CN101573443A - RNase H2 complex and genes therefor - Google Patents

Abstract

There is provided the genes for components of RNase H2, namely RNASEH2A, RNASEH2B and RNASEH2C, together with the proteins encoded thereby. Recombinant polynucleotides comprising these genes are described, optionally as vectors. Recombinant RNase H2 is also described together with an assay to assess the effect on activity of a test substance or of a modified (ie. mutated) component of the RNase H2.

Description

RNA enzyme H2 mixture and gene thereof

Invention field

The invention describes rnase (RNA enzyme) H2, gene and a kind of detection of agonist of the component coding of RNA enzyme H2 recombinant chou, antagonist and conditioning agent are used to handle immunoreactive detection method, for example treat virus infection and/or autoimmune disease.

Background technology

RNA enzyme H is a kind of enzyme complex, is used for the Yeast Nucleic Acid of restriction endonuclease degraded from the RNA/DNA mixture.RNA enzyme H is often used in and decomposes in the molecular biology and cDNA reverse transcription RNA segment afterwards.

Two class RNA enzyme H (1/I and 2/II) have special biochemical property.RNA enzyme H2 is the active main source of cell RNA enzyme H in human and the yeast.RNA enzyme H2 acts in dna replication dna and removes singlestranded RNA among lagging strand Okazaki fragment RNA primer and the excision duplex DNA-DNA.At yeast Rnh2Bp and RnhaCp and the Rnh2Ap of catalytic subunit copurification, and active essential by recombinant RNA enzyme H2 jointly.In the mankind, RNA enzyme H2 catalytic subunit, RNA enzyme H2A discerns by biochemical purification, and has clearly sequence homology with the yeast autoploid.Yet, there are not other yeast outer identified Rnh2Bp of autoploid and RnhaCp subunit, although a kind of second human protein AYP1 (called after RNA enzyme H2C now) and RNA enzyme H2A albumen are by copurification (Frank etc., PNAS U.S. 95:12872-7,1998).

Aicardi-Goutieres syndrome (AGS) is a kind of autosomal recessive disease of unknown etiology.The AGS clinical manifestation is serious neurological dysfunction, progressive microcephalus, spasm, myodystony posture and PMR.The Childhood that death often occurring in (Goutieres, Brain Dev 27:201-206,2005).Similar (the Aicardi of phenotype that the AGS proof is very strong to the congenital virus infection of brain; Goutieres, Ann Neurol 15:49-54,1984), AGS also follows the IFN-alpha levels significantly to raise, and is also similar to the host immune response behind the virus infection.So, too controversial about the nosetiology of AGS in academia, and particularly whether AGS is because to due to the result of the unusual adjusting of the gene susceptibility of viral pathogen or host immune response.

Crow etc., (Am J hum Genet 67:213-221,2000 and J Med Genet 40:183-187,2003) identify 2 kinds of AGS locus, be the AGS1 on the karyomit(e) 3p21 and be AGS2 (Ali etc. on the karyomit(e) 13q14.3, J Med Genet 43 (5): 444-50, in May, 2006 Epub on May 20th, 2005).

The contriver finds that now AGS2 is a component of human rna enzyme H2 mixture, and this gene is discerned fully.AGS2 has a ubiquitous expression pattern, does not have the domain structure of precognition, or human homology's gene is to infer function.A kind of distant homologous gene is positioned at S. yeast (identification of＜5% aminoacid sequence).The contriver also discerns AGS3 and AGS4.These three kinds of AGS expression of gene protein form the part of RNA enzyme H2 mixture jointly.

AGS has a lot of phenotypes similar to virus infection, and these similaritys are not limited to brain, and some case has additional neural characteristic (such as thrombopenia, hepatosplenomegaly and liver transaminase raise), shows that the genopathy that infects with other virusoids overlaps.

AGS and systemic lupus erythematosus (SLE) similarity is very remarkable (seeing Alarcan-riquelme, Nat Genet38:866-867, on July 27th, 2006) just.Particularly acra vasculitis dermatosis sees among the SLE, is apparent in corium-epidermis tie point and immunoglobulins deposition (seeing Crow etc., Nat Genet 38:917-920,2006) occurs.Reported the familial lupus erythematosus disease (seeing Lee-Kirsch etc., AmJ Hum Genet 79:731-737, on July 19th, 2006) of AGS1 gene recently with identical skin lesion localization.Additionally, high gamma sphaeroprotein (hypergammaglobinema) and autoantibody are in the news to see among the AGS and (see Crow etc., Nat Genet 38:917-920,2006).In addition, the CSF interferon A lpha of elevated levels is found among brain lupus erythematosus and the systemic SLE.The calcification of encephalic basal ganglion in brain SLE patient incidence up to 30%.

As the achievement that the contriver observes, AGS, SLE and virus infection have the potential cause of disease of common that relates to RNA enzyme H2.

Importantly, the contriver identifies among the AGS4, G37S in F39 family sudden change, and wherein plan-the TORCH (Pseudo-TORCH) and the AGS feature that have of affected individuals shows that these diseases have the common molecular basis.(plan-TORCH is another kind of genopathy, ill children's dominance symptom and toxoplasma gondii, and rubella, cytomegalovirus, the human herpes simplex vicus infects similar).Therefore, it is wideer than the AGS phenotypic spectrum of classics definition that the phenotypic spectrum of RNA enzyme H2 complex body sudden change is considered to, and the gene basis of many " congenital virus infection " case may also assert till now.

Summary of the invention

Therefore the present invention provides a kind of polynucleotide, contains nucleotide sequence SEQ ID No 1,3 or 5, or its homologous chromosomes.SEQ ID No 1 has provided AGS2 nucleotide sequence (RNA enzyme H2B); SEQ ID No 3 has provided AGS3 nucleotide sequence (RNA enzyme H2C); Provided AGS4 nucleotide sequence (RNA enzyme H2A) with SEQ ID No 5.

Polynucleotide sequence (the having unknown function) NM_024570 (AGS2/RNA enzyme H2B before had been called FLJ11712) in NCBI that is in the news; AF312034 (AGS3/RNA enzyme H2C before had been called AYP1); NM_006397 (AGS4/RNA enzyme H2A).

About being called the polynucleotide of " single homologous chromosomes " or " a plurality of homologous chromosomes ", we refer to that polynucleotide are by deletion, replace or add the nucleic acid class and revise and make to have at least 65% homologous chromosomes, for example, at least 74% homologous chromosomes of the nucleotide sequences that SEQ ID No 1,3 or 5 expresses.In one embodiment, in SEQ ID No 1,3 or 5 nucleotide sequences of expressing, polynucleotide will have 75% or 80% homologous chromosomes, preferred 85% homologous chromosomes.Term " single homologous chromosomes " or " a plurality of homologous chromosomes " comprise homologous gene, i.e. equivalent gene in different genera.

In one embodiment, for example, when by direct sequence arrangement and sequence alignment, homologous chromosomes will have 90% or more, SEQ ID No 1,3 or 5 nucleotide sequences 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of expressing.

Sequence homology can be by direct best-fit sequence permutation and comparison, or uses any suitable clustalw algorithm, decides such as BLAST.BLAST was described in J Mol Biol 25:403 (1990) by Altschul etc.The scoring of this ordering, S is calculated as replacement and gap scoring.When estimating what estimate that " substantive homologous chromosomes " refer to by BLAST is exactly to hang down desired value (E).Desired value is that different scorings are arranged and equated or be better than expection and occur in the database S by stochastic retrieval.The E value is low more, and it is remarkable more to mark.

Especially, the amino acid that do not influence of nucleotide sequence is expressed the definition that the modification of (owing to redundancy in the genetic code) falls into " homologous chromosomes ".Additionally, term " homologous chromosomes " also comprise a kind of can with a kind of polynucleotide that contain 15 from the hybridization of the adjacent base of any in SEQ ID No 1,3 or 5, preferably, under stringent condition.In one embodiment, polynucleotide and one are contained 20 or more a plurality of from any adjacent base (for example 25 to 50 adjacent bases) in SEQ ID No 1,3 or 5, preferably, and under stringent condition.

Stable Nucleotide hybridization is a kind of function of base pairing.Base pairing is that the degree under being subjected in the complementary two strands two polynucleotide chains and hybridization a situation arises influences.Especially, salt concn and temperature effect hybridization.Those of ordinary skills can expect that the double-stranded solvent temperatures of effective polynucleotide (E Tm) are controlled by following formula:

E Tm＝81.5+16.6(log M[Na+])+

(%G+C)-0.72 0.41 (% methane amide)

Carry out under stringent condition when hybridization, only have the height complementary base meeting is stayed in the double chain form.Term " stringent condition " about hybridization used herein refers to the cleaning condition 0.1X SSC under 60 to 68 ℃.Alternatively, cleaning condition can comprise the suitable concentration of a SDS, for example 0.1%SDS.

Polynucleotide can be DNA or RNA, also can be strand or two strands.Double-stranded DNA (for example, cDNA) is used all very convenient for great majority usually.Polynucleotide can be the form with carrier, for example expression vector.

Polynucleotide class of the present invention can be separated the polynucleotide class or can be recombinated.The polynucleotide class can be combined into to be expressed or cloning vector.The host cell that this carrier can be used for transfection or transformed host cell nuclear and cultivate at conventional substratum according to prior art.

Clone's DNA is combined with the host who is fit to, transfection or transformed host cell and selection transfection or mutant all are that those of ordinary skills can find a lot of methods that are fit to (to see in the prior art, Sambrook etc. for example, molecular cloning: laboratory manual, the third edition, press of cold spring harbor laboratory, 2001).

Proper host cell comprises bacterium, yeast, insect, Mammals and vegetable cell.Substantially, can select proper host cell with adaptive with used carrier.

In one embodiment, host cell can be people or non-human ES cell.

Aspect another, provide a kind of LCL to express the RNA enzyme H2 of sudden change in the present invention.

About proteinic term " sudden change " (for example, RNA enzyme H2A, RNA enzyme H2B or RNA enzyme H2C), we refer to that this type of protein amino acid sequence is different with the wild-type amino acid sequence.For RNA enzyme H2B, wild-type sequence shown in SEQ ID No2, for RNA enzyme H2C, wild-type sequence shown in SEQ ID No 4 and RNA enzyme H2A wild-type sequence shown in SEQ ID No 6.

The protein of sudden change can show change function or with the activity of wild-type protein qualitative correlation, but these and nonessential.Referring to contain the proteinic mixture of at least a composition about the term " sudden change " of protein complex (for example RNA enzyme H2) is aforesaid mutein.The mutein mixture can show the function of change or the activity relevant with the wild-type mixture, but and nonessential.

In one embodiment, the invention provides a kind of recombination of polynucleotide, contain the nucleotide sequence shown in SEQ ID No 1 or its homologous chromosomes and the protein of this sequence encoding.

In one embodiment, the invention provides a kind of recombination of polynucleotide, contain the nucleotide sequence shown in SEQ ID No 3 or its homologous chromosomes and the protein of this sequence encoding.

In one embodiment, the invention provides a kind of recombination of polynucleotide, contain the nucleotide sequence shown in SEQ ID No 5 or its homologous chromosomes and the protein of this sequence encoding.

In yet another aspect, the invention provides a kind of protein, contain SEQ ID No 2,4 or 6, or the aminoacid sequence shown in its homologous chromosomes.

About proteinic term " homologous chromosomes " (or " a plurality of homologous chromosomes "), we refer to deleted, alternative or interpolation amino acid has at least 65% homologous chromosomes, SEQ ID No 2 for example, at least 66% of aminoacid sequence homologous chromosomes shown in 4 or 6, or at least 70% homologous chromosomes for example.In one embodiment, protein has at least 75% or 80% homologous chromosomes of shown in SEQ ID No 2,4 or 6 aminoacid sequence, preferred 85% homologous chromosomes.In one embodiment, the homologous chromosomes of protein aminoacid sequence such as SEQ ID No 2,4 or 6 shown in will reach 90%, for example 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous chromosomes.Term " homologous chromosomes " comprises homologous protein not of the same race.

Additionally, the invention provides a kind of SEQ ID No 1, nucleotide sequence or its homologous chromosomes encoded protein matter shown in the SEQ ID No 3, a kind of among the SEQ ID No 5.Nucleotide sequence can be an a kind of part of polynucleotide, alternatively, is recombination of polynucleotide.Polynucleotide can form the part of big polynucleotide, in addition, also can form the part of carrier.

In one embodiment, the invention provides a kind of protein, contain the aminoacid sequence shown in SEQ ID No 2 or its homologous chromosomes.

In one embodiment, the invention provides a kind of protein, contain the aminoacid sequence shown in SEQ ID No 4 or its homologous chromosomes.

In one embodiment, the invention provides a kind of protein, contain the aminoacid sequence shown in SEQ ID No 6 or its homologous chromosomes.

In one embodiment, protein of the present invention can form a kind of chimeric (fusion) proteinic part.

For clarifying term used herein " protein ", just refer to a kind of peptide and polypeptide, be not the polymkeric substance that refers to any special size.

In yet another aspect, the invention provides a kind of RNA enzyme H2 of composite form, contain:

I) a kind of SEQ ID No 1, nucleotide sequence or its homologous chromosomes encoded protein matter shown in the SEQ ID No 3, a kind of among the SEQ ID No 5; Or

Ii) a kind of protein comprises SEQ ID No 2, the aminoacid sequence shown in SEQ ID No 4 or the SEQ ID No 6, or its homologous chromosomes.

In one aspect, the invention provides a kind of RNA enzyme H2 of composite form, comprise a kind of at least:

Iii) the RNA enzyme H2B of SEQ ID No 1 or the nucleotide sequence shown in it or its homologous chromosomes coding perhaps contains the RNA enzyme H2B of the aminoacid sequence shown in the SEQ ID No 2 or its homologous chromosomes;

Iv) the RNA enzyme H2C of the nucleotide sequence shown in the SEQ ID No 3 or its homologous chromosomes coding perhaps contains the RNA enzyme H2C of the aminoacid sequence shown in the SEQ ID No 4 or its homologous chromosomes;

V) the RNA enzyme H2A of the nucleotide sequence shown in the SEQ ID No 3 or its homologous chromosomes coding perhaps contains the RNA enzyme H2A of the aminoacid sequence shown in the SEQ ID No 6 or its homologous chromosomes.

In one embodiment, manifest i at least), ii) and two kinds of compositions iii) (for example, i) and iii); I) and ii); Or ii) and iii)).In one embodiment, show three kinds of component i), ii) and iii).The composition that alternatively, also can manifest other mixtures.

Recombinant RNA enzyme H2 mixture on molecular biology effectively, particularly cDNA product or other DNA-RNA hybrids be degraded or situation about suppressing under any other process.The double-stranded DNA cracking also comprises the ribonucleotide of single or several embeddings.Useful in the method for recombinant RNA enzyme H2 mixture in identifying the compound that to revise substrate specificity or revise complex activity.The compound that can revise complex activity can be for its any one composition or to the catalyzer (agonist) or the inactivator (antagonist) of whole mixture.Alternatively, the compound that can revise enzymic activity can act on the upwards conditioning agent of RNA enzyme H2.This detection method can be cell or protein detection method.Alternatively, one or more compositions of mixture can suddenly change from wild-type, for example the G37S of AGS4.The effect of this type of sudden change can be assessed or be identified on active or its substrate specificity of mixture.

Additionally, i), ii) or iii) arbitrary composition can be utilized separately for molecular biology or not have a kind of or the detection method of two kinds of composition remnants in addition.

RNA enzyme H2 is RNA enzyme H active main source in cell, therefore reducing enzymic activity may be the pathogenic basis that depends on the metabolic cell processes of RNA-DNA hybrid molecule and become autoimmune disease, include but not limited to AGS and SLE, and infected by microbes, particularly virus infection.Other infected by microbes comprise infectation of bacteria and fungal infection.About virus infection, what mention especially may be popular virus infection on a large scale, for example the viral on a large scale influenza of the high incidence that causes of the inappropriate inactivation of innate immune responses.

RNA enzyme H2 been proposed in and is used to remove Okazaki fragment RNA primer in the lagging strand dna replication dna.Although influence is not active in S. zymic Rnh2 deletion, it has caused the susceptibility to hydroxyurea really.

RNA enzyme H2 unlike 1 type RNA enzyme H, also can discern the single ribonucleotide in the intercalation of DNA, and this enzyme therefore can be very important in identification and processing inappropriate ribonucleotide that mixes in chromosomal DNA.This type of mistake is mixed and may be taken place more frequently under the situation of dNTPs removing, and provides an alternative explanation to hydroxyurea susceptibility for the Rnh2 mutant.

When single stranded RNA was relevant with chain in the double-stranded DNA, the DNA chain was shifted " the R-ring " that forms the dna single chain relatively.When rna binding protein matter ruptured, this ring can be transcribed the back at eukaryotic cell and be occurred.RNA enzyme H works with causing in the chromosomal DNA instability at this class formation of inhibition.

Therefore, the active removal of RNA enzyme H may be DNA synthetic result, removes the ribonucleotide that the DNA mistake is mixed, and/or suppresses the formation of R-ring.

Reverse transcription is HIV and other retroviruss necessary process of duplicating and the important drugs target spot of antiviral therapy.The degraded susceptible that the DNA-RNA hybrid molecule causes for endogenous RNA enzyme H in this process, and these therefore antagonism viruses play very vital role.Therefore, RNA enzyme H2 sudden change may damage host's antiviral function, and AGS may be the result of street virus pathogenic agent.

Alternatively, if weaken the level that active RNA enzyme H has improved endogenous RNA-DNA hybrid molecule, immune disorder may be AGS, and the basis of SLE also may be most important to autoimmune disease and virus infection.DsRNA and dsDNA are the agonists of known congenital immunity function, and activate I type Interferon, rabbit and produce (seeing Kawai etc., Nat Immunol7:131-137,2006 and Krieg etc., Annu Rev Immunol 20:709-760,2002).Defective in other nucleases has caused discharging the reduction from the clearance rate of the extracellular of apoptotic cell nucleic acid, as nucleic acid round-robin result, also is considered to trigger the multisystem autoimmune disease.The evidence of supporting comprise the heterozygote sudden change of having found deoxyribonuclease 1 in human patient SLE of several examples and deoxyribonuclease 1-/-mouse shows lupoid phenotype (seeing Napirei etc., Nat Genet 25:177-181,2000).In addition, deoxyribonuclease I I-/-IfnR-/-mouse shows the chronic polyarthritis of rheumatoid arthritis recently by report, (see Kawane etc., Nature 443:998-1002, in October, 2006), and believe that the RNA-DNA hybrid molecule may also produce stimulation to congenital immunity, and explained the plain alpha of high levels of interference of AGS diagnostic characteristic.Can raise the equally level of endogenous RNA-DNA hybrid molecule of RNA enzyme H2 dysfunction is then by stimulating Interferon, rabbit alpha to produce with the mechanism that is comparable to dsRNA and dsDNA.As the transgenic mouse model, produce the neuropathological feature that superfluous Interferon, rabbit alpha can explain many AGS in central nervous system subsequently, Interferon, rabbit alpha is at medium-term and long-term occur (the seeing Campbell etc., Brain Res 835:46-61,1999) of spongiocyte.

The contact of SLE and autoimmune disease be studies have shown that recently, for example, Kelly etc., Arthritis Rheum54:1557-1567,2006 show that getting in touch of dsRNA and TLR caused the IFN generation; Lovgren etc., ArthritisRheum 50:1861-1872,2004 show that in external SLE identified, the IFN derived need resisted-RNA Abs; Sigurdsson etc.Am J.Hum.Genet.76:528-537,2005 show that SLE is based on the dysfunction of IFN path; Graham etc., Proc.Natl.Acad.Sci USA 104:6758-6763,2007 show that sudden change of IFN regulatory factor and SLE risk increase relevant.

Innate immune responses in the influenza shows in nearest lesson with SLE has identical etiology.For example, Cheung etc., Lancet 360:1831-1837,2002 show that the TNF alpha of increase is relevant with H5N1 (97) pathology of rising; Kobasa etc., Nature 445:319-323,2007 show in 1918 influenzas of non-human primates and demonstrate the atypia innate immune responses; Lipatov etc., Journal of General Virology 86:1121-1130,2005 show that 9 models that infect the H5N1 influenza show the cytokine imbalance.

