  The amplified fragment thus obtained and a restriction enzyme KpnI-treated fragment of pBS / HC2 / F-Mulv (a fragment encoding the 63rd to 217th amino acid sequences of SEQ ID NO: 2 in the sequence listing including the RBD of the HC2ENV protein) From the plasmid obtained by ligation, a DNA fragment containing the F-MuLV / HC-RBD / F-MulVenv gene was excised again using the restriction enzyme NotI, incorporated into the expression vector pCMVβ, pC. HC2. Ver2. This expression plasmid expresses the chimeric env gene product protein shown below.

  In the chimeric env gene product protein, M, PRR, TM, and STOP are the same as described above. In the above chimeric env gene product protein, the start codon through the leader peptide part to immediately before the RBD, and the PRR through TM through the stop codon are derived from the F-MuLVenv gene, and the part containing the RBD is derived from the HC2 env gene. is there.

  In order to confirm the expression of these chimeric env genes as proteins, analysis by Western blotting was performed. Negative control empty pCMVβ plasmid, pC. HC2. ver1, pC. HC2.ver2, and the positive control F-MuLVenv full length pC. F-MuLV was introduced into a monkey-derived Cos7 cell line by a lipofection method using Lipofectamine 2000 (manufactured by Invitrogen), and the cells were collected after culturing for 24 hours. The collected cells are washed with a phosphate buffered balanced salt solution, lysed with a cell lysate (1% NP40, protease inhibitor cocktail, TNE buffer containing PMSF), and centrifuged (12,000 rpm, 10 minutes). Went. Each obtained supernatant sample was electrophoresed on 8% SDS-polyacrylamide gel, and transferred to an Immobilon-P (MILLIPORE) membrane. After blocking the transferred membrane with 10% skim milk solution, a 2,000-fold diluted solution of anti-Rauscher leukemia virus gp70 antibody was reacted as the primary antibody, and further, horseradish peroxidase (HRP) -labeled anti-sheep IgG was used as the secondary antibody. After reacting a 10,000-fold dilution of the antibody, binding of the antibody to the expressed protein was detected by a chemiluminescence method using ECLplus (Amersham Biosciences Corp.). The result is shown in FIG. As shown in the figure, the expression of the protein reacting with the anti-Rauscher leukemia virus gp70 antibody can be detected at the same position as the gp70 molecule in the purified F-MuLV particle. From any of the chimeric env genes, The product protein was shown to be translated.

(Detection of anti-HC2ENV protein antibody in plasma of IgA nephropathy patient)
Cells infected with the recombinant vaccinia virus expression vector expressing the recombinant HC2ENV protein obtained in Example 3 were fixed with 3.7% formaldehyde and then treated with 0.4% Triton X-100 to remove the cell membrane. This was reacted with a 14-fold diluted sample of IgA nephropathy patient plasma, and after washing, further reacted with a fluorescent-labeled anti-human immunoglobulin antibody, thereby anti-existing in the plasma of IgA nephropathy patient. HC2env gene product protein antibody, ie, anti-HC2ENV protein antibody was detected. An example of the result is shown in FIG. As shown in the figure, strong fluorescence accumulation due to antibody binding in IgA patient plasma was observed in the recombinant virus-infected cell population in the center of the figure.

(Comparative Example 1)
Recombinant expressing a fragment of the human intracellular signaling molecule HS-1 completely unrelated to the HC2env gene, instead of the cells infected with the recombinant vaccinia virus vector expressing the recombinant HC2ENV protein obtained in Example 3. Antibody binding in IgA patient plasma was detected in the same manner as in Example 5, except that vaccinia virus vector-infected cells were used. The result is shown in FIG. As shown in the figure, unlike Example 5, the accumulation of strong fluorescence was not recognized except that some weak fluorescence was observed on the left side of the figure.