Therefore, provide a kind of compound that can revise the active and substrate specificity of RNA enzyme H2 of great use:

A) improve the symptom of RNA enzyme H2 activity with opposing AGS.

B) improve RNA enzyme H2 activity with the opposing virus infection, especially, but be not limited to bacterium and virus infection.Mentioning especially may be that in virus replication, the RNA-DNA hybrid molecule forms in host cell by retrovirus (for example, HIV infects).Also has influenza popular bacterial strain on a large scale, unsuitable innate immune responses involves in the bacterial strain of this type of high incidence, for example, 1918 and the H5N1 bacterial strain (see Cheung etc., The Lancet 360:1831-1837,2002 and Lipatov etc., Journal of General Virology 86:1121-1130,2005).

C) improve RNA enzyme H2 activity to reduce chromosomal DNA stability, particularly suppress the formation of R-ring.

D) improve RNA enzyme H2 activity with the opposing autoimmune disease.Listed autoimmune disease includes, but is not limited to systemic lupus erythematosus (SLE), coeliac disease, crohn, diabetes (1 type), pulmonary apoplexy nephropathy syndrome, graves disease, Addison disease, rheumatic arthritis, psoriatic and multiple sclerosis.

E) improve RNA enzyme H2 activity to strengthen the efficient of mixture in the molecular biology reaction, for example in cDNA produces.

F) reduction RNA enzyme H2 activity promotes the efficient based on the gene therapy of virus.(RNA enzyme H2 activity will form the part of the host immune response that weakens this method application.The active reduction of RNA enzyme H2 will suppress this part to small part and suppress this host immune response).

G) reduce RNA enzyme H2 activity and come the immunne response (seeing Akwa etc., J.Immunol161:5016-26,1998) of stimulation of host for virus infection.

The present invention further provides the method for agonist or inhibitor or its composition of a kind of RNA of detection enzyme H2, or the conditioning agent of RNA enzyme H2 substrate specificity, described method comprises:

Vi) with RNA enzyme H2 recombinant chou or its component contact detection material, described component is RNA enzyme H2B, RNA enzyme H2C, RNA enzyme H2A or its any combination;

Vii) estimate RNA enzyme H2 or the activity of its component in sample, alternatively, continuity for some time; And

Viii) determine the effect of the detection material of RNA enzyme H2 or its composition activity.

Described method can be pointed to whole RNA enzyme H2 mixture, may be a kind of aforesaid reorganization mixture.Alternatively, described method can be modified to the composition (component) that points to RNA enzyme H2 mixture, for example, RNA enzyme H2B, RNA enzyme H2C, RNA enzyme H2A, each can be recombinated.

Above-mentioned detection method also can be by finishing RNA enzyme H2 recombinant chou or its component contact detection material (for example, RNA enzyme H2B, RNA enzyme H2C, RNA enzyme H2A or its any combination).The mutant forms of RNA enzyme H2 or its component can such as LCL or non-human transgenic animal, be expressed such as rat by clone.

In one embodiment, RNA enzyme H2 mixture presents in cell.

In one embodiment, detection method can directedly be identified a kind of inflammation modulators, promptly a kind of anti-inflammatory or pro-inflammatory mediator.In one embodiment, described detection method can directedly be identified antiseptic-germicide (for example, antiviral agent).

Detection method can be based on the detection method of cell, and endogenous RNA enzyme H2 activity is evaluated.Described method can identify that RNA enzyme H2 goes up the conditioning agent on indirect action or the RNA enzyme H2 uplink.

Detection method in Mammals (for example human) this excitomotor of RNA enzyme H2 or the effect of inhibitor, if only viral RNA enzyme H is had specificity, can form a kind of effective antiviral therapy at the agonist of RNA enzyme H of the virus of assessment or inhibitor.The influence of the antiviral medium that this type of is inferred on described RNA enzyme H2 mixture is vital in its in vitro effects of decision, and especially, may side effect significantly be arranged to mammalian hosts.

The step I i of aforesaid method) can pass through, the oligomeric nucleotide substrate that for example imports mark is finished.In one embodiment, the oligomeric nucleotide of mark by RNA enzyme H2 separately just discharges fluorescent mark.Because the approximate quenching of fluorescence agent molecule that is attached to relative nucleotide chain, complete oligomeric nucleotide does not show fluorescence.Therefore, fluorescence is the active standard of measure R NA enzyme H2.In this embodiment, oligomeric nucleotide serves as the substrate of RNA enzyme H2 or its component.Oligomeric nucleotide can be a double-stranded DNA, double-stranded RNA or double-stranded DNA-RNA hybrid molecule.

Step I i i) can still obtain by under the same conditions without any comparing RNA enzyme H2 activity under the sample.

This detection method can be carried out (for example, can be that kinetics is identified) after surpassing for some time, for example above 10 to 60 minutes.RNA enzyme H2 activity can be assessed by suitable (preferred conventional) time point in whole process, for example, and from per 2 minutes to per 20 minutes.In one embodiment, described detection method is per 5 minutes identified activities in 30 minutes.

The present invention further provides a kind of authentication method determines one or more modifications (for example amino acid mutation) in RNA enzyme H2 component effect, described detection method comprises:

Ix) provide RNA enzyme H2, described one of them kind composition is the sudden change relevant with its wild-type;

X) with described RNA enzyme H2 contact substrate;

Xi) assessment RNA enzyme H2 activity alternatively, surpasses for some time; And

Xii) decision RNA enzyme H2 goes up the active effect of revising composition.

In one embodiment, wild type rna enzyme H2B has the aminoacid sequence that SEQ ID No 2 expresses.Wild type rna enzyme H2C has the aminoacid sequence that SEQ ID No 4 expresses in one embodiment.In one embodiment, wild type rna enzyme H2A has the aminoacid sequence that SEQ ID No 6 expresses.

In one embodiment, RNA enzyme H2 expresses from clone, such as LCL.Alternatively, RNA enzyme H2 can express from non-human transgenic animal.

In one embodiment, RNA enzyme H2 mixture is presented in the cell.

The step I i of aforesaid method) can pass through, the oligomeric nucleotide substrate that for example imports mark is finished.In one embodiment, the oligomeric nucleotide of mark by RNA enzyme H2 separately just discharges fluorescent mark.Because the approximate quenching of fluorescence agent molecule that is attached to relative nucleotide chain, complete oligomeric nucleotide does not show fluorescence.Therefore, fluorescence is the active standard of measure R NA enzyme H2.In this embodiment, oligomeric nucleotide serves as the substrate of RNA enzyme H2 or its component.Oligomeric nucleotide can be a double-stranded DNA, double-stranded RNA or double-stranded DNA-RNA hybrid molecule.

Step I ii) can still obtain without any comparing RNA enzyme H2 activity under the sample by under the same conditions.

In one embodiment, a kind of modification composition of RNA enzyme H2 mixture is identified under the situation of other compositions existing or do not have.In this embodiment, the composition revised of RNA enzyme H2 mixture is to exist and do not have under the situation of other compositions certified.In this embodiment, the mutant form of the composition of the function that it may will use RNA enzyme H2 influence is useful, for example RNA enzyme H2A (AGS4) G37S or RNA enzyme H2B (AGS2) A177T, and carry out this detection method and will seek the active reconstruction of RNA enzyme H2.Alternatively, and carry out this detection method and will seek active the weakening of RNA enzyme H2.Alternatively, and carry out this detection method and will seek the active substrate specificity of RNA enzyme H2.

This detection method can be carried out (for example, can be that kinetics is identified) after surpassing for some time, for example above 10 to 60 minutes.RNA enzyme H2 activity can be assessed by suitable (preferred conventional) time point in whole process, for example, and from per 2 minutes to per 20 minutes.In one embodiment, described detection method is per 5 minutes identified activities in 30 minutes.

Can be used standard technique by the mouse of manual breakage specific gene to a kind of RNA enzyme H2.As IFN-α such as Akwa etc., the described transgenic mice of J Immunology 161:5016-5026 (1998), this type of mouse can be subjected to virus attack; And use mouse test mammalian rna enzyme H2 agonist, alternatively, by above-mentioned detection method in vivo the effect of identifying virus whether improved.The mouse of described manual breakage specific gene also is a part of the present invention.

In yet another aspect, the invention provides the RNA enzyme H2B in a kind of detection method detection patient genome, the sudden change of RNA enzyme H2C or RNA enzyme H2A gene, described method comprises:

I) will be from the molten born of the same parents of patient's white cell; And

Ii) estimate the RNA enzyme H2 activity of dissolving white cell.

No matter be by inhibition or the enhancing activity relevant with the wild-type mixture of normal range, detected sudden change will be to influence the active sudden change of RNA enzyme H2.

Alternatively, if detect the relevant activity of RNA enzyme H2 of minimizing or enhancing and normal range, just can carry out RNA enzyme H2B, RNA enzyme H2C or RNA enzyme H2A gene sequencing.

Described detection method, has the correct consulting of patient of suffering from child's AGS risk as diagnosing the AGS supplementary means or offering, can identify because RNA enzyme H2B, arbitrary as homozygote or heterozygote sudden change and manifest RNA enzyme H2 and reduce among RNA enzyme H2C or the RNA enzyme H2A.Described method may have the individual of autoimmune disease to evaluation, and congenital virus infection or enhanced virus susceptibility infection allow to diagnose and treatment (because the RNA enzyme H2 activity that reduces) is useful.

Described method can identify to suffer from SLE or other autoimmune diseases or have the patient who gives birth to child's risk of suffering from AGS or other autoimmune diseases equally.Described method can further provide the information of the degree of virus infection, or as the supplementary means of virus infection diagnosis, or identify infected by microbes susceptibility patient, particularly, but be not limited to virus infection.

In another alternative, no matter be by fingerprint or satellite imagery technology or to the order-checking of genome relevant portion, described method can simply be carried out gene type by the cdna sample that patient is provided.Gene information can be used as diagnosis AGS, SLE, and autoimmune disease to the supplementary means of viral susceptibility, is used to identify the active patient of RNA enzyme H2 with change, or identifies its genetic risk or tendency.

In the present invention aspect another, comprise can with by SEQ ID Nos 1,35 or its homologous chromosomes in the polyclone or the monoclonal antibody of arbitrary nucleotide sequence coded protein specific combination; A kind of protein contain SEQ ID Nos 2,46 or its homologous chromosomes in arbitrary aminoacid sequence; Or aforesaid recombinant RNA enzyme H2 mixture.In the antibody of the mutant form of arbitrary composition of RNA enzyme H2 mixture component (for example RNA enzyme H2A, G37S sudden change) is also included within.Monoclonal antibodies can pass through, for example have enzymic activity recombinant RNA enzyme H2 mixture immune mouse and use currently known methods to hybridize fusion thereafter.The clone can shield by ELISA, further uses the western blot assay of immunofluorescence checking and the epitope tagged composition of use cell expressing.Antibody can be used for the accessory molecule biological procedures and come purifying RNA enzyme H2 (for example at diagnostic test), comes support study dies RNA enzyme H2 mixture and function thereof.

In one embodiment, antibody is used antibody for treatment, and results from clone, such as hybridoma (seeing Kohler etc., Nature 256:495-497,1975 and Galfre Meth Enzymol 73:3,1981).The treatment that is fit to comprises rodent antibody with antibody, chimeric antibody, scFvs, humanized antibodies and human antibodies.Chimeric antibody is a genetic engineering antibody, comprises about 1/3rd non-human protein and about 2/3rds human proteins.The humanized antibodies is a genetic engineering antibody, contains minimum non-human protein on the human antibodies (being typically 5 to 10%) reverse host immune response is reduced to minimum (seeing Riechmann etc., Nature 332:323-327,1988).

Antibody fragments, such as F (ab ') 2, Fab and Fv fragment also are included in the term " antibody ".

The invention provides a kind of at least a RNA enzyme H2A that contains, the diagnostic kit of RNA enzyme H2B or RNA enzyme H2C primer.The primer that is fit to comprises that those are as shown in table 1, with suitable auxiliary agents bonded primer.The auxiliary agent that is fit to, the buffer reagent that comprises used herein, dNTPs, enzyme (for example TAQ polysaccharase), tagged compound and analogue.Test kit will be used in sample nucleic acid to seek the heredity distortion, such as can showing AGS, and SLE, autoimmune disease or to the susceptibility of infected by microbes, special but be not limited to the distortion (if can find) that the heredity of virus infection distributes.

In one embodiment, can be used to identify the RNA enzyme H2 that contains mutein to the special antibody of above-mentioned RNA enzyme H2 mixture.

In an alternative, can be used for the treatment of the special antibody of above-mentioned RNA enzyme H2 mixture.For example, can be used to improve with virus be the effectiveness of based gene treatment or the immune response of stimulation of host to antibody.

The invention provides a kind of generation and have mutant rna enzyme H2A, the method for the genomic transgenic nonhuman animal of RNA enzyme H2B or RNA enzyme H2C coding, described method comprises:

A) will comprise an encoding mutant RNA enzyme H2A, RNA enzyme H2B, or the recombination group of RNA enzyme H2C polynucleotide sequence group imports non-human zygote or non-human embryonic stem cell;

B) produce a transgenic nonhuman animal from described non-human zygote or non-human embryonic stem cell; And c) produce a kind of have encoding mutant RNA enzyme H2A, RNA enzyme H2B, or the genomic transgenic nonhuman animal of RNA enzyme H2C.

Mutant rna enzyme H2A, RNA enzyme H2B or RNA enzyme H2C polynucleotide encoding will preferably link to each other with promotor, so protein can be expressed by transgenic animal.

Have mutant rna enzyme H2A, the genomic non-human transgenic animal of RNA enzyme H2B or RNA enzyme H2C coding forms one aspect of the present invention.

Non-human transgenic animal can be any suitable animal and mention suitable example by mouse, rat, and primates, rabbit, dog, cat, fish, ox, pig and sheep are made.Yet, enumerate non-limit herein, other animals also can be used.

In one embodiment, non-human transgenic animal is a rodents.The example that is fit to comprises rat and mouse.

The embryonic stem cell that uses in the prior art can be used to method of the present invention, includes but not limited to from mouse species, and such as C57BL/6, CBA/, BALB/c, the embryonic stem cell that DBA/2 and SV129 extract.Preferred embryonic stem cell (Seong, E etc., Trends Genet.20,59-62,2004 of extracting from the C57BL/6 mouse; Wolfer etc., TrendsNeurosci.25:336-340,2002).

(comprise RNA enzyme H2A, RNA enzyme H2B or RNA enzyme H2C encoding sequence be directed into to make in the animal kind system that ins all sorts of ways and go a kind of transgenosis.For example, transgenosis can directly be injected the male nucleus of zygote and (be seen for example Hogan etc., Manipulating the Mouse Embryo, cold spring harbor laboratory, press of cold spring harbor laboratory (1994)), synthetic random integration is a locus that the different quantities transgenosis is duplicated, usually (see for example Costantini and Lacy at head to the afterbody ordered series of numbers, Nature, 294:92,1981).The zygote that is injected into is just transferred to false pregnancy again and is accepted in the uterus of parent.Among the synthetic offspring some have one or more genetically modified genes that are integrated into them that duplicate, usually at an integration site.These " are originated " animal and are bred then and become to set up animal transgenic lines and the reciprocal cross genetic background for selecting.Those of ordinary skills can understand the genetically modified benefit of importing on karyomit(e).Alternatively, transgenosis can be imported into animal by the gene target effect, for example by homologous recombination, at embryonic stem cell (ES).The genetically modified currently known methods of importing that is fit to comprises that blastocyst injects.In this regard, a target structure comprises the ready transgenosis of using prior art.

A target structure comprises can be imported into the host cell that uses existing method.Produce transgenic embryos and its shielding and can use known technology to carry out, Joyner etc. for example, Gene Targeting, A Practical Approach, OxfordUniversity press, 1993 describe.Transgenic animal or embryo's DNA can be by southern blotting or round pcr shielding.

Non-human transgenic animal can be healthy animal or can show disease symptoms or disorder by what transgenosis sudden change caused.These type of transgenic animal are well suited for doing the pharmaceutical research of medicine, also can be used as disease model, for example, and AGS, SLE, autoimmune disease or virus infection.This model may have AGS, SLE, the susceptibility of autoimmune disease or virus infection.

Below the present invention will pass through, non-limiting, example and numeral further described:

Description of drawings

Fig. 1 is neuroimaging and the clinical findings of AGS.(a) axial CT scan shows the calcification of basal ganglion.。(b) axial T2 MRI shows the high signal intensity in the substantia alba medullae spinalis, influences frontal lobe especially.As a comparison,, (c) axially CT scan suffers from the patient that congenital HIV infects, and shows the calcification of basal ganglion, sees Belman etc., Neurology 36:1192-1199 (1986).

Fig. 2 is the sketch that AGS2 critical region and RNA enzyme H2 (front called after FLJ11712) gene are described the position of the sudden change through identifying.。(a) gene mapping of karyomit(e) 13q14.1 is described the AGS2 position of modifying, and by overlapping next definite at the homozygote chromosome segment of stranger in blood family, and extends between karyomit(e) little satellite sign A C137880TG19 and D13S788.(b) physical map between the 571kbp critical zone comprises that 4 are decoded gene (UCSC Genome Browser collects in May, 2004).(c) RNA enzyme H2B (FLJ11712) elongates the 47kbp of gene order in 11 exons, and one 308 amino acid protein of encoding.Encoding sequence colourity is ash.The position of sudden change is indicated that by arrow the number of positions of sudden change is relevant with translation initiation site, and the corresponding runic that becomes.

The sketch in Fig. 3 AGS3 zone, RNA enzyme H2C (AYP1) gene, its sudden change and sequence are deposited in other species.。(a) gene mapping of karyomit(e) 11q13.1..Critical region is a linkage disequilibrium data definition in the Pakistani family, and between D11S4205 and D11S987.RNA enzyme H2C (AYP1) gene is positioned at the 1.4kbp that 65.2Mbp crosses over the localized telomeric gene order in kinetochore on the minus strand.(b) RNA enzyme H2C (AYP1) gene structure comprises 4 exons (encoding sequence grey), and one 164 aminoacid protein of encoding.The position of sudden change is indicated that by arrow the number of positions of sudden change is relevant with translation initiation site, and the corresponding runic that becomes.。(c) two kinds of sudden changes all are arranged in and are retained in mammiferous remnants.The proteinic series arrangement of mammalian rna enzyme H2C zone directly faces sudden change.Hs, Homosapiens; Bt, Bos Taurus; Cf Canis familiaris; Mm, Mus musculus; Rn, the Rattusnorvegicus. substituted amino acid shows in above series arrangement.(d, e) the sequence electrophoretogram (d) of RNA enzyme H2C (AYP1) sudden change c.428A＞T (e) c.205C＞T.

Fig. 4 RNA enzyme H2A gene, chromosome position, gene structure and sudden change position.(a) gene mapping of karyomit(e) 19p13.13.RNA enzyme H2A is positioned at two s-generation collateral relative by blood children (F39 of family) zone, and two children have homozygote SNPs (SNP A-1509361,1606327,1606325), defines potential genetic linkage between a SNP A-1515950 and the A-1508018.(b) RNA enzyme H2A gene structure.RNA enzyme H2A is positioned at 12.8Mbp (UCSC Genome Browser May2004 compilation), crosses over the 7kbp of chromosome sequence.It comprises 8 exons and one 299 amino acid protein of encoding.(c) G37S sudden change occur in complete preservation from bacterium the remnants to the mankind.Hs, human (Homo sapiens); Cf, domesticated dog (Canis familiaris); Mm, house mouse (Mus musculus); Rn, Rattus norvegicus (Rattus norvegicus); Ce, beautiful new rhabditis axei (Caenorhabditis elegans); Sp, fission yeast mycelia (Schizosaccharomyces pombe); Sc, S. cervisiae (Saccharomyces cerevisiae); Ec, intestinal bacteria (Escherichia coli; Ph, Pyrococcus horikoshii). (d) the advance notice tertiary structure of modeling dissolved crystalline structure RNA enzyme H2A catalytic site on 2 type RNA enzyme H protein.The G37 remnants (center) of sudden change are positioned at roughly near active site and substrate in conjunction with remaining (Chapados etc., J Mol Biol 307:541-556 (2001)).

Fig. 5 is when being expressed as mammalian cell, and people RNA enzyme H2B (FYJ11712), RNA enzyme H2C (AYP1) and RNA enzyme H2A form enzymic activity Type II ribonuclease H mixture.(a) people RNA enzyme H2 mixture and its S. yeast counterpart of diagram suggestion.(b) epitope tagged FYJ11712 of T7 and AYP1 can with by the common immunoprecipitation of RNA enzyme H2A (IP) of myc-mark.(c, d) RNA enzyme H2A/B/C mixture manifests ribonuclease H activity.(c) oligomeric nucleotide heteroduplex is used to measure enzymic activity.Oligonucleotide C, can be by the degradable substrate of any ribonuclease H, it is a kind of oligomeric nucleotide of 3 '-fluorescein-labeled RNA of hybridizing with the oligomeric nucleotide of DABCYL DNA (follow fluorescently-labeled 3 ' chip separation up to enzymatic cleavage, DABCYL is with regard to the cancellation fluorescent agent) of complementary 5 ' mark.Oligonucleotide B, a kind of can only be by the substrate of II type ribonuclease H degraded.It has list for having with the position 15 of fluorescein in the oligomeric nucleotide chain of 3 '-mark, the dna double chain of Yeast Nucleic Acid.Oligonucleotide A is a kind of RNA:DNA heterozygote, be similar to oligonucleotide C, but antienzyme arranged, as with 2 ' O methyl RNA Nucleotide synthetic, 3 ' the fluorescein-labeled oligomeric nucleotide.(d) II type ribonuclease H activity can be by the HEK293T extract immunoprecipitation from the RNA enzyme H2A/B/C that contains the epi-position mark.Myc IP is by shared with mouse-anti-myc antibody; LgG IP is by controlling immunoprecipitation with normal mice lgG immunoglobulin (Ig).With the carrier that blank pCGT-Dest and pcDNA3.1mychis carrier infect, cell.Error line, s.e.m.

Sudden change among Fig. 6 RNA enzyme H2A has reduced the activity of RNA enzyme H.(a) the RNA enzyme H2A protein of usefulness mutant form, from the immunoprecipitation of the RNA enzyme H2 mixture of HEK293T cell, show that the mixture that RNA enzyme H2A G37S sudden change can not occur rupturing forms in conjunction with the RNA enzyme H2B of wild-type mark and the common transfection of RNA enzyme H2C.Enter input, IP, immunoprecipitate, WT, wild-type.(b) sudden change among the RNA enzyme H2A has reduced enzymic activity.The fluorescent rna enzyme H detection method of same immunoprecipitation complex is shown in (a).Error line, s.e.m.

Microsatellite gene type in two stranger in blood families of Fig. 7 extracts between the critical zone of AGS2.Frame is played in the zone of homozygote mark.

Fig. 8 is from the multiple sequence of (a) RNA enzyme H2B/Rnh2Bp (b) RNA enzyme H2C/Rnh2Cp homologue of representative eucaryon kind.It is upwards given with its substitution amino acid that the amino acid that replaces among patient AGS is shown as black and white.(b) aminoacid sequence that comprises kluyveromyces spp (Kluyveromyces waltii) is used for setting up homologue between people AYP1 and S. cereuisiae fermentum Rnh2Cp.Use the CHROMA (Goodstadt etc., Bioinformatics 17:845-846 (2001)) and the location of eucaryon sequence 80% consensus to be shown.Come self-align cut remnants to be shown in to scrape in the arc and at interval (-) only be used to locate purpose, do not represent any deletion.The GenInfo identifier is presented on the right, location.

The consistent abbreviation (amino acid): a, aromatic series (FHWY); B, big (EFHIKLMQRWY); C, charged (DEHKR); H, hydrophobicity (ACFGHILMTVWY); L, aliphatics (ILV); P, polarization (CDEHKNQRST); S, little (ACDGNPSTV); *, Ser/Thr (ST); +, positive charge (HKR); With-, negative charge (DE). plant abbreviation; Ag, anopheles costalis (Anopheles gambiae); Am, honeybee (Apis mellifera); An, aspergillus nidulans (Aspergillusnidulans); Bt, European ox (Bos taurus); Ca, Candida albicans (Candida albicans); Ce, beautiful new rhabditis axei (Caenorhabditis elegans); Cf, domesticated dog (Canis familiaris); Cg, Candida glabrata (Candida glabrata); Cp, little Cryptosporidium (Cryptosporidium parvum); Dd, discoideum net post bacterium (Dictyostelium discoideum); Dh, Hansen cryptococcus (Debaryomyceshansenii); Dm, drosophila melanogaster (Drosophila melanogaster); Dr, chub mackerel (Danio rerio); Eg, Eremothecium (Eremothecium gossypii); Gz, Gibberella zeae (Gibberella zeae); Hs, human (Homo sapiens); Kl, kluyveromyces spp (Kluyveromyces lactis); Kw, kluyveromyces spp (Kluyveromyces waltii); Mm, house mouse (Mus musculus); Nc, Neurospora crassa (Neurosporacrassa); Os, paddy rice (Oryza sativa); Rn, Rattus norvegicus (Rattus norvegicus); Sb, saccharomyces bayanus (Saccharomyces bayanus); Sca, Saccharomycodes (Saccharomyces castellii); Sce, S. cervisiae (Saccharomyces cerevisiae); Skl, Saccharomycodes (Saccharomyces kluyveri); Sku, Saccharomycodes (Saccharomyces kudriavzevii); Sm, Saccharomycodes (Saccharomyces mikatae); Spa, Saccharomyces paradoxus (Saccharomyces paradoxus); Spo, fission yeast mycelia (Schizosaccharomycespombe); Tb, Bruce trypanosome (Trypanosoma brucei); Tc, Oswaldocruzia (Trypanosoma cruzi); Tn, the green filefish (Tetraodon nigroviridis) of spot; Xt, xenopus (Xenopus tropicalis); And, Yl, ascomycetous yeast belongs to (Yarrowia lipolytica).

The AGS3 locus collection of illustrative plates of Fig. 9 karyomit(e) 11q13.2.Microsatellite gene type in six Asia families.Suppose that ancestral haplotype shows with runic, and the homozygote zone marker is played frame.

Active polyinosine-the polycytidylic acid of Figure 10 RNA enzyme H (poly (I:C)) is handled the HCT116 cell.A: anti-oligomeric nucleotide substrates enzymes, B:RNA enzyme H specific substrate, C: ribonuclease H substrate.

Figure 11 uses the western blotting antagonism of affinity purifying polyclonal antibody to raise from anti--RNA enzyme H2A/B/C mixture antagonism HeK293T cytolysis thing of sheep, and the RNA enzyme H2A/B/C complex proteins matter of wherein using epitope tagged carrier is by overexpression.Antibody can detect the mark mammalian rna enzyme H2A of overexpression, H2B and H2C (all being low-frequency band).* be to infer endogenous band.

Figure 12 RNA enzyme H2BA177T target structure is introduced ES cell sketch.The target structure contains the homologous sequence of 2.5kb arm from RNA enzyme H2B genomic locus, follows PGK-Xin Meisu box to embed between the exon 6 and 7, and the side is loxP site (trilateral).A kind of Nucleotide changes (arrow shows) and is imported into and produces common patient AGS and suddenly change at L-Ala to the Threonine of codon 177.The homologous recombination that this structure is used to success makes up ES clone, and nowadays this clone be used to blastocyst and inject and make up " gene is knocked in " AGS mouse model.

Figure 13 is used for wild-type LCL (WT LCL) what be stale-proof dynamically, detects the active method of RNA enzyme H2 on patient's AGS of RNA enzyme H2A G37S and RNA enzyme H2BA177T the lymphoblastoid cell lines.Clone contain shown in RNA enzyme H2 subunit on homozygote sudden change, reduced enzymic activity, relevant with the wild-type lymphoblastoid cell lines (WT).

The terminal fluorescent rna enzyme of Figure 14 H detection method shows that patient's AGS lymphoblastoid cell lines has reduced the enzymic activity of RNA enzyme H2.

Figure 15 a. dissolved recombinant RNA enzyme H2 protein complex SDS PAGE gel electrophoresis; B. the GST that recombinates, wild-type GST-RNA enzyme H2 mixture and have the enzymic activity of the GST-RNA enzyme H2 mixture of GS37 sudden change on the A subunit.

Embodiment

Embodiment 1: patient and experimenter

All experimenters in the research realize the Case definition of AGS, have and early send out this neurological feature of encephalopathic, the common infection in utero of passive research, the encephalic calcification in an exemplary distribution, a kind of CSF lymphocytosis＞5 cells/mm3 and/or＞IFN-α among the 2IU/ml CSF.Based on agreement, blood sample is the children that take from infection, its parents and infect the compatriot.Chromosomal DNA is taken from peripheral blood leucocyte by standard method.The research card is studied Ethics Committee and is passed through Scotland multicenter study Ethics Committee (04:MRE00/19) approval through healthy office/United Kingdom NHS of teaching hospital trust now.

Gene type and linkage analysis

The genome range of arranging by SNP scans the Affymetrix Human Mapping10K Xba1422.0 that is to use MRC Geneservice (Cambridge, Britain)

The high-density gene type of AGS2 and AGS3 locus is to finish (Jackson etc. as previously mentioned, Am J Hum Genet 63:541-546 (1998)) use from Marshfield linkage map (http://research.marshfieldclinic.org/genetics) and people's gene group browser sequence (May 2004 freeze, http://genome.ucsc.edu/) through identifying the definite little satellite sign of karyomit(e) of microsatellites of making a fresh start.In a model of gene frequency autosomal recessive inheritance with disease gene frequency complete penetrance (estimating at 1 to 1000) and hypothesis equal sign, linkage analysis is to use GENEHUNTER (version 2.0 β) (Kruglyak etc., Am J Hum Genet 56:1347-1363 (1996)).

Information biology

Database retrieval has utilized PSI-BLAST (http://www.ncbi.nlm.nih.gov/blast/; Altschul etc., Nucleic Acids Res.25:3389-3402 (1997), nonredundant protein sequence database and an E-value comprise threshold value 2x10 ^-3

Relatively the protein structure model is to utilize SWISS-MODEL (Schwede etc., Nucleic Acids Res 31:3381-3385 (2003) use default setting and RNA enzyme H2A protein (NP_006388) precognition structure by WHAT_CHECK (Nature 381:272 (1996) checking such as Hooft.Four known structure homology archeobacteria RNA enzyme H2 protein (showing 35-40% sequence identity NP_006388) are used as best prediction Available templates and use, 1uaxA1io2A 1x1pA and 1ekeB.Reactive site contacts remaining (Chapados etc., J Mol Biol.307:541-556 (2001)) with the expectation substrate and is explained, uses using VMD (v1.8.3) (Humphrey etc., J Mol Graph 14:33-38,27-28 (1996)).

Sudden change detects

Primer is designed to amplify RNA enzyme H2B, the coding exon (primer sequence is as shown in table 1) of RNA enzyme H2C and RNA enzyme H2A.Purifying PRC amplifies product and is to use painted terminator chemical action (Applied Biosystems) and the electrophoresis on an ABI3700 capillary analysis instrument to move (Applied Biosystems), or carry out on Megabace 500 (Amersham Pharmacia) the capillary analysis instrument.Mutation analysis uses Mutation Surveyor (Softgenetics) to carry out.

Table 1

Gene		Primer sequence	The PCR condition
				RNA enzyme H2AB			Amplitaq Gold except as otherwise noted
Exons 1	F	catggagggccgctggg SEQ ID No 7	60 ℃/35 circulations (Bioxact Taq plus Hispec)
					R	gctgtcccagagagaatcc SEQ ID No 8
2	F	aagtagaagaaaggaaaacagg SEQ ID No 9	55 ℃/35 circulations
					R	agctattcagaaagagcaagg SEQ ID No10
3	F	gagatactggatagtcttttgg SEQ ID No 11	55 ℃/35 circulations
					R	cggtgacagacccacaacc SEQ ID No 12
4	F	tgtttttcactttgagattaagg SEQ ID No 13	55 ℃/35 circulations
					R	gatcat taaaaggagagagagg SEQ ID No 14
5	F	cccagccatgagttaatgtg SEQ ID No 15	55 ℃/35 circulations
					R	acattcagtgtgtctgtaagc SEQ ID No 16
6	F	gtatatgatacaaccttaggag SEQ ID No 17	55 ℃/35 circulations
					R	cagacttactgccttctctgaa SEQ ID No 18
	R2	cttctctgaaaatttcttagag SEQ ID No 19
					R3	ctctggattcagacttactg

		SEQ ID No 20
				7	F	taaatggtctgaaggccacc SEQ ID No 21	55 ℃/35 circulations
	R	atgaggcttctgtgatattaag SEQ ID No 22
				8	F	gggtcagaatttgaattcagg SEQ ID No 23	55 ℃/35 circulations
	F2	tttttgctttcactcctcc SEQ ID No 24
					R	tcataaacagtgatatgataagc SEQ ID No 25
9	F	taaaagtaggacttactaagtc SEQ ID No 26	55 ℃/35 circulations
					R	acaccttaaaatatattagaacc SEQ ID No 27
10	F	ggtctaaattatacatgttgggt SEQ ID No 28	55 ℃/35 circulations
					R	aaactgtaatcacaatcaagcgt SEQ ID No 29
11	F	tgaaacttgagacatgcagtc SEQ ID No 30	57 ℃/35 circulations
					R	aacatgaaattggcctcttcc SEQ ID No 31
RNA enzyme H2A			ProMega Taq; Boehringer-Mannheim expansion buffering 1; 72 ℃/45 secs
				Exons 1-3	F	GCTCCTGCAGTATTAGTTCTTG SEQ ID No 32	60 ℃/40 circulations
	R	CCAGTTAATCTCATTCTAGGAAC SEQ ID No 33
					Seq8f	GAAGCAGTGATGATAGAACAG SEQ ID No 34
	Seq8r	CTGTTCTATCATCACTGCTTC SEQ ID No 35
				Exon 4	F	GTTCCTAGAATGAGATTAACTGG SEQ ID No 36	56 ℃/40 circulations
	R	AGACGTATCTGATGCTGAATCT SEQ ID No 37
					Seq9r	CTTGACCTCCGGTGATCTA SEQ ID No 38
Exon 5-6	F	AAAGCAGCGTGATAGAGAAAG SEQ ID No 39	63 ℃/40 circulations
					R	AAACCTCAGAAGTGTGCTCAG

		SEQ ID No 40
					Seq10f	ATCCCTGATTGATCCATCC SEQ ID No 41
Exon 7-8	F	TGAATGTTATGGTGCAACTTG SEQ ID No 42	56 ℃/40 circulations
					R	Tacgtgtggttctccttaaaca SEQ ID No 43
RNA enzyme H2C			As RNA enzyme H2A+0.5M trimethyl-glycine; 72C/90 second
				All exons	F	GGAAAACCCTAGGACTTGAAC SEQ ID No 44	62 ℃/40 circulations
	R	AGCTGGTTTACTGCTGTGAAG SEQ ID No 45
					Seq1f	CAGGACTCGAAGTGTCGTT SEQ ID No 46
	Seq1r	AACGACACTTCGAGTCCTG SEQ ID No 47
					Seq2r	tccctttgctcacgaagtc SEQ ID No 48
	Seq3f	CCTGGAGGATTCTACTGGGT SEQ ID No 49
					Seq3r	ACTACCCAGTAGAATCCTCCAG SEQ ID No 50
	Seq4f	GCCACACTGCATCTGCTG SEQ ID No 51

Vector construction

Gateway Vector system (Invitrogen) is used to make up mammalian expression vector.RNA enzyme H2B, the encoding sequence of RNA enzyme H2C and RNA enzyme H2A is amplified (CSODF031YM15, CR602872, Invitrogen and clone IRAUp969G0361D, BC011748, RZPD, difference) from plasmid clone and the clone is pDONR221 ^TMAYP1 buys pENTRY carrier (clone IOH27907, the Human Ultimate that controls oneself and make ^TMFull ORF GatewayShuttle Clone, NM_032193, Invitrogen).PcDNA3.1mychis-Dest and pCGT-Dest use Gateway Vector conversion system by inserting Gateway reading frame box to the polyclone position of pcDNA3.1mychis (Invitrogen) and pCGT (Van Aelst etc., EmboJ 15:3778-3786 (1996)).Site-directed mutagenesis is by use Stratagene Quikchange test kit to carry out RNA enzyme H2A pENTRY clone according to manufacturer's specification sheets.

Immunoprecipitation and Western blotting

The HEK293T cell is the common transfection of 1 μ g structure of using Lipofectamine (Invitrogen) momently with each according to manufacturer's specification sheets.After 24 hours, cell is dissolved in 50mM Tris (pH 7.8) in the time of 4 ℃, 280mM NaCl, 0.5%NP40,0.2mM EDTA, 0.2mM EGTA, 10% glycerol, 0.1mM sodium orthovanadate, 1 μ M DTT and 1 μ M PMSF solution 10 minutes.The dissolved cell is by 20mM ethyl piperazidine ethyl sulfonic acid (pH 7.9), and 10mM KCl, 1mM EGTA, 10% glycerol and 0.1mM sodium orthovanadate damping fluid are diluted to 1: 1 and surpass 10 minutes, extract centrifuging purifying (15800x g, 10min, 4 ℃).According to manufacturer's agreement, use anti--myc antibody (clone 9B11 of 1 μ g mouse, CellSignalling) or 1 μ g mouse IgG (Santa Cruz), make 500 μ g protein solutes use a-protein/G PLUS agarose (Santa Cruz) immunoprecipitation.Western blotting uses anti--myc monoclonal antibody of mouse and uses mouse monoclonal to resist-T7 antibody (Novagen) 1/5000 1/1000.

RNA enzyme H measuring method

10 μ M oligonucleotide (Eurogentec) are at 95 ℃ 60mM KCl, thermal treatment sex change 5 minutes and progressively be cooled to room temperature among the 50mM TrisHCl pH8.Fluorescent rna enzyme H measuring method is at the 60mM KCl of 100 μ l capacity, 50mM TrisHClpH8,10mM MgCl ₂, the oligomeric nucleotide mixture of 0.25 μ M, at 96 preferably in the flat underside, at 37 ℃ orbital shakers with 60rpm rotating speed concussion 3 hours.1/10 capacity of each immunoprecipitation is used to each reaction, uses the e. coli rna enzyme H (Invitrogen) of 2.5 units as positive control.Use VICTOR ²1420 fluorescence multiaspect counters (Perkin Elmer) are read fluorescence, use the discharging filter 100msec that excites filter and 535nm of a 480nm to read fluorescence.

The result

AGS2 gene locus purifying and evaluation RNA enzyme H2B (FLJ11712)

The little satellite marker gene of high-density karyomit(e) somatotype is used for one group of 10 family and comes purifying AGS2 gene locus.(F8 and F10 Fig. 7) are found the little overlapping region that manifests pure and mild son sign in two stranger in blood families.Autozygous chromosome segment (Broman etc. might be represented in this zone, Am J Hum Genet 65:1493-1550 (1999) and Gibson etc., Hum Mol Genet 15:789-795 (2006)), these chromosome segments have the height possibility at some and contain and occur (Lander etc., Science 265:2049-2054 (1987)) in few euchromosome degenerative disease of Disease-causing gene.Use the overlapping region of these homozygote marks, the AGS2 critical region is optimized to the 571kbp zone on the karyomit(e) 13q14.3. between genetic marker AC1378890TG19 and the D13S788 (Fig. 2).The decoding genes encoding exon of all four critical regions is sorted.