  As can be seen from the results of Examples 1, 3, 5 and Comparative Example 1, there is an antibody (IgA) that causes an immune reaction with the protein that is the expression product of the gene of the present invention in the plasma of IgA nephropathy patients. . Therefore, the onset of IgA nephropathy occurs when the gene of the present invention is expressed to produce a protein that is an expression product thereof, and when the human has an antibody that reacts with this protein by an autoimmune mechanism, It can be said that the complex is deposited in the glomerular mesandium region and causes a chronic inflammatory response.

(Binding of RBD of HC2ENV protein to human cells)
In order for a viral vector that expresses the HC2env gene to be practical as a vector for gene transfer into human cells, the HC2ENV protein, which is the HC2env gene product, must have a binding property to the human or primate cell surface. It is. Thus, it was confirmed that the HC2ENV protein has surface binding activity against primate cells.

  Similar to the method described in Example 4, using the primer pair HC-start-RI and HC2-Sma-3 (SEQ ID NOs: 35 and 36 in the sequence listing) from the start codon of the HC2env gene to the receptor binding domain (RBD) ) Was amplified by the PCR method. Next, pCSI.3 is used so that the 3 'end (C-terminus of the amino acid sequence) of the amplified fragment continues in-frame to the 5' end of the green fluorescent protein EGFP gene. EGFP vector (Manel, N., Kim, FJ, Kinet, S., Taylor, N., Sitbon, M., and Battini, JL The ubiquitous glucose transporter GLUT-1 is a receptor for HTLV. Cell 115 (4 ): 449-59, 2003.], and a plasmid expressing a chimeric protein in which the RBD portion of the HC2env gene was bound to EGFP was prepared.

  As a negative control, pEGFP-C1 (manufactured by Invitrogen) expressing only EGFP is prepared, and as a positive control, pHTLV2-EGFP expressing a chimeric protein in which the RBD portion of the human T cell leukemia virus HTLV-IIenv gene is bound to EGFP. (See the same document).

  Each of these plasmids was singly introduced into human 293FT cells by transfection using the calcium phosphate method, and cultured for 48 hours. The cells were detached and centrifuged, and the resulting transfected cell pellet was proteolytically degraded. The suspension was suspended in a phosphate buffered saline containing an enzyme inhibitor cocktail (Roche Diagnostics), and freeze-thawing was repeated 4 times with liquid nitrogen and a 42 ° C. warm bath. Thereafter, the cell-derived genomic DNA was pulverized by repeatedly passing the suspension through an injection needle, sonicated twice on ice for 30 seconds, and then centrifuged at 7,000 × g for 10 minutes to dissolve the supernatant. A cell extract was used. The protein concentration of these extracts was equalized by the fluorescence intensity of EGFP, and binding to the target cells was performed according to the method described in the aforementioned Manel, N et al. As target cells, primate CV-1 cells and COS7 cells were used as examples.

  The result of the analysis by the fluorescence cell sorter is shown in FIG. As shown in the figure, a molecule obtained by labeling the RBD portion of the HC2ENV protein, which is the HC2env gene product, with EGFP clearly shows significant surface-binding activity against the target cells, CV-1 cells and COS7 cells. It was confirmed.

(Gene transfer into human cells using a viral vector expressing the HC2env gene)
It was confirmed that a viral vector expressing the HC2env gene can be used for gene introduction into human cells. A retrovirus vector for gene transfer was prepared as shown in FIG. That is, first, a gene introduction plasmid 5 containing a gene 4 to be introduced, a packaging signal (Ψ sequence), an LTR, and the like is prepared. Next, this gene introduction plasmid 5 is introduced into the packaging cell 6. The packaging cell 6 is a cell that has a gene containing all or part of the HC2env gene and expresses as a surface protein 7 a protein containing all or part of the HC2ENV protein that is the gene product. Then, the recombinant retroviral vector 8 germinated from the packaging cell 6 is recovered.