RNA enzyme H2B (FLJ11712) is the AGS2 gene

Change among 7 FLJ11712 of missense sequence in the family that filters out through identifying, yet, tangible pathogenicity bo sudden change do not had at DLEU7, GUCY1B2 or FLJ30707.The same period of family that has the sudden change in this gene in bigger 18 of summations through identifying inferior screen mutation FLJ11712 in the group (table 2, Fig. 2).Great majority sudden changes all is a missense, wherein have 2 be recycled discovery in agnate (A177T, V185G).All missense genes have caused non-conservation to replace the remnants (Fig. 8) that remain in the Mammals, and except remaining change (Y219H) of single reservation, it is remnants that keep back Dictostelium.Nonsense mutation is identified in two families, the terminator codon in exon 2 (F17) and the donor splicing site sudden change in 6 (F15).In these cases, the individuality of catching an illness all is to have second to sport the compound heterozygote that missense changes.Observed mutation spectrum shows that the sudden change result will be time equipotential factor allel but not loses the FLJ11712 protein function fully.Sudden change separates with disease in all families, and might father and mother all be the heterozygote of sudden change.The sudden change of at least 160 control allelotrope heredity.Have only most of common sudden changes, A177T, be one at the found heterozygote individuality of tested 241 samples.

Table 2-is the sudden change of the FLJ11712 through identifying in 18 AGS (Alcardl-Goutleres syndrome) family

Nucleotide is that start codon (ATG) A from nucleotide sequence NM_024570 begins to encode.Abbreviation: fs, frameshift.Hom, the individual pure and mild son of catching an illness; Het, the individual heterozygote of catching an illness; Sudden change through identifying in the M, parent; Sudden change through identifying in the P, male parent, nps, no parents' sample.

RNASEH2B (FLJ11712) is the homologous chromosomes of yeast Rnh2Bp

Previous 308 aminoacid proteins of not determining function of FLJ11712 coding.Semiquantitative RT-PCR is presented in the human tissue of wider range and may detect, and shows ubiquitous expression (data not shown).

In four repeated retrieval (Fig. 8), one is used PSI-BLAST (Altschuhl or the like, Nucleic Acids Res25:3389-3402 (1997)) database search discloses people FLJ11712 and saccharomyces cerevisiae protein, the significant similarity of Rnh2Bp (Rnh202).Rnh2Bp is the main component (Jeong etc., Nucleic Acids Res 32:407-414 (2004)) of yeast rna enzyme H2 mixture.This observation shows that FLJ11712 may have effect in mammiferous being equal in the mixture, and has improved the possibility that additional RNA enzyme H composition can also suddenly change in AGS.

RNA enzyme H2C (AYP1) is the AGS3 gene

A kind of SNP ordered series of numbers genome scanning is applied to 6 blood relation families, 5 Pakistanis (F30-34) and 1 Bangladeshi (F35).Follow the new locus through identifying of time microsatellite gene type (Fig. 9), AGS3, at karyomit(e) 11q13.2, follow the multiple spot lod score (MarshfieldGenetic Map) of a maximum of 4.54 that is positioned at 66.8cM, between markers D11S4205 and D11S913.Alternatively, the genotype in 5 Pakistani families shows that (the black matrix genotype Fig. 9), follows the interval of modifying of 4.9cm between D11S4205 and D11S987 to arrive the little pure and mild subregion of AGS3 critical interval to ancestral haplotype in the F30 of family.

Especially, AYP1 with the protein (Frank etc., Proc Natl Acad Sci USA 95:12872-12877 (1988)) of human rna enzyme H2A copurification, is positioned at this critical interval (Fig. 3) on the biochemistry of encoding.AYP1 therefore be sorted and nonconservative missense mutation be in 6 families (see Table 3 and Fig. 9) through identifying.Homozygote that is arranged in codon 69 (R69W) mutates now all from 5 Pakistani families of the total significant ancestral haplotype of tool individuality of catching an illness.One second difference homozygote sudden change, K1431 appears in the Bangladesh family.Sudden change separates with the disease in the family, and sudden change all is not detected at least 172 Asia control allelotrope, and the AYP1 copying cutting that RT-PCR expresses shows the expression pattern for the similar extensive distribution of FLJ11712.

Table 3.AYP1 sudden change is suffered from AGS (Aicardi-Goutieres syndrome) at 6, and family after testing

Nucleotide is from initiator codon (ATG) open numbering (for protein is transcribed thing AF312034).Abbreviation: Hom, the homozygote in the individuality of catching an illness; M, the sudden change among mother through identifying; P, the sudden change among the father through identifying.

Concurrent, inventors formulate human homology's karyomit(e) that AYP1 is S. yeast Rnh2Cp, second subunit of a yeast rna enzyme H2 (Jeong etc., Nucleic Acids Res 32:4407-414 (2004)).The open reading frame of a data library searching kluyveromyces spp is identified human AYP1 and S. yeast Rnh2Cp (Fig. 8) in 4 search iteration.

RNA enzyme H2A is the AGS4 gene

RNA enzyme H2A is that 8 exon genes is crossed over the gene order that karyomit(e) 19P13.13 goes up 7kbp, and one 299 aminoacid protein of encoding.RNA enzyme H2A does not concentrate jointly with any gene mapping AGS locus.Yet evaluation SNP-ordered series of numbers genome scanning data are identified the RNA enzyme H2A locus homozygote zonule (seeing SANCHIS etc., J PEDIATR 146:701-705,2005) (Fig. 4 A) that is positioned at aforementioned white race Hispanic blood relation AGS family.Sequence order-checking to 2 sick children in this family identifies a simple zygote C.

The A transversion causes the non-conservative missense mutation (Fig. 4 b) of a G37S.This amino acid is to keep Zi human to archeobacteria (Fig. 4 c) fully, and be arranged in the circle on expection and the b-layer of the bottom that separates the bonded substrate, near the catalytic site (Fig. 4 d) of RNA enzyme H2 J MolBiol 307:541-546 such as (, 2001) Chapados.Sudden change is not controlled in the allelotrope and parents are detected in the homozygote sudden change 178 Caucasians.

RNA enzyme H2B (FLJ11712), RNA enzyme H2C (AYP1) and next exogenic mixture RNA enzyme H2A form have RNA enzyme H2 activity

With the sequence homology of S. zymic Rnh2Ap-Rnh2Bp-Rnh2Cp mixture prediction RNA enzyme H2B, RNA enzyme H2C and RNA enzyme H2A protein will form to have the active protein complex of RNA enzyme H2 (Fig. 5 a).In order to determine this point, these three genes with channel system between netting be cloned to epitope tagged Mammals expression vector (pCGT-Dest (T7) and pCDNA3.1myehis-Dest), and all three structures momently cotransfection be the HEK293 cell.At anti--mould immuno-precipitation of C-end mark FLJ11712-mould, destroy the AYP1 of the terminal T7-mark of N-and the RNA enzyme H2A (Fig. 5 b) of T7-mark and confirmed that these three subunits are in external interference effect.

Aforementioned fluorometric assay (Parniak etc., Anal Biochem 33-39,2003) is used test, and this is used for the mixture of enzymic activity.Fluorescently-labeled oligonucleotide is DNA oligonucleotide complementation (Fig. 5 c) with the epipolic DABCYL-mark of cancellation by thermal treatment.Fluorescence-oligonucleotide enzyme process assay products has caused discharging fluorescein from adjacent quencher molecule, produces fluorescence mark (Fig. 5 c).

The contriver finds that ' oligonucleotide C ', a RNA:DNA mixture are confirmed that by the effective cracking of immunoprecipitation complex this mixture manifests RNA enzyme H activity (Fig. 5 d).In addition, immunoprecipitation complex has necessary ' 2 type ' RNA enzyme H activity, and is same, and its identified one embeds monokaryon ribosomal ribonucleic acid in the RNA-DNA mixture, and effectively ' oligonucleotide B ' is with the colibacillary RNA enzyme of E. H--' 1 type ' RNA enzyme H in cracking.As expection, because ' oligonucleotide A ' uses anti-2-O ' methyl RNA nuclease chemical action synthetic, and is therefore not cleaved.

Sudden change in the RNA enzyme H2 mixture reduces enzymic activity

Selected pathogenic mutation (FLJ11712, A177T, T1631; AYP1 R69W, K1431; RNaseH2A G37S) is introduced into its that use site-directed mutagenesis and represent gene, and pass through transient transfection HEK293 cell expressing.Immunoprecipitation is implemented assessing its effect to stable composite, and enzymic activity determined (Fig. 6 a and 6b).These experimental results show that RNa enzyme H2A G37S sudden change for not influence of stable composite, but expection is positioned at the sudden change of catalytic site, has significantly reduced enzymic activity (Fig. 6 b).As for the AYP1 sudden change, the AYP subunit in the ruined mixture and the amount of other subunits have reduced.This reduces relevant with enzymic activity.This shows that reducing active enzyme is the result that sudden change cracking mixture forms.

Embodiment 2: RNA enzyme H2 activity in poly-(I:C) HCT116 cell

The result of contriver's observation is in AGS, and natural immunity is to be activated inadequately, shows that RNA enzyme H2 is relevant with innate immune responses.In order to test this poly-(I:C), the synthetic of a kind of dsRNA is used with the merit isomer, and the cytokine product of itself and virus infection and known protein kinase of replying by dsRNA-(PKR) or Toll sample acceptor 3 (TLR3) produces inducing action and replys in stimulating innate immunity.Half-merge the HCT116 colon cancer cell will be used in RPMI1640 and have poly-(I:C) (Invivogen, the U.S.) of 25mg/ml treatment in 10%FCS and 1% penicillin/streptomycin through a time period.The preparation of protein cleavage thing is finished from cell and fluorescent rna enzyme H2 mensuration.

The results are shown among Figure 10.The dual growth of 2 hours enzymic activity is observed, shows that RNA enzyme H2 is positioned at the catchment of path, and may be the effector of innate immune responses therefore.

Embodiment 3: polyclonal antibody

Polyclonal antibody is by expressing a RNA enzyme H2 immunogen as bacterial expression protein (seeing " Antibodies:ALaboratory Manual " ed Harlow and Lane, Cold Spring Harbor Laboratory Press (1December 1988) ISBN 978-0879693145).This marking protein uses a GST mark purifying on the B subunit.The GST mark then uses PreScission proteolytic enzyme (Amersham) and generates protein and is injected into sheep (Eurogentec) and comes cracked.The Freund adjuvant is used to injection.One first injection uses whole adjuvants to make, and is that time auxiliary all uses adjuvant subsequently.

Consequent sheep blood serum uses the chromatography column that contains the conjugated antigen mixture to come the immunoaffinity purifying.Antigenic compound is consistent with the immunogen of use.Western blotting (seeing Figure 11) shows that antibody can detect the subunit with correct molecular weight of overexpression, and for example, those antibody have the specificity at RNA enzyme H2A/H2B and H2C subunit.Additional band (*) may be represented a kind of in the endogenous protein.The result that bands of a spectrum are identified in western blotting confirms by anti--traget antibody.

The immunogen sequence of using is:

Be used to resist-RNA enzyme H2B (having remaining linker) from the proteolytic enzyme cutting

LEVLFQGPLGSPEFPSTSLYKKAGSTMAAGVDCGDGVGARQHVFLVSEYLKDASKKMKNGLMFVKLVNPCSGEGAIYLFNMCLQQLFEVKVFKEKHHSWFINQSVQSGGLLHFATPVDPLFLLLHYLIKADKEGKFQPLDQVVVDNVFPNCILLLKLPGLEKLLHHVTEEKGNPEIDNKKYYKYSKEKTLKWLEKKVNQTVAALKTNNVNVSSRVQSTAFFSGDQASTDKEEDYIRYAHGLISDYIPKELSDDLSKYLKPEPSASLPNPPSKKIKLSDEPVEAKEDYTKFNTKDLKTEKKNSKMTAAQKALAKVDKSGMKSIDTFFGVKNKKKIGKV(SEQ ID No 52).

Be used for RNA enzyme H2C

MESGDEAAIERHRVHLRSATLRDAVPATLHLLPCEVAVDGPAPVGRFFTPAIRQGPEGLEVSFRGRCLRGEEVAVPPGLVGYVMVTEEKKVSMGKPDPLRDSGTDDQEEEPLERDFDRFIGATANFSRFTLWGLETIPGPDAKVRGALTWPSLAAAIHAQVPED(SEQ ID No 53).

Be used for anti-RNA enzyme H2A

MDLSELERDNTGRCRLSSPVPAVCRKEPCVLGVDEAGRGPVLGPMVYAICYCPLPRLADLEALKVADSKTLLESERERLFAKMEDTDFVGWALDVLSPNLISTSMLGRVKYNLNSLSHDTATGLIQYALDQGVNVTQVFVDTVGMPETYQARLQQSFPGIEVTVKAKADALYPVVSAASICAKVARDQAVKKWQFVEKLQDLDTDYGSGYPNDPKTKAWLKEHVEPVFGFPQFVRFSWRTAQTILEKEAEDVIWEDSASENQEGLRKITSYFLNEGSQARPRSSHRYFLERGLESATSL(SEQ ID No 54).

Embodiment 4: lymphoblastoid cell lines (LCL)

Lymphoblastoid cell lines (LCL) is to set up successive from patient's AGS main B cell and increase hatching cell system by infecting with EBV.The appropriate methodology that is used to set up LCLs is a techniques well known, for example, and Penno etc., Methods in Cell Science 15 (1): 43-47,1993.LCLs has been converted to typical sufferer sudden change in subunit.

Following LCLs is fabricated:

Table 4

Title	Protein	Sudden change
			AGS PIO	RNA enzyme H2A	G37S
AJ003	RNA enzyme H2B	A177T
			AJ010	RNA enzyme H2B	A177T
AJ011	RNA enzyme H2B	A177T/+
			AJ012	RNA enzyme H2B	A177T/+
AJ039	RNA enzyme H2B	Q58X/A 177T
			AJ055	RNA enzyme H2B	K162T

AJ055 RNA enzyme H2B K162T

The enzymic activity of cell can be measured by described fluorescent rna enzyme H activity test method.(Figure 13) is the same with the titration end point detection method, and kinetics measuring method also can be measured (for example every five minutes) at the enterprising line time of the Perkin Elmer VictorIII with fixed temperature regulation (37oC) by z and finish.The exemplary measuring method on RNA enzyme H2 suddenlys change active lymphoblastoid cell lines as shown in figure 13.

Embodiment 5: recombinant chou RNA enzyme H2

Recombinant chou, enzymic activity ground RNA enzyme H2 protein complex (is seen Figure 15 a) by using a kind of expression to contain the synthetic and purifying of polycistronic bacterium that 3 kinds of genes constitute.The activity and the wild-type mixture of sudden change can be measured by enzyme assay, shown in Figure 15 b.

Embodiment 6:RNA enzyme H2 suppresses virus replication

The RNA-DNA intermediate is most important for virus replication.Therefore, active to adjusted endogenous RNA enzyme H2 is that useful host response suppresses virus replication by expection.We support this expection on evidence, being presented at the HCT116 cell follows in the several hrs of dsRNA (polyI:C) (the immune signalling system activator of a kind of effective endogeny) treatment, enzymic activity has remarkable rising, the immune signalling system activator of a kind of effective endogeny (see figure 10).

Sequence table

＜110〉directorate of Medical Research

＜120〉RNA enzyme H2 mixture and gene thereof

<130>P45264.WO.01/JSL/BOU

<150>GB 0611444.1

<151>2006-06-09

<160>246

<170>PatentIn version 3.4

<210>1

<211>1515

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>1

actagcggag ccgcgaggga gaggccgcgg ccccttcccg ttgcctgcgg ccaccggccg 60

gcattcagag cccctcgcct ggcgctaaat ttaaaaacgt aacacgagca gcaggctggt 120

ctcggaaacg aaacgaaatt cggtccctgg gcctcctccc gggcgctgcc ggtccctcag 180

cgcgccgcgc cacccggaac agacccttct cccgccattt tcggcggggc tgggagactg 240

aggcccgcgg cgctgagcct gcggcgcccc ggaagaggcg ggcggcatgg ccgctggcgt 300

ggactgcggg gacggggttg gcgcccggca gcacgtgttc ctggtttcag aatatttaaa 360

agatgcttca aagaagatga aaaatgggct aatgtttgta aaactggtta acccctgttc 420

aggagaagga gccatttact tgttcaatat gtgtctacag cagctgcttg aagtaaaagt 480

tttcaaggaa aaacaccatt cttggtttat aaatcaatca gttcaatcag gaggtcttct 540

ccattttgcc acacctgtgg atcctctatt tctgcttctc cactacctca taaaggctga 600

taaggagggg aagtttcagc cccttgatca agttgtggtg gataacgtgt ttccaaattg 660

catcttgttg ctgaaacttc ctggacttga gaagttactt catcatgtga cagaggaaaa 720

aggtaatcca gaaatagaca acaagaaata ttacaagtac agcaaagaga agacattaaa 780

gtggctggaa aaaaaggtta atcaaactgt ggcagcatta aaaaccaata atgtgaatgt 840

cagttcccgg gtacagtcaa ctgcattttt ctctggtgac caagcttcca ctgacaagga 900

agaggattat attcgttatg cccatggtct gatatctgac tacatcccta aagaattaag 960

tgatgactta tctaaatact taaagcttcc agaaccttca gcctcattgc caaatcctcc 1020

atcaaagaaa ataaagttat cagatgagcc tgtagaagca aaagaagatt acactaagtt 1080

taatactaaa gatttgaaga ctgaaaagaa aaatagcaaa atgactgcag ctcagaaggc 1140

tttggctaaa gttgacaaga gtggaatgaa aagtattgat accttttttg gggtaaaaaa 1200

taaaaaaaaa attggaaagg tttgaaactt tgaaaataaa atctagcaaa aatatttgct 1260

ttttacatgt ttcagtttgt ccttcctgac tgttaatgac tacctttggt tgggggaagg 1320

aagaggccaa tttcatgttc tcttaaacat ttctttgcat ttggtttttg tgttcctgaa 1380

caaaatatgg gaaagtgtct aacttcatgg ctatggcctt ttggagtctc atctgacata 1440

atgaaaagta atcacttgaa gagaattaac atatagcatc atgattttct caataaactg 1500

atgtgtgaca atgtt 1515

<210>2

<211>312

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>2

Met Ala Ala Gly Val Asp Cys Gly Asp Gly Val Gly Ala Arg Gln His

1 5 10 15

Val Phe Leu Val Ser Glu Tyr Leu Lys Asp Ala Ser Lys Lys Met Lys

20 25 30

Asn Gly Leu Met Phe Val Lys Leu Val Asn Pro Cys Ser Gly Glu Gly

35 40 45

Ala Ile Tyr Leu Phe Asn Met Cys Leu Gln Gln Leu Leu Glu Val Lys

50 55 60

Val Phe Lys Glu Lys His His Ser Trp Phe Ile Asn Gln Ser Val Gln

65 70 75 80

Ser Gly Gly Leu Leu His Phe Ala Thr Pro Val Asp Pro Leu Phe Leu

85 90 95

Leu Leu His Tyr Leu Ile Lys Ala Asp Lys Glu Gly Lys Phe Gln Pro

100 105 110

Leu Asp Gln Val Val Val Asp Asn Val Phe Pro Asn Cys Ile Leu Leu

115 120 125

Leu Lys Leu Pro Gly Leu Glu Lys Leu Leu His His Val Thr Glu Glu

130 135 140

Lys Gly Asn Pro Glu Ile Asp Asn Lys Lys Tyr Tyr Lys Tyr Ser Lys

145 150 155 160

Glu Lys Thr Leu Lys Trp Leu Glu Lys Lys Val Asn Gln Thr Val Ala

165 170 175

Ala Leu Lys Thr Asn Asn Val Asn Val Ser Ser Arg Val Gln Ser Thr

180 185 190

Ala Phe Phe Ser Gly Asp Gln Ala Ser Thr Asp Lys Glu Glu Asp Tyr

195 200 205

Ile Arg Tyr Ala His Gly Leu Ile Ser Asp Tyr Ile Pro Lys Glu Leu

210 215 220

Ser Asp Asp Leu Ser Lys Tyr Leu Lys Leu Pro Glu Pro Ser Ala Ser

225 230 235 240

Leu Pro Asn Pro Pro Ser Lys Lys Ile Lys Leu Ser Asp Glu Pro Val

245 250 255

Glu Ala Lys Glu Asp Tyr Thr Lys Phe Asn Thr Lys Asp Leu Lys Thr

260 265 270

Glu Lys Lys Asn Ser Lys Met Thr Ala Ala Gln Lys Ala Leu Ala Lys

275 280 285

Val Asp Lys Ser Gly Met Lys Ser Ile Asp Thr Phe Phe Gly Val Lys

290 295 300

Asn Lys Lys Lys Ile Gly Lys Val

305 310

<210>3

<211>514

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>3

gcgtcgcgta ggagggagga tggagagcgg cgacgaagcg gccatcgaga ggcaccgcgt 60

ccacttgcgc tccgccacat tgcgcgacgc cgtacccgcc acactgcatc tgctgccctg 120

cgaggttgcg gtggacgggc ccgccccggt ggggcgcttc ttcacgcccg ccatccgcca 180

gggccccgag ggactcgaag tgtcgtttcg gggccgctgt ctacggggag aggaggtggc 240

ggtgccgcct ggcctcgtgg gatacgtgat ggtgacagaa gagaagaagg tgtcgatggg 300

gaagccagac cccttgcggg attccgggac tgacgaccaa gaggaggagc cgctggagcg 360

ggacttcgac cgcttcattg gagccactgc caacttcagc cgcttcaccc tgtggggtct 420

ggagaccatc cctggcccgg atgccaaagt gcgtggggcc ttaacttggc ccagccttgc 480

ggcagcgatt cacgcacagg tgcccgagga ctga 514

<210>4

<211>164

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>4

Met Glu Ser Gly Asp Glu Ala Ala Ile Glu Arg His Arg Val His Leu

1 5 10 15

Arg Ser Ala Thr Leu Arg Asp Ala Val Pro Ala Thr Leu His Leu Leu

20 25 30

Pro Cys Glu Val Ala Val Asp Gly Pro Ala Pro Val Gly Arg Phe Phe

35 40 45

Thr Pro Ala Ile Arg Gln Gly Pro Glu Gly Leu Glu Val Ser Phe Arg

50 55 60

Gly Arg Cys Leu Arg Gly Glu Glu Val Ala Val Pro Pro Gly Leu Val

65 70 75 80

Gly Tyr Val Met Val Thr Glu Glu Lys Lys Val Ser Met Gly Lys Pro

85 90 95

Asp Pro Leu Arg Asp Ser Gly Thr Asp Asp Gln Glu Glu Glu Pro Leu

100 105 110

Glu Arg Asp Phe Asp Arg Phe Ile Gly Ala Thr Ala Asn Phe Ser Arg

115 120 125

Phe Thr Leu Trp Gly Leu Glu Thr Ile Pro Gly Pro Asp Ala Lys Val

130 135 140

Arg Gly Ala Leu Thr Trp Pro Ser Leu Ala Ala Ala Ile His Ala Gln

145 150 155 160

Val Pro Glu Asp

<210>5

<211>1148

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>5

gcgccgagac ccgctcctgc agtattagtt cttgcagctg gtggtggcgg ctgaggcggc 60

atggatctca gcgagctgga gagagacaat acaggccgct gtcgcctgag ttcgcctgtg 120

cccgcggtgt gccgcaagga gccttgcgtc ctgggcgtcg atgaggcggg caggggcccc 180

gtgctgggcc ccatggtcta cgccatctgt tattgtcccc tgcctcgcct ggcagatctg 240

gaggcgctga aagtggcaga ctcaaagacc ctattggaga gcgagcggga aaggctgttt 300

gcgaaaatgg aggacacgga ctttgtcggc tgggcgctgg atgtgctgtc tccaaacctc 360

atctctacca gcatgcttgg gcgggtcaaa tacaacctga actccctgtc acatgataca 420

gccactgggc ttatacagta tgcattggac cagggcgtga acgtcaccca ggtattcgtg 480

gacaccgtag ggatgccaga gacataccag gcgcggctgc agcaaagttt tcccgggatt 540

gaggtgacgg tcaaggccaa agcagatgcc ctctacccgg tggttagtgc tgccagcatc 600

tgtgccaagg tggcccggga ccaggccgtg aagaaatggc agttcgtgga gaaactgcag 660

gacttggata ctgattatgg ctcaggctac cccaatgatc ccaagacaaa agcgtggttg 720

aaggagcacg tggagcctgt gttcggcttc ccccagtttg tccggttcag ctggcgcacg 780

gcccagacca tcctggagaa agaggcggaa gatgttatat gggaggactc agcatccgag 840

aatcaggagg gactcaggaa gatcacatcc tacttcctca atgaagggtc ccaagcccgt 900

ccccgttctt cccaccgata tttcctggaa cgcggcctgg agtcagcaac cagcctctag 960

cagctgcctc tacgcgctct acctgcttcc ccaacccaga cattaaaatt gtttaaggag 1020

aaccacacgt aggggatgta cttttgggac agaagcaagg tgggagtgtg ctctgcagcc 1080

gggtccagct acttcctttt ggaaccttaa atagaatggg tgttggttga ttaattttat 1140

ttaaaaaa 1148

<210>6

<211>299

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>6

Met Asp Leu Ser Glu Leu Glu Arg Asp Asn Thr Gly Arg Cys Arg Leu

1 5 10 15

Ser Ser Pro Val Pro Ala Val Cys Arg Lys Glu Pro Cys Val Leu Gly

20 25 30

Val Asp Glu Ala Gly Arg Gly Pro Val Leu Gly Pro Met Val Tyr Ala

35 40 45

Ile Cys Tyr Cys Pro Leu Pro Arg Leu Ala Asp Leu Glu Ala Leu Lys

50 55 60

Val Ala Asp Ser Lys Thr Leu Leu Glu Ser Glu Arg Glu Arg Leu Phe

65 70 75 80

Ala Lys Met Glu Asp Thr Asp Phe Val Gly Trp Ala Leu Asp Val Leu

85 90 95

Ser Pro Asn Leu Ile Ser Thr Ser Met Leu Gly Arg Val Lys Tyr Asn

100 105 110

Leu Asn Ser Leu Ser His Asp Thr Ala Thr Gly Leu Ile Gln Tyr Ala

115 120 125

Leu Asp Gln Gly Val Asn Val Thr Gln Val Phe Val Asp Thr Val Gly

130 135 140

Met Pro Glu Thr Tyr Gln Ala Arg Leu Gln Gln Ser Phe Pro Gly Ile

145 150 155 160

Glu Val Thr Val Lys Ala Lys Ala Asp Ala Leu Tyr Pro Val Val Ser

165 170 175

Ala Ala Ser Ile Cys Ala Lys Val Ala Arg Asp Gln Ala Val Lys Lys

180 185 190

Trp Gln Phe Val Glu Lys Leu Gln Asp Leu Asp Thr Asp Tyr Gly Ser

195 200 205

Gly Tyr Pro Asn Asp Pro Lys Thr Lys Ala Trp Leu Lys Glu His Val

210 215 220

Glu Pro Val Phe Gly Phe Pro Gln Phe Val Arg Phe Ser Trp Arg Thr

225 230 235 240

Ala Gln Thr Ile Leu Glu Lys Glu Ala Glu Asp Val Ile Trp Glu Asp

245 250 255

Ser Ala Ser Glu Asn Gln Glu Gly Leu Arg Lys Ile Thr Ser Tyr Phe

260 265 270

Leu Asn Glu Gly Ser Gln Ala Arg Pro Arg Ser Ser His Arg Tyr Phe

275 280 285

Leu Glu Arg Gly Leu Glu Ser Ala Thr Ser Leu

290 295

<210>7

<211>17

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the RNA enzyme H2B exons 1 forward primer of chemosynthesis

<400>7

catggagggc cgctggg 17

<210>8

<211>19

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the RNA enzyme H2B exons 1 reverse primer of chemosynthesis

<400>8

gctgtcccag agagaatcc 19

<210>9

<211>22

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the RNA enzyme H2B exon 2 forward primer of chemosynthesis

<400>9

aagtagaaga aaggaaaaca gg 22

<210>10

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the RNA enzyme H2B exons 1 reverse primer of chemosynthesis

<400>10

agctattcag aaagagcaag g 21

<210>11

<211>22

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the RNA enzyme H2B exon 3 forward primer of chemosynthesis

<400>11

gagatactgg atagtctttt gg 22

<210>12

<211>19

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the RNA enzyme H2B exon 3 reverse primer of chemosynthesis

<400>12

cggtgacaga cccacaacc 19

<210>13

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜223〉RNA enzyme H2B exon 4 forward primers of chemosynthesis

<400>13

tgtttttcac tttgagatta agg 23

<210>14

<211>22

<212>DNA

＜213〉artificial sequence

<220>

＜223〉RNA enzyme H2B exon 4 reverse primers of chemosynthesis

<400>14

gatcattaaa aggagagaga gg 22

<210>15

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉RNA enzyme H2B exon 5 forward primers of chemosynthesis

<400>15

cccagccatg agttaatgtg 20

<210>16

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉RNA enzyme H2B exon 5 reverse primers of chemosynthesis

<400>16

acattcagtg tgtctgtaag c 21

<210>17

<211>22

<212>DNA

＜213〉artificial sequence

<220>

＜223〉RNA enzyme H2B exon 6 forward primers of chemosynthesis

<400>17

gtatatgata caaccttagg ag 22

<210>18

<211>22

<212>DNA

＜213〉artificial sequence

<220>

＜223〉RNA enzyme H2B exon 6 reverse primers of chemosynthesis

<400>18

cagacttact gccttctctg aa 22

<210>19

<211>22

<212>DNA

＜213〉artificial sequence

<220>

＜223〉RNA enzyme H2B exon 6 reverse primers 2 of chemosynthesis

<400>19

cttctctgaa aatttcttag ag 22

<210>20

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉RNA enzyme H2B exon 6 reverse primers 3 of chemosynthesis

<400>20

ctctggattc agacttactg 20

<210>21

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the RNA enzyme H2B exon 7 forward primer of chemosynthesis

<400>21

taaatggtct gaaggccacc 20

<210>22

<211>22

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the RNA enzyme H2B exon 7 reverse primer of chemosynthesis

<400>22

atgaggcttc tgtgatatta ag 22

<210>23

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉RNA enzyme H2B exon 8 forward primers of chemosynthesis

<400>23

gggtcagaat ttgaattcag g 21

<210>24

<211>19

<212>DNA

＜213〉artificial sequence

<220>

＜223〉RNA enzyme H2B exon 8 forward primers 2 of chemosynthesis

<400>24

tttttgcttt cactcctcc 19

<210>25

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜223〉RNA enzyme H2B exon 8 reverse primers of chemosynthesis

<400>25

tcataaacag tgatatgata agc 23

<210>26

<211>22

<212>DNA

＜213〉artificial sequence

<220>

＜223〉RNA enzyme H2B exon 9 forward primers of chemosynthesis

<400>26

taaaagtagg acttactaag tc 22

<210>27

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜223〉RNA enzyme H2B exon 9 reverse primers of chemosynthesis

<400>27

acaccttaaa atatattaga acc 23

<210>28

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜223〉RNA enzyme H2B exons 10 forward primer of chemosynthesis

<400>28

ggtctaaatt atacatgttg ggt 23

<210>29

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜223〉RNA enzyme H2B exons 10 reverse primer of chemosynthesis

<400>29

aaactgtaat cacaatcaag cgt 23

<210>30

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉RNA enzyme H2B exons 11 forward primer of chemosynthesis

<400>30

tgaaacttga gacatgcagt c 21

<210>31

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉RNA enzyme H2B exons 11 reverse primer of chemosynthesis

<400>31

aacatgaaat tggcctcttc c 21

<210>32

<211>22

<212>DNA

＜213〉artificial sequence

<220>

＜223〉RNA enzyme H2A exons 1-3 forward primer of chemosynthesis

<400>32

gctcctgcag tattagttct tg 22

<210>33

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜223〉RNA enzyme H2A exons 1-3 reverse primer of chemosynthesis

<400>33

ccagttaatc tcattctagg aac 23

<210>34

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the RNA enzyme H2A exons 1-3seq8r primer of chemosynthesis

<400>34

gaagcagtga tgatagaaca g 21

<210>35

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the RNA enzyme H2A exons 1-3seq8r primer of chemosynthesis

<400>35

ctgttctatc atcactgctt c 21

<210>36

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜223〉RNA enzyme H2A exon 4 forward primers of chemosynthesis

<400>36

gttcctagaa tgagattaac tgg 23

<210>37

<211>22

<212>DNA

＜213〉artificial sequence

<220>

＜223〉RNA enzyme H2A exon 4 reverse primers of chemosynthesis

<400>37

agacgtatct gatgctgaat ct 22

<210>38

<211>19

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the RNA enzyme H2A exon 4seq9r primer of chemosynthesis

<400>38

cttgacctcc ggtgatcta 19

<210>39

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the RNA enzyme H2A exon 5-6 forward primer of chemosynthesis

<400>39

aaagcagcgt gatagagaaa g 21

<210>40

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the RNA enzyme H2A exon 5-6 reverse primer of chemosynthesis

<400>40

aaacctcaga agtgtgctca g 21

<210>41

<211>19

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the RNA enzyme H2A exon 5-6seq10f primer of chemosynthesis

<400>41

atccctgatt gatccatcc 19

<210>42

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉RNA enzyme H2A exon 7-8 forward primer of chemosynthesis

<400>42

tgaatgttat ggtgcaactt g 21

<210>43

<211>22

<212>DNA

＜213〉artificial sequence

<220>

＜223〉RNA enzyme H2A exon 7-8 reverse primer of chemosynthesis

<400>43

tacgtgtggt tctccttaaa ca 22

<210>44

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉forward primer of all exons of RNA enzyme H2C of chemosynthesis

<400>44

ggaaaaccct aggacttgaa c 21

<210>45

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reverse primer of all exons of RNA enzyme H2C of chemosynthesis

<400>45

agctggttta ctgctgtgaa g 21

<210>46

<211>19

<212>DNA

＜213〉artificial sequence

<220>

＜223〉all exon seq1f primers of the RNA enzyme H2C of chemosynthesis

<400>46

caggactcga agtgtcgtt 19

<210>47

<211>19

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the RNA enzyme H2C exon seq1r primer of chemosynthesis

<400>47

aacgacactt cgagtcctg 19

<210>48

<211>19

<212>DNA

＜213〉artificial sequence

<220>

＜223〉all exon seq2r primers of the RNA enzyme H2C of chemosynthesis

<400>48

tccctttgct cacgaagtc 19

<210>49

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉all exon seq3f primers of the RNA enzyme H2C of chemosynthesis

<400>49

cctggaggat tctactgggt 20

<210>50

<211>22

<212>DNA

＜213〉artificial sequence

<220>

＜223〉all exon seq3r primers of the RNA enzyme H2C of chemosynthesis

<400>50

actacccagt agaatcctcc ag 22

<210>51

<211>18

<212>DNA

＜213〉artificial sequence

<220>

＜223〉all exon seq4f primers of the RNA enzyme H2C of chemosynthesis

<400>51

gccacactgc atctgctg 18

<210>52

<211>337

<212>PRT

＜213〉artificial sequence

<220>

＜223〉the immunogen sequence of anti-RNA enzyme H2B (tool proteolytic cleavage joint)

<400>52

Leu Glu Val Leu Phe Gln Gly Pro Leu Gly Ser Pro Glu Phe Pro Ser

1 5 10 15

Thr Ser Leu Tyr Lys Lys Ala Gly Ser Thr Met Ala Ala Gly Val Asp

20 25 30

Cys Gly Asp Gly Val Gly Ala Arg Gln His Val Phe Leu Val Ser Glu

35 40 45

Tyr Leu Lys Asp Ala Ser Lys Lys Met Lys Asn Gly Leu Met Phe Val

50 55 60

Lys Leu Val Asn Pro Cys Ser Gly Glu Gly Ala Ile Tyr Leu Phe Asn

65 70 75 80

Met Cys Leu Gln Gln Leu Phe Glu Val Lys Val Phe Lys Glu Lys His

85 90 95

His Ser Trp Phe Ile Asn Gln Ser Val Gln Ser Gly Gly Leu Leu His

100 105 110

Phe Ala Thr Pro Val Asp Pro Leu Phe Leu Leu Leu His Tyr Leu Ile

115 120 125

Lys Ala Asp Lys Glu Gly Lys Phe Gln Pro Leu Asp Gln Val Val Val

130 135 140

Asp Asn Val Phe Pro Asn Cys Ile Leu Leu Leu Lys Leu Pro Gly Leu

145 150 155 160

Glu Lys Leu Leu His His Val Thr Glu Glu Lys Gly Asn Pro Glu Ile

165 170 175

Asp Asn Lys Lys Tyr Tyr Lys Tyr Ser Lys Glu Lys Thr Leu Lys Trp

180 185 190

Leu Glu Lys Lys Val Asn Gln Thr Val Ala Ala Leu Lys Thr Asn Asn

195 200 205

Val Asn Val Ser Ser Arg Val Gln Ser Thr Ala Phe Phe Ser Gly Asp

210 215 220

Gln Ala Ser Thr Asp Lys Glu Glu Asp Tyr Ile Arg Tyr Ala His Gly

225 230 235 240

Leu Ile Ser Asp Tyr Ile Pro Lys Glu Leu Ser Asp Asp Leu Ser Lys

245 250 255

Tyr Leu Lys Pro Glu Pro Ser Ala Ser Leu Pro Asn Pro Pro Ser Lys

260 265 270

Lys Ile Lys Leu Ser Asp Glu Pro Val Glu Ala Lys Glu Asp Tyr Thr

275 280 285

Lys Phe Asn Thr Lys Asp Leu Lys Thr Glu Lys Lys Asn Ser Lys Met

290 295 300

Thr Ala Ala Gln Lys Ala Leu Ala Lys Val Asp Lys Ser Gly Met Lys

305 310 315 320

Ser Ile Asp Thr Phe Phe Gly Val Lys Asn Lys Lys Lys Ile Gly Lys

325 330 335

Val

<210>53

<211>164

<212>PRT

＜213〉artificial sequence

<220>

＜223〉the immunogen sequence of anti-RNA enzyme H2B

<400>53

Met Glu Ser Gly Asp Glu Ala Ala Ile Glu Arg His Arg Val His Leu

1 5 10 15

Arg Ser Ala Thr Leu Arg Asp Ala Val Pro Ala Thr Leu His Leu Leu

20 25 30

Pro Cys Glu Val Ala Val Asp Gly Pro Ala Pro Val Gly Arg Phe Phe

35 40 45

Thr Pro Ala Ile Arg Gln Gly Pro Glu Gly Leu Glu Val Ser Phe Arg

50 55 60

Gly Arg Cys Leu Arg Gly Glu Glu Val Ala Val Pro Pro Gly Leu Val

65 70 75 80

Gly Tyr Val Met Val Thr Glu Glu Lys Lys Val Ser Met Gly Lys Pro

85 90 95

Asp Pro Leu Arg Asp Ser Gly Thr Asp Asp Gln Glu Glu Glu Pro Leu

100 105 110

Glu Arg Asp Phe Asp Arg Phe Ile Gly Ala Thr Ala Asn Phe Ser Arg

115120125

Phe Thr Leu Trp Gly Leu Glu Thr Ile Pro Gly Pro Asp Ala Lys Val

130 135 140

Arg Gly Ala Leu Thr Trp Pro Ser Leu Ala Ala Ala Ile His Ala Gln

145 150 155 160

Val Pro Glu Asp

<210>54

<211>299

<212>PRT

＜213〉artificial sequence

<220>

＜223〉the immunogen sequence of anti-RNA enzyme H2A

<400>54

Met Asp Leu Ser Glu Leu Glu Arg Asp Asn Thr Gly Arg Cys Arg Leu

1 5 10 15

Ser Ser Pro Val Pro Ala Val Cys Arg Lys Glu Pro Cys Val Leu Gly

20 25 30

Val Asp Glu Ala Gly Arg Gly Pro Val Leu Gly Pro Met Val Tyr Ala

35 40 45

Ile Cys Tyr Cys Pro Leu Pro Arg Leu Ala Asp Leu Glu Ala Leu Lys

50 55 60

Val Ala Asp Ser Lys Thr Leu Leu Glu Ser Glu Arg Glu Arg Leu Phe

65 70 75 80

Ala Lys Met Glu Asp Thr Asp Phe Val Gly Trp Ala Leu Asp Val Leu

85 90 95

Ser Pro Asn Leu Ile Ser Thr Ser Met Leu Gly Arg Val Lys Tyr Asn

100 105 110

Leu Asn Ser Leu Ser His Asp Thr Ala Thr Gly Leu Ile Gln Tyr Ala

115 120 125

Leu Asp Gln Gly Val Asn Val Thr Gln Val Phe Val Asp Thr Val Gly

130 135 140

Met Pro Glu Thr Tyr Gln Ala Arg Leu Gln Gln Ser Phe Pro Gly Ile

145 150 155 160

Glu Val Thr Val Lys Ala Lys Ala Asp Ala Leu Tyr Pro Val Val Ser

165 170 175

Ala Ala Ser Ile Cys Ala Lys Val Ala Arg Asp Gln Ala Val Lys Lys

180 185 190

Trp Gln Phe Val Glu Lys Leu Gln Asp Leu Asp Thr Asp Tyr Gly Ser

195 200 205

Gly Tyr Pro Asn Asp Pro Lys Thr Lys Ala Trp Leu Lys Glu His Val

210 215 220

Glu Pro Val Phe Gly Phe Pro Gln Phe Val Arg Phe Ser Trp Arg Thr

225 230 235 240

Ala Gln Thr Ile Leu Glu Lys Glu Ala Glu Asp Val Ile Trp Glu Asp

245 250 255

Ser Ala Ser Glu Asn Gln Glu Gly Leu Arg Lys Ile Thr Ser Tyr Phe

260 265 270

Leu Asn Glu Gly Ser Gln Ala Arg Pro Arg Ser Ser His Arg Tyr Phe

275 280 285

Leu Glu Arg Gly Leu Glu Ser Ala Thr Ser Leu

290 295

<210>55

<211>26

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>55

Leu Glu Thr Ile Pro Gly Pro Asp Ala Lys Val Arg Gly Ala Leu Thr

1 5 10 15

Trp Pro Ser Leu Ala Ala Ala Ile His Ala

20 25

<210>56

<211>26

<212>PRT

＜213〉European ox (Bos taurus)