  In the retroviral vector construction system shown in FIG. 16, as the HC2env gene expression vector to be introduced into the packaging cell 6, the plasmid pC. HC2. ver2 was prepared and used as a gag / pol expression vector in the literature [Kim, FJ, Manel, N., Boublik, Y., Battini, J.-L., and Sitbon. M. Human T-cell leukemia virus type 1 envelope. pCLEco- (ΔScaCla) derived from murine leukemia virus described in J. Virol. 77: 963-969, 2003] -mediated syncytium formation can be activated in resistant mammalian cell lines by a carboxy-terminal truncation of the envelope cytoplasmic domain. Prepared. Moreover, the β-galactosidase expression plasmid pCLLacZ described in the same document was prepared as the gene introduction plasmid 5 into which the gene 4 to be introduced was incorporated. Here, as the HC2env gene expression vector to be introduced, the plasmid pC. HC2. The reason for using ver2 is that HC2ENV protein 7 in which the sequence of the SU / TM cleavage site is optimized by exchanging the region below PRR with the same region of the env gene product of F-MuLV is used.

  These three plasmids were co-transfected into 293T cells by the calcium phosphate method, and the culture medium was changed the next day. After further culture for one day, the culture supernatant was recovered and used as a virus solution. The virus solution was added with polybrene to human HeLa or 293T cells previously seeded in a 12-well culture plate, and the culture solution was replaced the next day. After further culturing for one day, the cells were fixed and washed with 0.5% glutaraldehyde, added with LacZ staining solution, reacted at 37 ° C. overnight, washed with water, dried and then observed with a stereomicroscope. The micrograph is shown in FIG. As shown by the arrows in the figure, a large number of black colored cells appear in the cytoplasm due to the enzymatic activity of LacZ, and a retroviral vector expressing the HC2env gene product HC2ENV protein on the particle surface is human HeLa cells. Were confirmed to have gene transfer activity.

  The HC2env gene of the present invention and its expression product HC2ENV protein can be used, for example, for diagnosis and treatment of IgA nephropathy, and can also be used for retrovirus vectors for gene transfer.

FIG. 1 is a diagram showing an example of a fluorescence micrograph in which an antigen molecule deposited in the glomeruli of an IgA nephropathy patient is detected with a mouse monoclonal antibody 12H5.1 (Example 1). FIG. 2 shows the amino acid sequence of the mouse endogenous xenobiotic retrovirus env gene product and the nucleotide sequence of the gene (Example 1). FIG. 3 shows the results of Southern blotting performed on a human genomic DNA library (Example 1). FIG. 4 is a diagram showing an example of a database search result indicating the position of the HC2env gene of the present invention in the human genome. FIG. 5 is a diagram showing the sequences and structures of the 3′LTR and 5′LTR of the HC2 provirus in which the HC2env gene of the present invention is present. FIG. 6 is a table summarizing the results of 5'RACE, 3'RACE and full-length cDNA amplification of the HC2env gene of the present invention (Example 1). FIG. 7 is a photograph of an example of the results of confirming the expression of the HC2env gene of the present invention in human tissues by RT-PCR (Example 2). FIG. 8 is a schematic diagram showing a recombinant vaccinia virus expression vector incorporating the HC2env gene of the present invention. FIG. 9 is a diagram showing an example of a fluorescence micrograph in which the expression of the HC2env gene of the present invention is detected by the indirect fluorescent antibody method (Example 3). FIG. 10 is a photograph of an example of the result of confirming the expression of a chimeric ENV protein containing the HC2ENV protein of the present invention by Western blotting (Example 4). FIG. 11 is a diagram showing another example of a fluorescence micrograph obtained by detecting the expression of the HC2env gene of the present invention by the indirect fluorescent antibody method (Example 5). FIG. 12 is a view showing still another example of a fluorescence micrograph in which the expression of the HC2env gene of the present invention is detected by the indirect fluorescent antibody method (Comparative Example 1). FIG. 13 is a diagram showing an example of surface binding activity of the HC2ENV protein of the present invention on primate cells (Example 6). FIG. 14 is a flowchart showing an example of a method for detecting a single nucleotide polymorphism of the HC2env gene of the present invention. FIG. 15 is a schematic diagram of an example of the human antibody measurement method of the present invention. FIG. 16 is a schematic diagram showing an example of a method for producing a retrovirus vector for gene introduction of the present invention. FIG. 17 is a photograph showing an example of the result of gene introduction into human cells using the retrovirus vector for gene introduction of the present invention (Example 7).