<400>56

Leu Glu Thr Ile Pro Gly Pro Asp Ala Lys Val Arg Gly Ala Leu Thr

1 5 10 15

Trp Pro Ser Leu Ala Ala Ala Ile His Ala

20 25

<210>57

<211>26

<212>PRT

＜213〉domesticated dog (Canis familiaris)

<400>57

Leu Glu Ser Ile Pro Gly Pro Asp Ala Lys Leu Arg Gly Ala Leu Ser

1 5 10 15

Trp Pro Ser Leu Ala Ala Ala Ile His Ala

20 25

<210>58

<211>26

<212>PRT

＜213〉house mouse (Mus musculus)

<400>58

Leu Glu Thr Val Pro Gly Pro Asp Ala Lys Val His Arg Ala Leu Gly

1 5 10 15

Trp Pro Ser Leu Ala Ala Ala Ile His Ala

20 25

<210>59

<211>26

<212>PRT

＜213〉Rattus norvegicus (Rattus norvegicus)

<400>59

Leu Glu Thr Val Pro Gly Pro Asp Ala Lys Val His Arg Ala Leu Gly

1 5 10 15

Trp Pro Ser Leu Ala Ala Ala Asp Asp Ser

20 25

<210>60

<211>19

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>60

Leu Glu Val Ser Phe Arg Gly Arg Cys Leu Arg Gly Glu Glu Val Ala

1 5 10 15

Val Pro Pro

<210>61

<211>19

<212>PRT

＜213〉European ox (Bos taurus)

<400>61

Leu Glu Ala Ser Phe Arg Gly Arg Ser Leu Arg Gly Glu Glu Val Val

1 5 10 15

Val Pro Pro

<210>62

<211>19

<212>PRT

＜213〉domesticated dog (Canis familiaris)

<400>62

Leu Glu Val Ser Phe Arg Gly Arg Lys Leu Arg Gly Glu Glu Val Val

1 5 10 15

Val Pro Pro

<210>63

<211>19

<212>PRT

＜213〉house mouse (Mus musculus)

<400>63

Leu Gln Ala Ser Phe Arg Gly Arg Gly Leu Arg Gly Glu Glu Val Ala

1 5 10 15

Val Pro Pro

<210>64

<211>19

<212>PRT

＜213〉Rattus norvegicus (Rattus norvegicus)

<400>64

Leu Gln Val Ser Phe Arg Gly Arg Gly Leu Arg Gly Glu Asp Val Ala

1 5 10 15

Val Pro Pro

<210>65

<211>14

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>65

atgccaaagt gcgt 14

<210>66

<211>14

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>66

atgccatagt gcgt 14

<210>67

<211>13

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>67

tgtctacggg gag 13

<210>68

<211>14

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>68

tgtctatggg gaga 14

<210>69

<211>17

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>69

Val Leu Gly Val Asp Glu Ala Gly Arg Gly Pro Val Leu Gly Pro Met

1 5 10 15

Val

<210>70

<211>17

<212>PRT

＜213〉domesticated dog (Canis familiaris)

<400>70

Val Leu Gly Val Asp Glu Ala Gly Arg Gly Pro Val Leu Gly Pro Met

1 5 10 15

Val

<210>71

<211>17

<212>PRT

＜213〉house mouse (Mus musculus)

<400>71

Val Leu Gly Val Asp Glu Ala Gly Arg Gly Pro Val Leu Gly Pro Met

1 5 10 15

Val

<210>72

<211>17

<212>PRT

＜213〉Rattus norvegicus (Rattus norvegicus)

<400>72

Val Leu Gly Val Asp Glu Ala Gly Arg Gly Pro Val Leu Gly Pro Met

1 5 10 15

Val

<210>73

<211>17

<212>PRT

＜213〉drosophila melanogaster (Drosophila melanogaster)

<400>73

Met Leu Gly Val Asp Glu Ala Gly Arg Gly Pro Val Leu Gly Pro Met

1 5 10 15

Val

<210>74

<211>17

<212>PRT

＜213〉beautiful new rhabditis axei (Caenorhabditis elegans)

<400>74

Val Leu Gly Ile Asp Glu Ala Gly Arg Gly Pro Val Leu Gly Pro Met

1 5 10 15

Val

<210>75

<211>17

<212>PRT

＜213〉fission yeast mycelia (Schizosaccharomyces pombe)

<400>75

Arg Leu Gly Val Asp Glu Ala Gly Arg Gly Pro Val Leu Gly Pro Met

1 5 10 15

Val

<210>76

<211>17

<212>PRT

＜213〉S. cervisiae (Saccharomyces cerevisiae)

<400>76

Ile Met Gly Ile Asp Glu Ala Gly Arg Gly Pro Val Leu Gly Pro Met

1 5 10 15

Val

<210>77

<211>17

<212>PRT

＜213〉intestinal bacteria (Escherichia coli)

<400>77

Val Ala Gly Val Asp Glu Val Gly Arg Gly Pro Leu Val Gly Ala Val

1 5 10 15

Val

<210>78

<211>17

<212>PRT

＜213〉super hyperthermophilic archaeon strain (Pyrococcus horikoshii)

<400>78

Val Ala Gly Val Asp Glu Ala Gly Arg Gly Pro Val Ile Gly Pro Leu

1 5 10 15

Val

<210>79

<211>16

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>79

tgaggcgggc aggggc 16

<210>80

<211>16

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>80

tgaggcgagc aggggc 16

<210>81

<211>18

<212>RNA

＜213〉artificial sequence

<220>

＜223〉3 of chemosynthesis ' end is with fluorescently-labeled oligonucleotide, band 2-O methyl RNA Nucleotide.Oligo A

Part, the RNA:DNA heterozygote

<400>81

gaucugagcc ugggagcu 18

<210>82

<211>18

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the NDA oligonucleotide of chemosynthesis, the part of Oligo A, RNA:DNA heterozygote.

<400>82

ctagactcgg accctcga 18

<210>83

<211>18

<212>DNA

＜213〉artificial sequence

<220>

＜223〉oligonucleotide chain of chemosynthesis (Oligo B), 3 ' end fluorescent mark

<400>83

gatctgagcc tgggagct 18

<210>84

<211>18

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the DNA oligonucleotide of chemosynthesis, the part of Oligo B, RNA:DNA heterozygote.

<400>84

ctagactcgg accctcga 18

<210>85

<211>18

<212>RNA

＜213〉artificial sequence

<220>

＜223〉3 ' end fluorescently-labeled RNA oligonucleotide (part of Oligo C)

<400>85

gaucugagcc ugggagcu 18

<210>86

<211>18

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the DNA oligonucleotide (part of Oligo C) of 5 ' end DABCYL mark

<400>86

ctagactcgg accctcga 18

<210>87

<211>18

<212>RNA

＜213〉artificial sequence

<220>

＜223〉NDA oligonucleotide (the double-stranded fragment of RNA:DNA)

<400>87

gaucugagcc ugggagcu 18

<210>88

<211>18

<212>DNA

＜213〉artificial sequence

<220>

＜223〉NDA oligonucleotide (the double-stranded fragment of RNA:DNA)

<400>88

ctagactcgg accctcga 18

<210>89

<211>18

<212>DNA

＜213〉artificial sequence

<220>

＜223〉anti-RNA enzyme H degraded product

<400>89

ctagactcgg accctcga 18

<210>90

<211>13

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>90

Arg Gln His Val Phe Leu Val Ser Glu Tyr Leu Lys Asp

1 5 10

<210>91

<211>152

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>91

Gly Leu Met Phe Val Lys Leu Val Asn Pro Cys Ser Gly Glu Gly Ala

1 5 10 15

Ile Tyr Leu Phe Asn Met Cys Leu Gln Gln Leu Leu Glu Val Lys Val

20 25 30

Phe Lys Glu Lys His His Ser Trp Phe Ile Asn Gln Ser Val Gln Ser

35 40 45

Gly Gly Leu Leu His Phe Ala Thr Pro Val Asp Pro Leu Phe Leu Leu

50 55 60

Leu His Tyr Leu Ile Lys Ala Asp Lys Glu Gly Lys Phe Gln Pro Leu

65 70 75 80

Asp Gln Val Val Val Asp Asn Val Phe Pro Asn Cys Ile Leu Leu Leu

85 90 95

Lys Leu Pro Gly Leu Glu Lys Leu Leu His His Val Thr Glu Glu Lys

100 105 110

Gly Asn Pro Glu Ile Asp Asn Lys Lys Tyr Tyr Lys Tyr Ser Lys Glu

115 120 125

Lys Thr Leu Lys Trp Leu Glu Lys Lys Val Asn Gln Thr Val Ala Ala

130 135 140

Leu Lys Thr Asn Asn Val Asn Val

145 150

<210>92

<211>5

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>92

Phe Ser Gly Asp Gln

1 5

<210>93

<211>36

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>93

Glu Glu Asp Tyr Ile Arg Tyr Ala His Gly Leu Ile Ser Asp Tyr Ile

1 5 10 15

Pro Lys Glu Leu Ser Asp Asp Leu Ser Lys Tyr Leu Lys Leu Pro Glu

20 25 30

Pro Ser Ala Ser

35

<210>94

<211>23

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>94

Ser Lys Lys Ile Lys Leu Ser Asp Glu Pro Val Glu Ala Lys Glu Asp

1 5 10 15

Tyr Thr Lys Phe Asn Thr Lys

20

<210>95

<211>31

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>95

Lys Lys Asn Ser Lys Met Thr Ala Ala Gln Lys Ala Leu Ala Lys Val

1 5 10 15

Asp Lys Ser Gly Met Lys Ser Ile Asp Thr Phe Phe Gly Val Lys

20 25 30

<210>96

<211>13

<212>PRT

＜213〉Rattus norvegicus (Rattus norvegicus)

<400>96

Arg Gln Leu Val Phe Leu Leu Pro Glu Tyr Leu Lys Asp

1 5 10

<210>97

<211>149

<212>PRT

＜213〉Rattus norvegicus (Rattus norvegicus)

<400>97

Gly Leu Leu Phe Val Lys Leu Val Asn Pro His Ser Gly Glu Gly Ala

1 5 10 15

Thr Tyr Leu Ile Asp Ala Cys Leu Gln Lys Leu Phe Glu Ile Lys Val

20 25 30

Phe Lys Glu Lys His His Ser Trp Phe Ile Asn Gln Ser Val Gln Ser

35 40 45

Gly Gly Leu Leu His Phe Ala Thr Pro Met Asp Pro Leu Phe Leu Leu

50 55 60

Leu His Tyr Leu Ile Lys Ala Gly Lys Glu Gly Lys Tyr Gln Pro Leu

65 70 75 80

Asp Gln Val Val Val Asp Asp Lys Phe Pro Asp Cys Thr Leu Leu Leu

85 90 95

Arg Phe Pro Glu Leu Glu Lys Ser Leu Arg His Val Thr Glu Glu Lys

100 105 110

Glu Val Asn Ser Lys Lys Tyr Tyr Lys Tyr Ser Thr Glu Lys Thr Leu

115 120 125

Lys Trp Leu Glu Lys Lys Val Asn Gln Thr Val Ala Ala Leu Lys Ala

130 135 140

Asn His Val Asn Val

145

<210>98

<211>5

<212>PRT

＜213〉Rattus norvegicus (Rattus norvegicus)

<400>98

Phe Ser Gly Val Gln

1 5

<210>99

<211>36

<212>PRT

＜213〉Rattus norvegicus (Rattus norvegicus)

<400>99

Glu Glu Asp Tyr Val Arg Tyr Ala His Gly Leu Ile Ser Asp Tyr Ile

1 5 10 15

Pro Lys Glu Leu Ser Asp Asp Leu Ser Lys Leu Leu Lys Leu Pro Glu

20 25 30

Pro Pro Ala Ser

35

<210>100

<211>23

<212>PRT

＜213〉Rattus norvegicus (Rattus norvegicus)

<400>100

Ala Lys Lys Leu Lys Leu Ala Asp Glu Pro Val Glu Ala Lys Glu Asp

1 5 10 15

Tyr Thr Lys Phe Asn Thr Lys

20

<210>101

<211>31

<212>PRT

＜213〉Rattus norvegicus (Rattus norvegicus)

<400>101

Lys Lys Asn Ser Lys Met Thr Ala Ala Gln Lys Ala Leu Ala Lys Val

1 5 10 15

Asp Lys Ser Gly Met Lys Ser Ile Asp Ala Phe Phe Gly Ala Lys

20 25 30

<210>102

<211>13

<212>PRT

＜213〉house mouse (Mus musculus)

<400>102

Arg Gln Leu Val Phe Leu Leu Pro Glu His Leu Lys Asp

1 5 10

<210>103

<211>149

<212>PRT

＜213〉house mouse (Mus musculus)

<400>103

Ser Leu Leu Phe Val Lys Leu Ala Asn Pro His Ser Gly Glu Gly Ala

1 5 10 15

Thr Tyr Leu Ile Asp Met Cys Leu Gln Gln Leu Phe Glu Ile Lys Val

20 25 30

Phe Lys Glu Lys His His Ser Trp Phe Ile Asn Gln Ser Val Gln Ser

35 40 45

Gly Gly Leu Leu His Phe Ala Thr Pro Met Asp Pro Leu Phe Leu Leu

50 55 60

Leu His Tyr Leu Leu Lys Ala Gly Lys Glu Gly Lys Tyr Gln Pro Leu

65 70 75 80

Asp Gln Val Val Val Asp Asp Thr Phe Pro Asp Cys Thr Leu Leu Leu

85 90 95

Arg Phe Pro Glu Leu Glu Lys Ser Leu Arg His Val Thr Glu Glu Lys

100 105 110

Glu Val Asn Ser Lys Lys Tyr Tyr Lys Tyr Ser Ser Glu Lys Thr Leu

115 120 125

Lys Trp Leu Glu Lys Lys Val Asn Gln Thr Val Val Ala Leu Lys Ala

130 135 140

Asn Asn Val Asn Val

145

<210>104

<211>5

<212>PRT

＜213〉house mouse (Mus musculus)

<400>104

Phe Ser Gly Gly Gln

1 5

<210>105

<211>36

<212>PRT

＜213〉house mouse (Mus musculus)

<400>105

Glu Glu Asp Tyr Val Arg Tyr Ala His Gly Leu Ile Ser Asp Tyr Ile

1 5 10 15

Pro Lys Glu Leu Ser Asp Asp Leu Ser Lys Phe Leu Lys Leu Pro Glu

20 25 30

Pro Pro Ala Ser

35

<210>106

<211>23

<212>PRT

＜213〉house mouse (Mus musculus)

<400>106

Ser Lys Lys Leu Lys Leu Ser Asp Glu Pro Val Glu Ala Lys Glu Asp

1 5 10 15

Tyr Thr Lys Phe Asn Thr Lys

20

<210>107

<211>31

<212>PRT

＜213〉house mouse (Mus musculus)

<400>107

Lys Lys Asn Ser Lys Met Thr Ala Ala Gln Lys Ala Leu Ala Lys Val

1 5 10 15

Asp Lys Ser Gly Met Lys Ser Ile Asp Ala Phe Phe Gly Ala Lys

20 25 30

<210>108

<211>13

<212>PRT

＜213〉xenopus (Xenopus tropicalis)

<400>108

Glu Gln Trp Val Leu Leu Ala Pro Glu Ser Leu Ser Asp

1 5 10

<210>109

<211>150

<212>PRT

＜213〉xenopus (Xenopus tropicalis)

<400>109

Lys Thr Ile Phe Ala Lys Leu Arg Ala Pro Cys Thr Asp Lys Gly Ser

1 5 10 15

Met Phe Leu Phe Ile Asp Ser Gly Gln Gln Ile Cys Glu Val Lys Ala

20 25 30

Phe His Glu Glu Tyr Arg Ser Trp Phe Ile Gly Gln Thr Val Gln Gln

35 40 45

Asp Gly Arg Leu Leu Ile Ala Thr Pro Ile Asp Pro Met Phe Leu Val

50 55 60

Leu His Tyr Leu Ile Lys Ala Asp Lys Glu Gln Gly Lys Phe Gln Pro

65 70 75 80

Val Glu Gln Ile Val Val Asp Glu Glu Phe Pro Ser Cys Ser Met Leu

85 90 95

Leu Gln Cys Thr Pro Val Ser Lys Ser Leu His His Val Thr Glu Glu

100 105 110

Lys Glu Ile Gly Ser Lys Lys Phe Tyr Lys Tyr Ser Lys Glu Lys Thr

115 120 125

Leu Val Trp Leu Lys Lys Lys Val Glu Leu Thr Val Lys Val Leu Lys

130 135 140

Ser Ser Asn Ile Cys Val

145 150

<210>110

<211>5

<212>PRT

＜213〉xenopus (Xenopus tropicalis)

<400>110

Ile Arg Asn Thr Gln

1 5

<210>111

<211>36

<212>PRT

＜213〉xenopus (Xenopus tropicalis)

<400>111

Glu Glu Asp Tyr Thr Arg Tyr Ala His Gly Leu Ile Ser Glu Tyr Leu

1 5 10 15

Thr Glu Asp Leu Arg Glu Asp Leu Ser Lys Tyr Leu Gly Leu Pro Asp

20 25 30

Leu Ser Ser Pro

35

<210>112

<211>58

<212>PRT

＜213〉xenopus (Xenopus tropicalis)

<400>112

Val Lys Lys Arg Lys Val Ser Glu Ala Pro Val Glu Ala Glu Glu Asp

1 5 10 15

Tyr Thr Lys Phe Asn Ser Asp Ser Lys Asn Lys Lys Ser Asn Ser Lys

20 25 30

Met Thr Ala Ala Gln Lys Ser Leu Ala Lys Val Asp Lys Ser Gly Met

35 40 45

Lys Asn Ile Ser Ala Phe Phe Ser Pro Lys

50 55

<210>113

<211>13

<212>PRT

＜213〉the green filefish (Tetraodon nigroviridis) of spot

<400>113

Asp Ser Trp Val Val Ile Ala Ala Asp Ser Ala Val Asp

1 5 10

<210>114

<211>149

<212>PRT

＜213〉the green filefish (Tetraodon nigroviridis) of spot

<400>114

Asp Pro Ser Leu Val Thr Leu Arg Asn Pro Ser Thr Gly Ala Gly Ser

1 5 10 15

Leu Tyr Met Leu Ser Ser Gly Asp Trp Arg Leu Phe Glu Val Lys Ala

20 25 30

Phe Glu Glu Asp Phe His Ser Trp Phe Val Gly Gln Ala Val Gln Arg

35 40 45

Asp Gly Lys Leu Leu Phe Ile Thr Pro Met Asp Pro Leu Tyr Leu Ile

50 55 60

Leu Pro Tyr Leu Met Lys Ser Gly Ile Glu Gly Lys Phe Gln Pro Ala

65 70 75 80

Asp Gln Val Val Met Asp Glu Glu Phe Pro Ala Cys Ser Arg Leu Leu

85 90 95

Gly Cys Thr Arg Ser Leu Ala Ser Leu His His Val Thr Glu Glu Lys

100 105 110

Glu Val Gly Asn Leu Lys Phe His Arg Tyr Ser Gln Glu Lys Thr Leu

115 120 125

Ser Trp Leu Arg Lys Lys Val Glu Arg Thr Val Ala Ala Leu Lys Lys

130 135 140

Arg Ser Ile Ser Val

145

<210>115

<211>5

<212>PRT

＜213〉the green filefish (Tetraodon nigroviridis) of spot

<400>115

Ile Arg Ala Lys Pro

1 5

<210>116

<211>36

<212>PRT

＜213〉the green filefish (Tetraodon nigroviridis) of spot

<400>116

Gln Glu Asp Tyr Leu Arg Tyr Ala His Gly Leu Ile Ser Glu Tyr Ile

1 5 10 15

Ser Glu Asp Leu Ser Lys Ala Leu Tyr Lys His Leu Glu Leu Pro Glu

20 25 30

Ile Ser Ser Pro

35

<210>117

<211>58

<212>PRT

＜213〉the green filefish (Tetraodon nigroviridis) of spot

<400>117

Ser Lys Lys Arg Lys Leu Ser Asp Lys Ser Val Glu Ala Glu Glu Asp

1 5 10 15

Tyr Thr Lys Phe Asn Ser Ala Asp Phe Ala Arg Lys Pro Pro Lys Lys

20 25 30

Met Thr Ala Ala Gln Lys Ser Leu Ala Lys Val Asp Lys Thr Gly Met

35 40 45

Lys Pro Met Ser Ser Phe Phe Ser Pro Lys

50 55

<210>118

<211>14

<212>PRT

＜213〉honeybee (Apis mellifera)

<400>118

Asn Thr Trp Val Phe Leu Met Lys Gly Asp Asn Leu Ser Asn

1 5 10

<210>119

<211>144

<212>PRT

＜213〉honeybee (Apis mellifera)

<400>119

Val Pro Asp Val Ile Lys Leu Arg His Pro Ala Thr Asn Gln Pro Ala

1 5 10 15

Met Phe Val Phe Ser Pro Gly Asn Val Thr Val Gln Glu Val Leu Thr

20 25 30

Phe Asp Glu Asn Lys Arg Ser Trp Phe Ile Asp Asp Asn Val Lys Ser

35 40 45

Asp Gly Lys Leu His Leu Ser Thr Pro Ile Asp Pro Ile Phe Leu Ile

50 55 60

Leu Pro Tyr Leu Arg Lys Ser Gln Gln Ala Gln Pro Leu Glu Gln Cys

65 70 75 80

Leu Trp Asp Glu Asp Phe Pro Glu Met Thr Arg Leu Thr Gln Cys Gln

85 90 95

Asn Leu Lys Leu Ile Leu Ile Ala Asp Arg Lys Gly Asp Glu Ser Leu

100 105 110

Gln Ala Tyr Lys Phe Asn Glu Glu Lys Thr Leu Ile Trp Leu Arg Lys

115 120 125

Lys Val Glu Arg Val Ile Glu Val Leu Lys Gln Lys Gly Ile His Val

130 135 140

<210>120

<211>5

<212>PRT

＜213〉honeybee (Apis mellifera)

<400>120

Val Lys Ser Thr Lys

1 5

<210>121

<211>58

<212>PRT

＜213〉honeybee (Apis mellifera)

<400>121

Glu Met Glu Tyr Leu Lys Tyr Ala His Gly Ile Val Ser Glu Tyr Leu

1 5 10 15

Ala Glu Asp Leu Ser Lys Lys Leu Ala Gln Tyr Leu Asn Ile Pro Asp

20 25 30

Glu Thr Glu Asn Lys Lys Arg Lys Leu Asn Ser Pro Lys Asp Asn Gly

35 40 45

Asp Glu Lys Arg Ser Lys Lys Asp Thr Ser

50 55

<210>122

<211>15

<212>PRT

＜213〉honeybee (Apis mellifera)

<400>122

Pro Glu Ser Thr Lys Leu Glu Lys Ser Gln Lys Thr Ile Gly Lys

1 5 10 15

<210>123

<211>15

<212>PRT

＜213〉honeybee (Apis mellifera)

<400>123

Ala Ala Ser Gly Ser Lys Ser Ile Thr Ser Phe Phe Lys Lys Lys

1 5 10 15

<210>124

<211>13

<212>PRT

＜213〉drosophila melanogaster (Drosophila melanogaster)

<400>124

Leu Lys Lys Val Phe Phe Met Ser Glu Glu Leu Leu Pro

1 5 10

<210>125

<211>145

<212>PRT

＜213〉drosophila melanogaster (Drosophila melanogaster)

<400>125

His Leu Arg Leu Glu Met Phe Phe His Pro Gly His Gly Lys Glu Ala

1 5 10 15

Leu Phe Ile Thr His Pro Asp Gly Arg Met Met Glu Leu Val Ala Phe

20 25 30

Thr Glu Pro Arg Arg Ser Trp Phe Val Asp Ser Glu Val Cys Ser Asn

35 40 45

Gly Arg Ile Tyr Met Thr Ala Pro Val Asp Pro Thr Phe Leu Ala Leu

50 55 60

His His Leu Arg Lys His Cys Ala Gln Arg Ala Ile Ser Leu Asp Asn

65 70 75 80

Ile Ala Val Glu Glu Ala Ser Thr Ser Arg Leu Leu Asn Glu Ile Leu

85 90 95

Asp Pro Gly Asn Leu Lys Cys Val Ala Asp Val Lys Ser Ser Gly Glu

100 105 110

Gln Lys Phe Tyr Lys Tyr Asn Gln Glu Arg Thr Leu Ala Trp Leu Ala

115 120 125

Leu Lys Thr Arg Gln Val Ala Lys Ile Leu Lys Glu Lys Gln Val His

130 135 140

Cys

145

<210>126

<211>5

<212>PRT

＜213〉drosophila melanogaster (Drosophila melanogaster)

<400>126

Val Arg Ser Glu Lys

1 5

<210>127

<211>36

<212>PRT

＜213〉drosophila melanogaster (Drosophila melanogaster)

<400>127

Glu Met Asp Tyr Thr Arg Met Ala Cys Asp Tyr Val Gly Arg Tyr Leu

1 5 10 15

Gly Ala Asp Leu His Gly Leu Leu Thr Ser Tyr Leu His Ile Pro Ser

20 25 30

Glu Ile Gln Ala

35

<210>128

<211>24

<212>PRT

＜213〉drosophila melanogaster (Drosophila melanogaster)

<400>128

Ser Gln Lys Arg Lys Ser Val Ala Gly Lys Asn Glu Gly Ser Asp Ser

1 5 10 15

Lys Lys Ile Lys Leu Asn Glu Ser

20

<210>129

<211>30

<212>PRT

＜213〉drosophila melanogaster (Drosophila melanogaster)

<400>129

Leu Lys Glu Arg Ser Leu Thr Ala Lys Glu Lys Ala Leu Ala Lys Gly

1 5 10 15

Ala Lys Gly Thr Lys Ser Ile Ala Ser Phe Phe Lys Ala Lys

20 25 30

<210>130

<211>155

<212>PRT

＜213〉beautiful new rhabditis axei (Caenorhabditis elegans)

<400>130

Ser Arg Lys Val Val Ile Ser Lys Glu Gly Asn Leu Pro Asn Ser Arg

1 5 10 15

Ile Ile Lys Leu Arg His Pro Lys Glu Gly Leu Ala Leu Phe Arg Ile

20 25 30

Ser Glu Thr Cys Phe Asp Glu Ile Phe Ala Val Asn Asp Gly His Arg

35 40 45

Ser Phe Phe Tyr Gly Glu Ser Val Ile Glu Asn Gly Thr Ile His Ile

50 55 60

Phe Thr Pro Tyr Asn Leu Val Phe Ile Cys Leu Pro Tyr Leu Gln Lys

65 70 75 80

Thr Ala Gly Lys Phe Val Glu Ile Gly Glu Ile Leu Val Asp Asp Gln

85 90 95

Val Asp Gly Ile Lys Glu Leu Ala Glu Asn Glu Arg Ile Leu Lys Ala

100 105 110

Leu Glu Ala Val Ser Asp Val Lys Asp Val Leu Asp Val Lys Leu Tyr

115 120 125

Arg Leu Asn Asp Gln Lys Met Leu Asp Trp Met Arg Arg Lys Phe Asp

130 135 140

Ser Leu Lys Lys Leu Leu Glu Ser Asp Ala His

145 150 155

<210>131

<211>90

<212>PRT

＜213〉beautiful new rhabditis axei (Caenorhabditis elegans)

<400>131

Pro Glu Ala Phe Asp Arg Tyr Val Phe Thr Phe Leu Ser Glu Tyr Leu

1 5 10 15

Ser Ser Asp Leu Ala Leu Ile Val Lys Asp His Leu Asn Ile Gln Asp

20 25 30

Ala Thr Val Ser Glu Asn Ile Asp Met Ser Met Lys Arg Lys Ala Val

35 40 45

Asp Asp Asp Glu Leu Glu Asp Lys Lys Gln Pro Val Ala Lys Lys Pro

50 55 60

Lys Glu Ser Val Gln Ser Lys Lys Leu Gln Ala Ala Ser Lys Gly Thr

65 70 75 80

Lys Ser Ile Ser Ser Phe Phe Gly Lys Lys

85 90

<210>132

<211>85

<212>PRT

＜213〉paddy rice (Oryza sativa)

<400>132

Pro Pro Arg Leu Leu Val Ala Pro Arg Pro Ser Asp Gly Asn Cys Gln

1 5 10 15

Gly Asn Val Leu Ser Leu Arg His Pro Arg Ser Asp Glu Glu Thr Gly

20 25 30

Tyr Leu Phe Ile Asp Gly Gln Leu His Glu Phe Asn Trp Phe Lys Glu

35 40 45

Arg Phe Gly Ser Trp Phe Leu Gly Asp Tyr Val Cys Glu Asp Gly Ser

50 55 60

Leu Tyr Tyr Cys Thr Val Val Asp Pro Ile Phe Ile Leu Leu Pro Ile

65 70 75 80

Leu Lys Ala Ala Arg

85

<210>133

<211>74

<212>PRT

＜213〉paddy rice (Oryza sativa)

<400>133

Pro Gly Lys Phe Arg Gln Leu Asp Glu Ile Leu Tyr Val Glu Gly Tyr

1 5 10 15

Pro Gly Tyr Gln His Leu Met Gly Ile Ala Gly Asn His Ile Asp Leu

20 25 30

Val Cys Glu Val Lys Glu Val Ala Asn Val Lys Phe Phe Arg Leu Asp

35 40 45

Asp Ser Lys Val Leu Ser Trp Leu Cys Cys Lys Val His Asn Leu Lys

50 55 60

Glu Val Phe Pro Lys Leu Gly Lys Asn Tyr

65 70

<210>134

<211>72

<212>PRT

＜213〉paddy rice (Oryza sativa)

<400>134

Glu Lys Glu Leu Leu Lys Asp Ala Val Gln Ile Ile Arg Glu Tyr Leu

1 5 10 15

Asn Asp Glu Pro Trp Leu Thr Leu Leu Cys Lys Lys Leu Gln Leu Asp

20 25 30

Ile Lys Glu Ile Ile Glu Ala Asn Lys Thr Ser Glu Ala Ser Phe Cys

35 40 45

Ala Glu Asn Ser Pro Val Pro Phe His Pro Ala Glu Glu Lys Leu Gly

50 55 60

Ser Ser Ser Thr Arg Ser Ser Lys

65 70

<210>135

<211>15

<212>PRT

＜213〉paddy rice (Oryza sativa)

<400>135

Ala Glu Val Glu Ser Lys Asn Ile Lys Asp Met Phe Arg Arg Val

1 5 10 15

<210>136

<211>13

<212>PRT

＜213〉discoideum net post bacterium (Dictyostelium discoideum)

<400>136

Asn Asp Arg Val Phe Ile Ile Gln Gln Pro Lys Asn Asp

1 5 10

<210>137

<211>149

<212>PRT

＜213〉discoideum net post bacterium (Dictyostelium discoideum)

<400>137

Asp Tyr Glu Ile Leu Thr Leu Pro Ser Pro Lys His Lys Ser Val Lys

1 5 10 15

Cys Arg Tyr Ile Leu Asp Lys Glu Asn Asp Arg Ile Leu Glu Ile Asn

20 25 30

Lys Phe Asn Ser Lys Pro Ser Ser Trp Phe Ile Asp Asn Gly Val Arg

35 40 45

His Asp Gly Ser Met Tyr Leu Ser Ser Asp Ile Asp Pro Leu Phe Leu

50 55 60

Leu Ile Pro Phe Leu Glu Lys Tyr Lys Ser Leu His Lys Asn Glu Tyr

65 70 75 80

Leu Glu Ile Ser Ser Ile Ile Asn Asp Ser Tyr Tyr Cys Asn Leu Ser

85 90 95

Lys Ile Pro Phe Lys His Ser Gln Leu Ser Leu Ile Cys Asp Ser Lys

100 105 110

Glu Ile Ala Gly Ser Lys Leu Tyr Arg Leu Glu Asp Glu Lys Leu Leu

115 120 125

Ile Trp Leu Arg Cys Lys Val Lys Asn Ile Ser Asn Tyr Leu Lys Glu

130 135 140

Thr Asn Thr Asp Ile

145

<210>138

<211>94

<212>PRT

＜213〉discoideum net post bacterium (Dictyostelium discoideum)

<400>138

Val Lys Lys Asp Leu Ser Trp Glu Thr Leu Tyr Thr Met Ser Ile Gly

1 5 10 15

Phe Val Ser Glu Tyr Leu Ser Glu Ser Asn Cys Lys Met Leu Asn Gln

20 25 30

Ser Phe Asn Leu Leu Ser Ala Thr Ala Gln Gln Thr Ile Lys Ser Glu

35 40 45

Ser Glu Tyr Leu Val Tyr Thr Glu Arg Ile Ile Asp Glu Pro Glu Pro

50 55 60

Pro Lys Ser Lys Arg Gly Ala Ser Lys Lys Ala Pro Ala Lys Lys Ser

65 70 75 80

Pro Val Lys Gly Gly Gly Ile Ser Asn Phe Leu Ser Leu Lys

85 90

<210>139

<211>101

<212>PRT

＜213〉fission yeast mycelia (Schizosaccharomyces pombe)

<400>139

Gln Gly Lys Ile Phe Ile Leu Pro Lys Asp Ser Thr Asn Gln Gln Phe

1 5 10 15

Val Glu Leu Ser His Pro Leu Thr Gly Arg Pro Leu Arg Tyr Leu Leu

20 25 30

Thr Asn Asp His Leu Leu Gln Ile Leu Gln Val Gly Asp Ser Ser Lys

35 40 45

Gln Arg Ser Trp Phe Val Gly Asp His Val Val Ser Asp Gly Tyr Leu

50 55 60

Tyr Val Cys Thr Pro Ile Asp Leu Leu Ala Leu Val Leu Pro Ile Ile

65 70 75 80

Gln Glu Leu Thr Trp Ser Arg Arg Lys Glu Pro Asn Arg Tyr Val Ser

85 90 95

Phe Glu Asp Phe Ile

100

<210>140

<211>60

<212>PRT

＜213〉fission yeast mycelia (Schizosaccharomyces pombe)

<400>140

Arg Val Ser Glu Val Leu Ser Pro Asn Leu Leu Asn Thr Leu His Arg

1 5 10 15

Ile Cys Lys Val Asn Glu Pro Val Gly Ser Leu Pro Lys Thr Phe Gln

20 25 30

Leu Asp Glu Ser Ser Val Val Lys Ile Leu Leu Arg Lys Ala Lys Val

35 40 45

Ala Gln Glu Asn Leu Pro Pro Ser Ile Val Thr Glu

50 55 60

<210>141

<211>5

<212>PRT

＜213〉fission yeast mycelia (Schizosaccharomyces pombe)

<400>141

Pro Leu Asp Leu Arg

1 5

<210>142

<211>59

<212>PRT

＜213〉fission yeast mycelia (Schizosaccharomyces pombe)

<400>142

Glu Leu Ser Cys Lys Trp His Ala Ala Ser Leu Val Cys Glu Asp Leu

1 5 10 15

Gln Pro Glu Trp Tyr Asn Lys Leu Ala Tyr Trp Gln Glu Glu Leu Ala

20 25 30

Pro Leu His Ala Tyr Thr Lys Asn Leu Glu Glu Ser Arg Lys Ile Leu

35 40 45

Val Glu Lys Glu Ala Leu Leu Asn Ser Lys Lys

50 55

<210>143

<211>16

<212>PRT

＜213〉fission yeast mycelia (Schizosaccharomyces pombe)

<400>143

Asp Ile Thr Ser Ser Leu Leu Lys Lys Pro Asn Arg Lys Gln Ala Thr

1 5 10 15

<210>144

<211>15

<212>PRT

＜213〉fission yeast mycelia (Schizosaccharomyces pombe)

<400>144

Ser Gly Glu Gly Met Thr Lys Ile Ser Ser Phe Phe Thr Lys Lys

1 5 10 15

<210>145

<211>52

<212>PRT

＜213〉S. cervisiae (Saccharomyces cerevisiae)

<400>145

Glu Glu Arg Leu Ile Ile Leu Pro Asp Asp Tyr Glu Thr Ser Lys Thr

1 5 10 15

Ile Asn Thr Phe Thr Leu Pro Pro Pro Ser Asn Ile Thr Ser Lys Pro

20 25 30

Arg Ile Glu Leu Phe Glu Asn Ile Asn Gly Lys Leu Tyr Glu Ile Arg

35 40 45

Ser Phe Gln Phe

50

<210>146

<211>21

<212>PRT

＜213〉S. cervisiae (Saccharomyces cerevisiae)

<400>146

Ile Lys Ser Thr Phe Ile Val Asn Thr Ser Asp Pro Thr Asp Gly Tyr

1 5 10 15

Val Phe Asn Ser Ser

20

<210>147

<211>16

<212>PRT

＜213〉S. cervisiae (Saccharomyces cerevisiae)

<400>147

Leu Tyr Asp Ile Ala Phe Ser Leu Ile Gly Phe Tyr Tyr Arg Asn Ser

1 5 10 15

<210>148

<211>15

<212>PRT

＜213〉S. cervisiae (Saccharomyces cerevisiae)

<400>148

Asn Ser Lys Thr Asn Glu Lys Phe Leu Thr Val Arg Asp Tyr His

1 5 10 15

<210>149

<211>104

<212>PRT

＜213〉S. cervisiae (Saccharomyces cerevisiae)