Explanation of symbols

1: HC2ENV protein of the present invention or part thereof 2: Antibody in serum or body fluid of subject 3: Labeled secondary antibody 4: Gene transfer target gene 5: Gene transfer plasmid 6: Packaging cell 7: Surface protein 8: Book Retroviral vector for gene transfer of the invention

Sequence number 3: Primer 0112295 ORF-L
SEQ ID NO: 4: Primer 0112295 ORF-R
Sequence number 5: Primer L
Sequence number 6: Primer R
Sequence number 7: Primer HC2env011L1
Sequence number 8: Primer HC2env011R
SEQ ID NOs: 9 to 16: coding strand primer SEQ ID NOs: 17 to 24: non-coding strand primer
SEQ ID NO: 25: primer for env probe SEQ ID NO: 28: primer T7
SEQ ID NO: 29: Primer Sp6
Sequence number 30: Primer HC2env3RACE1
Sequence number 31: Primer HC2pol3RACE1
Sequence number 32: Primer 5RACEpol-env
Sequence number 33: Primer HC2PV-L
Sequence number 34: Primer HC2PV-R
Sequence number 35: Primer HC2-start-RI
Sequence number 36: Primer HC2-Sma-3
Sequence number 37: Primer clone57-5kpn
Sequence number 38: Primer clone57-3kpn

Claims (28)

An env gene of human endogenous retrovirus HC2, comprising a polynucleotide of any one of (1) to (6) below:
(1) A polynucleotide comprising the base sequence of SEQ ID NO: 1 in the sequence listing.
(2) A polynucleotide comprising a nucleotide sequence in which one or several bases are deleted, substituted or added in SEQ ID NO: 1 in the sequence listing, and a nucleotide sequence complementary to the polynucleotide of (1) above A polynucleotide that hybridizes under stringent conditions.
(3) A polynucleotide comprising a nucleotide sequence in which one or several bases are deleted, substituted or added in SEQ ID NO: 1 in the sequence listing, wherein the polynucleotide has homology with the polynucleotide of (1) above. A polynucleotide which is 80% or more.
(4) A polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2 in the sequence listing.
(5) Anti-gp70 antibody 12H5.1, which is a mouse monoclonal antibody, or an anti-Rauscher consisting of an amino acid sequence in which one or several amino acid residues are deleted, substituted or added in SEQ ID NO: 2 in the sequence listing A polynucleotide encoding a protein capable of antigen-antibody reaction with leukemia virus gp70 antibody.
(6) A polynucleotide comprising a base sequence complementary to the polynucleotide of any one of (1) to (5). In the nucleotide sequence of SEQ ID NO: 1 in the sequence listing, the 69th (T → A), the 524th (T → C), the 586th (C → G), the 592nd (G → T), the 887th position (C → T), 970th (C → T), 1000th (C → T), 1140th (A → G), 1143th (T → C) and 1212th (C → A) The gene according to claim 1, comprising at least one single nucleotide polymorphism selected from the group consisting of: The gene according to claim 1 or 2, wherein the polynucleotide is at least one of DNA and RNA. A polynucleotide comprising the whole or a part of the polynucleotide according to any one of (1) to (6) according to claim 1. The polynucleotide according to claim 4, wherein the polynucleotide is used as a primer or probe of the gene according to claim 1 or 2. A polynucleotide, which is a base of any one of SEQ ID NOs: 3 to 24 and 30 to 36 in the sequence listing used as a primer or probe for amplifying or detecting all or part of the gene according to claim 1 or 2 A polynucleotide comprising a sequence. A probe for detecting the gene according to claim 2, wherein the probe is complementary to all or part of the polynucleotide of any one of (1) to (6), and the single nucleotide sequence in SEQ ID NO: 1 A probe consisting of a base sequence containing a type of base. MRNA which is a transcription product of the gene according to claim 1 or 2. A protein that is an expression product of the gene according to claim 1 or 2, wherein the protein is the following (7) or (8).
(7) A protein comprising the amino acid sequence of SEQ ID NO: 2 in the sequence listing.
(8) The anti-gp70 antibody 12H5.1, which is a mouse monoclonal antibody, or an anti-Rauscher consisting of an amino acid sequence in which one or several amino acid residues are deleted, substituted or added in SEQ ID NO: 2 in the sequence listing A protein that reacts with leukemia virus gp70 antibody as an antigen antibody. An IgA nephropathy marker from a gene according to claim 1 or 2, an mRNA according to claim 8, a protein according to claim 9, and a human antibody that undergoes an antigen-antibody reaction using the protein according to claim 9 as an antigen An IgA nephropathy marker comprising at least one selected from the group consisting of: A method for measuring gene expression of the gene according to claim 1 or 2, wherein the amount of mRNA according to claim 8 or the protein according to claim 9 is measured. The measurement method according to claim 11, wherein the measurement of the mRNA is a measurement using the polynucleotide according to any one of claims 4 to 6 as a primer or a probe. The method for measuring gene expression according to claim 11, wherein the measurement of the protein is measurement using an antibody having the protein according to claim 9 as an antigen. A method for measuring a human antibody using the protein according to claim 9 as an antigen, wherein the protein according to claim 9 and the human antibody are reacted with each other, and the human antibody and the human antibody are used as an antigen. A method for measuring a human antibody, comprising reacting a labeled antibody with an antigen-antibody and measuring a complex of the protein according to claim 9, the human antibody and the labeled antibody. The method for measuring a human antibody according to claim 14, wherein the protein is immobilized on a solid phase. The method for measuring a human antibody according to claim 14 or 15, wherein the human antibody is a human antibody in plasma. An IgA nephropathy test kit for measuring gene expression of the gene according to claim 1 or 2 by the gene expression measurement method according to any one of claims 11 to 13, wherein the kit is used as a primer or a probe. A test kit for IgA nephropathy, comprising an antibody comprising the polynucleotide according to any one of Items 4 to 6 or the protein according to Claim 9 as an antigen. An IgA nephropathy test kit for measuring a human antibody having the protein according to claim 9 as an antigen by the human antibody measurement method according to any one of claims 14 to 16, comprising the protein according to claim 9 and A test kit for IgA nephropathy, comprising a labeled antibody using the human antibody as an antigen. The test kit for IgA nephropathy according to claim 18, wherein the protein according to claim 9 is immobilized on a solid phase. A pharmaceutical product for treating or preventing IgA nephropathy, comprising a base sequence complementary to all or part of the gene according to claim 1 or 2, wherein the gene transcription or protein of claim 1 or 2 A pharmaceutical comprising a polynucleotide capable of suppressing expression. A pharmaceutical product for treating or preventing IgA nephropathy, wherein the antigenic determinant (epitope) for a human antibody having the expression product of the env gene of human endogenous retrovirus HC2 in the amino acid sequence of the protein of claim 9 as an antigen A pharmaceutical comprising a polypeptide comprising the amino acid sequence of An expression vector for introducing a gene containing all or part of the gene according to claim 1 or 2 into a target cell, the gene containing all or part of the gene according to claim 1 or 2, An expression vector comprising a regulatory sequence necessary for expression of the gene, wherein the gene is operably linked to the regulatory sequence. A retrovirus vector for gene transfer, wherein the coat protein of the virus particle contains all or part of the protein according to claim 9. 24. A gene introduction method using a retrovirus vector for gene introduction, wherein the retrovirus vector for gene introduction is the retrovirus vector for gene introduction according to claim 23. A method for producing a transgenic cell, comprising a step of introducing a target gene into a target cell by the gene introduction method according to claim 24. A gene introduction kit comprising a packaging cell transformed with the expression vector according to claim 22 and a gene introduction plasmid having a ψ sequence. A gene introduction kit comprising the expression vector according to claim 22 and a gene introduction plasmid having a ψ sequence. 28. The gene introduction kit according to claim 27, further comprising a plasmid expressing at least one of gag and pol genes.

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