<400>149

Asp Lys Asn Trp Glu Asn Ile Ser Leu Ser Arg Leu Lys Ser Gly Leu

1 5 10 15

Ala Lys Val Ser Glu Thr Ile Glu Glu Ala Gly Asp Val Tyr Tyr Lys

20 25 30

Ile Thr Ser Ala Met Ile Thr Gln Phe Leu Leu Gly Lys Val Ser Lys

35 40 45

Ile Val Glu Asn Phe Pro Pro Ser Ile Pro Thr Leu Lys Asn Ala Pro

50 55 60

Thr Glu Ile Lys Gln Cys Tyr Lys Val Val Met Ala Thr Asn Leu Leu

65 70 75 80

Val Ser Leu Ile Pro Arg Ala Ala Tyr His Asn Leu Leu Thr Phe Ser

85 90 95

Pro Thr Met Asp Ser Gly Cys Leu

100

<210>150

<211>22

<212>PRT

＜213〉S. cervisiae (Saccharomyces cerevisiae)

<400>150

Glu Asn Tyr Glu Thr Thr Asn Glu Leu Gln Asn Ala Glu Arg Glu Leu

1 5 10 15

Leu Met Lys Ser Ala Met

20

<210>151

<211>16

<212>PRT

＜213〉S. cervisiae (Saccharomyces cerevisiae)

<400>151

Ser Asn Gly Arg Val Ser Leu Pro Val Lys Lys Val Thr Lys Lys Ile

1 5 10 15

<210>152

<211>15

<212>PRT

＜213〉S. cervisiae (Saccharomyces cerevisiae)

<400>152

Val Ala Ile Gly Lys Gly Ala Ile Asp Gly Phe Phe Lys Arg Lys

1 5 10 15

<210>153

<211>24

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>153

Leu His Leu Leu Pro Cys Glu Val Ala Val Asp Gly Pro Ala Pro Val

1 5 10 15

Gly Arg Phe Phe Thr Pro Ala Ile

20

<210>154

<211>31

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>154

Leu Glu Val Ser Phe Arg Gly Arg Cys Leu Arg Gly Glu Glu Val Ala

1 5 10 15

Val Pro Pro Gly Leu Val Gly Tyr Val Met Val Thr Glu Glu Lys

20 25 30

<210>155

<211>35

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>155

Asn Phe Ser Arg Phe Thr Leu Trp Gly Leu Glu Thr Ile Pro Gly Pro

1 5 10 15

Asp Ala Lys Val Arg Gly Ala Leu Thr Trp Pro Ser Leu Ala Ala Ala

20 25 30

Ile His Ala

35

<210>156

<211>24

<212>PRT

＜213〉house mouse (Mus musculus)

<400>156

Leu His Leu Leu Pro Cys Asp Val Leu Val Ser Arg Pro Ala Pro Val

1 5 10 15

Asp Arg Phe Phe Thr Pro Ala Val

20

<210>157

<211>32

<212>PRT

＜213〉house mouse (Mus musculus)

<400>157

Leu Gln Ala Ser Phe Arg Gly Arg Gly Leu Arg Gly Glu Glu Val Ala

1 5 10 15

Val Pro Pro Gly Phe Ala Gly Phe Val Met Val Thr Glu Glu Lys Gly

20 25 30

<210>158

<211>35

<212>PRT

＜213〉house mouse (Mus musculus)

<400>158

Ser Phe Ser His Phe Thr Leu Trp Gly Leu Glu Thr Val Pro Gly Pro

1 5 10 15

Asp Ala Lys Val His Arg Ala Leu Gly Trp Pro Ser Leu Ala Ala Ala

20 25 30

Ile His Ala

35

<210>159

<211>24

<212>PRT

＜213〉Rattus norvegicus (Rattus norvegicus)

<400>159

Leu His Leu Leu Pro Cys Asp Val Leu Val Ser Arg Pro Ala Pro Val

1 5 10 15

Glu Arg Phe Phe Thr Pro Ala Val

20

<210>160

<211>32

<212>PRT

＜213〉Rattus norvegicus (Rattus norvegicus)

<400>160

Leu Gln Val Ser Phe Arg Gly Arg Gly Leu Arg Gly Glu Asp Val Ala

1 5 10 15

Val Pro Pro Gly Phe Ala Gly Phe Val Met Val Thr Glu Glu Met Gly

20 25 30

<210>161

<211>35

<212>PRT

＜213〉Rattus norvegicus (Rattus norvegicus)

<400>161

Ser Phe Ser His Phe Thr Leu Trp Gly Leu Glu Thr Val Pro Gly Pro

1 5 10 15

Asp Ala Lys Val His Arg Ala Leu Gly Trp Pro Ser Leu Ala Ala Ala

20 25 30

Asp Asp Ser

35

<210>162

<211>24

<212>PRT

＜213〉European ox (Bos taurus)

<400>162

Leu His Leu Leu Pro Cys Glu Val Pro Val Asn Arg Pro Thr Pro Val

1 5 10 15

Gly Arg Phe Phe Thr Pro Ala Ile

20

<210>163

<211>31

<212>PRT

＜213〉European ox (Bos taurus)

<400>163

Leu Glu Ala Ser Phe Arg Gly Arg Ser Leu Arg Gly Glu Glu Val Val

1 5 10 15

Val Pro Pro Gly Phe Val Gly Tyr Val Val Thr Glu Glu Lys Ala

20 25 30

<210>164

<211>35

<212>PRT

＜213〉European ox (Bos taurus)

<400>164

Ser Phe Ser Ser Phe Thr Val Trp Gly Leu Glu Ser Ile Pro Gly Pro

1 5 10 15

Asp Ala Lys Leu Arg Gly Ala Leu Ser Trp Pro Ser Leu Ala Ala Ala

20 25 30

Ile His Ala

35

<210>165

<211>24

<212>PRT

＜213〉domesticated dog (Canis familiaris)

<400>165

Leu His Leu Leu Pro Cys Glu Val Leu Val Asn Arg Pro Ala Pro Val

1 5 10 15

Asp Arg Phe Phe Thr Pro Ala Ile

20

<210>166

<211>32

<212>PRT

＜213〉domesticated dog (Canis familiaris)

<400>166

Leu Glu Val Ser Phe Arg Gly Arg Lys Leu Arg Gly Glu Glu Val Val

1 5 10 15

Val Pro Pro Gly Leu Val Gly Tyr Val Met Ala Met Glu Glu Gln Gly

20 25 30

<210>167

<211>35

<212>PRT

＜213〉domesticated dog (Canis familiaris)

<400>167

Ser Phe Ser Arg Phe Thr Leu Trp Gly Leu Glu Thr Ile Pro Gly Pro

1 5 10 15

Asp Ala Lys Val Arg Gly Ala Leu Thr Trp Pro Ser Leu Ala Ala Ala

20 25 30

Ile His Ala

35

<210>168

<211>24

<212>PRT

＜213〉chub mackerel (Danio rerio)

<400>168

Ile His Leu Leu Pro Cys Glu Val Glu His Asp Gly Pro Ala Glu Val

1 5 10 15

Phe Lys Phe Phe Asn Pro Thr Val

20

<210>169

<211>32

<212>PRT

＜213〉chub mackerel (Danio rerio)

<400>169

Thr Thr Val Ser Phe Arg Gly Arg Gly Leu Lys Gly Gln Glu Leu Gln

1 5 10 15

Cys Pro Gln Gly Tyr Thr Gly Val Val Leu Lys Glu Val Gln Lys Pro

20 25 30

<210>170

<211>35

<212>PRT

＜213〉chub mackerel (Danio rerio)

<400>170

Val Phe His His Phe Thr Tyr Trp Asn Leu Glu Thr Pro Pro Thr Ser

1 5 10 15

Asp Asp Gly Val Val Met Ala Met Ala Trp Pro Glu Leu Ala Glu Ala

20 25 30

Ile His Gly

35

<210>171

<211>24

<212>PRT

＜213〉drosophila melanogaster (Drosophila melanogaster)

<400>171

Val His Tyr Leu Pro Ala Lys Ile Asp Gly Asp Gly Glu Ala Asn Val

1 5 10 15

Lys Asn Tyr Phe Asn Asn Tyr Thr

20

<210>172

<211>31

<212>PRT

＜213〉drosophila melanogaster (Drosophila melanogaster)

<400>172

Thr Asn Ala Leu Arg Gly Phe Pro Leu Met Gly Glu Lys Leu Lys val

1 5 10 15

Pro Glu Gly Tyr Cys Gly Leu Val Leu Gln Glu Thr Glu Lys Pro

20 25 30

<210>173

<211>35

<212>PRT

＜213〉drosophila melanogaster (Drosophila melanogaster)

<400>173

Val Phe Gln Asp Phe Thr Tyr Trp Asn Tyr Asp Lys Val Pro Ser Asn

1 5 10 15

Gly Asp Pro Tyr Arg Gln Ala Leu Pro Leu Pro Asp Val Ala Gln Ala

20 25 30

Leu Ser Gln

35

<210>174

<211>24

<212>PRT

＜213〉anopheles costalis (Anopheles gambiae)

<400>174

Leu Gln Tyr Ile Pro Ala Thr Ile Lys Gly Asp Gly Pro Ala Asn Leu

1 5 10 15

Glu Gln Phe Phe Thr Pro Tyr Thr

20

<210>175

<211>32

<212>PRT

＜213〉anopheles costalis (Anopheles gambiae)

<400>175

Leu Cys Asn Ala Leu Arg Gly Tyr Pro Leu Arg Gly Lys Ala Thr Ser

1 5 10 15

Leu Pro Ala Gly Tyr Thr Gly Val Met Leu Gln Glu Thr Lys Lys Pro

20 25 30

<210>176

<211>35

<212>PRT

＜213〉anopheles costalis (Anopheles gambiae)

<400>176

Ala Phe Arg Asp Phe Thr Tyr Trp Asn Tyr Asp Arg Gln Pro Ser Arg

1 5 10 15

Asn Asp Pro Met Ala Lys Ala Leu Gly Trp Leu Gln Leu Ala Glu Val

20 25 30

Leu His Gly

35

<210>177

<211>57

<212>PRT

＜213〉Gibberella zeae (Gibberella zeae)

<400>177

Ala Asn Ile Leu Pro Cys Arg Ile His His Asp Gly Pro Ile Glu Pro

1 5 10 15

Thr Ser Thr Tyr Trp Thr Pro Ala Ala Thr Asp Ala Tyr Phe Arg Gly

20 25 30

Arg Lys Leu Gln Gly Lys Ile Val Lys Leu Pro Lys Asp Cys Arg Gly

35 40 45

Ile Val Val Glu Arg Val Ser Gln Gln

50 55

<210>178

<211>14

<212>PRT

＜213〉Gibberella zeae (Gibberella zeae)

<400>178

Glu Phe Asp Glu Met Val Val Trp Gly His Glu Ala Val Ala

1 5 10

<210>179

<211>18

<212>PRT

＜213〉Gibberella zeae (Gibberella zeae)

<400>179

Pro Tyr Val Arg Ser Met Glu Glu Trp Leu Gln Val Ala Asp Lys Ile

1 5 10 15

His Ser

<210>180

<211>25

<212>PRT

＜213〉Neurospora crassa (Neurospora crassa)

<400>180

Pro Asn Leu Leu Pro Cys Arg Ile His His Ser Gly Ser Val Glu Pro

1 5 10 15

Thr Asp Ser Phe Trp Asn Pro Arg Cys

20 25

<210>181

<211>32

<212>PRT

＜213〉Neurospora crassa (Neurospora crassa)

<400>181

Lys Val Ser Tyr Phe Arg Gly Arg Lys Leu His Gly Lys Thr Leu Pro

1 5 10 15

Leu Pro Glu Gly Tyr Lys Gly Val Val Ala Ser Lys Val Phe Pro Lys

20 25 30

<210>182

<211>14

<212>PRT

＜213〉Neurospora crassa (Neurospora crassa)

<400>182

Glu Phe Glu Glu Val Val Val Trp Gly His Glu His Leu Ala

1 5 10

<210>183

<211>18

<212>PRT

＜213〉Neurospora crassa (Neurospora crassa)

<400>183

Ala Tyr Val Arg Gly Val Glu Glu Trp Val Gly Phe Ala Glu Arg Val

1 5 10 15

His Ser

<210>184

<211>25

<212>PRT

＜213〉aspergillus nidulans (Aspergillus nidulans)

<400>184

Pro Asn Ile Leu Pro Cys Arg Ile His Tyr Asp Gly Ser Val Glu Ser

1 5 10 15

Leu Asn Arg Tyr Trp Thr Pro Ser Thr

20 25

<210>185

<211>32

<212>PRT

＜213〉aspergillus nidulans (Aspergillus nidulans)

<400>185

Gln Thr Ala Tyr Phe Arg Gly Arg Arg Leu Arg Gly Arg Arg Val Gln

1 5 10 15

Leu Pro Glu Gly Tyr Glu Gly Val Val Val Thr His Thr Glu Arg Glu

20 25 30

<210>186

<211>14

<212>PRT

＜213〉aspergillus nidulans (Aspergillus nidulans)

<400>186

Thr Phe Gly Asp Tyr Val Val Trp Gly His Glu Val Ile Pro

1 5 10

<210>187

<211>18

<212>PRT

＜213〉aspergillus nidulans (Aspergillus nidulans)

<400>187

Ser Phe Val Lys Gly Val Glu Glu Trp Val Lys Phe Ala Glu Val Met

1 5 10 15

His Ser

<210>188

<211>24

<212>PRT

＜213〉little Cryptosporidium (Cryptosporidium parvum)

<400>188

Phe Asn Ile Leu Pro Phe Gln Ile Leu Phe Asp Gly Arg Leu Pro Lys

1 5 10 15

Glu Asn Pro Phe Asn Arg Leu Tyr

20

<210>189

<211>30

<212>PRT

＜213〉little Cryptosporidium (Cryptosporidium parvum)

<400>189

Ser Lys Ser Phe Arg Gly Arg Lys Leu Asn Gly Arg Ile Ile His Leu

1 5 10 15

Ser Lys Ser Ser His Ser Ile Leu Ile Leu Ser Lys Asn Asn

20 25 30

<210>190

<211>35

<212>PRT

＜213〉little Cryptosporidium (Cryptosporidium parvum)

<400>190

Ser Phe Asn Gln Val Thr Tyr Trp Asn Tyr Asp Asp Glu Ile Lys Gln

1 5 10 15

Ser Asp Asp Ile Pro Gln Phe Leu Asn Val Ile His Asn Phe Asn Ile

20 25 30

Leu His Arg

35

<210>191

<211>24

<212>PRT

＜213〉Candida albicans (Candida albicans)

<400>191

Ala Ser Val Val Pro Met Asn Ile Gln Tyr Ser Gly Pro Ala Asn Thr

1 5 10 15

Glu Asp Tyr Phe Ala Pro Ser Lys

20

<210>192

<211>32

<212>PRT

＜213〉Candida albicans (Candida albicans)

<400>192

Val Ala Tyr Phe Arg Gly Cys Lys Leu Val Gly Lys Thr Leu Glu Leu

1 5 10 15

Asp Lys Tyr Asp Val Lys Gly Tyr Val Ile Asn Lys Ser Glu His Leu

20 25 30

<210>193

<211>35

<212>PRT

＜213〉Candida albicans (Candida albicans)

<400>193

Asn Phe Gln Lys Leu Thr Val Tyr Gly His Asp Thr Leu Pro Ser Arg

1 5 10 15

Thr Asn Gln Trp Ser Leu Ile Pro Glu Tyr Leu Ser Ile Ser Asn Ile

20 25 30

Val Asn Glu

35

<210>194

<211>24

<212>PRT

＜213〉Hansen cryptococcus (Debaryomyces hansenii)

<400>194

Gly Asn Ile Val Pro Cys His Ile Gln Tyr Asn Gly Ala Ala Asn Thr

1 5 10 15

Lys Asp Tyr Phe Thr Pro Ser Lys

20

<210>195

<211>31

<212>PRT

＜213〉Hansen cryptococcus (Debaryomyces hansenii)

<400>195

Val Ala Tyr Tyr Arg Gly Cys Lys Leu Val Gly Lys Asn Met Asn Ile

1 5 10 15

Ser Asn Asp Tyr Ser Gly Tyr Leu Ile Asn Lys Ser Glu Ser Leu

20 25 30

<210>196

<211>35

<212>PRT

＜213〉Hansen cryptococcus (Debaryomyces hansenii)

<400>196

Lys Phe Glu Glu Ile Thr Val Tyr Gly His Asp Thr Val Pro Glu Leu

1 5 10 15

Thr Ser Gln Trp Gly Leu Ile Gln Glu Trp Lys Asp Ile Ser Glu Val

20 25 30

Ile His Ser

35

<210>197

<211>24

<212>PRT

＜213〉fission yeast mycelia (Schizosaccharomyces pombe)

<400>197

Ala Ser Leu Leu Pro Cys His Ile Ser Tyr Asp Gly Pro Ala Pro Val

1 5 10 15

Phe Glu Tyr Phe His Asp Lys Ile

20

<210>198

<211>30

<212>PRT

＜213〉fission yeast mycelia (Schizosaccharomyces pombe)

<400>198

Thr Val Cys Leu Arg Gly Arg Glu Leu Asn Gly Glu Glu Leu Asp Leu

1 5 10 15

Pro Glu Asn Tyr Thr Gly Gln Val Val Leu Cys Asp Asp Gly

20 25 30

<210>199

<211>14

<212>PRT

＜213〉fission yeast mycelia (Schizosaccharomyces pombe)

<400>199

Thr Phe Glu Lys Val Met Leu Trp Asn Arg Asp Asn Met Arg

1 5 10

<210>200

<211>18

<212>PRT

＜213〉fission yeast mycelia (Schizosaccharomyces pombe)

<400>200

Gln Trp Lys Arg Gly Val Leu Glu Trp Ile Gln Phe Ala Ser Lys Val

1 5 10 15

Lys Phe

<210>201

<211>24

<212>PRT

＜213〉paddy rice (Oryza sativa)

<400>201

Val His Leu Leu Pro Cys Gly Ile Lys Gln Asn Gly Ala Ala Ala Val

1 5 10 15

Ser Asp Tyr Phe Lys Pro Lys Asp

20

<210>202

<211>29

<212>PRT

＜213〉paddy rice (Oryza sativa)

<400>202

Glu Ala Phe Phe Arg Gly Arg Lys Leu Gln Gly Ala Thr Ile Ser Leu

1 5 10 15

Pro Asp Gly Tyr Arg Gly Tyr Val Leu Glu Lys Arg Ser

20 25

<210>203

<211>35

<212>PRT

＜213〉paddy rice (Oryza sativa)

<400>203

Glu Phe Gln Asn Ile Thr Tyr Trp Asn His Asp Thr Thr Pro Ser Ala

1 5 10 15

Glu Asp Pro Leu Pro Arg Cys Phe His Leu Leu Thr Val Ala Asn Ala

20 25 30

Met His Lys

35

<210>204

<211>25

<212>PRT

＜213〉Oswaldocruzia (Trypanosoma cruzi)

<400>204

Val His Ser Leu Pro Phe Lys Thr Thr Phe Arg Gly Thr Thr Asp Ile

1 5 10 15

Glu Glu Asn Phe Ser Arg His Ile Glu

20 25

<210>205

<211>32

<212>PRT

＜213〉Oswaldocruzia (Trypanosoma cruzi)

<400>205

Leu Arg Asn Thr Leu Arg Gly Arg Thr Leu Ile Gly Arg Glu Val Val

1 5 10 15

Leu Pro Pro Ser Tyr Val Leu Ala Val Thr Ser Phe Met Pro Arg Gln

20 25 30

<210>206

<211>32

<212>PRT

＜213〉Oswaldocruzia (Trypanosoma cruzi)

<400>206

Thr Glu Asp Arg Phe Cys Val Trp Glu His Asp Lys Gln Pro Glu Arg

1 5 10 15

Val Asp Ala Leu Ala His Trp Leu Glu Leu Ser Arg Val Leu His Ser

20 25 30

<210>207

<211>25

<212>PRT

＜213〉Bruce trypanosome (Trypanosoma brucei)

<400>207

Ile His Ser Leu Pro Val Lys Thr Asn Phe Arg Gly Thr Thr Asp Val

1 5 10 15

Thr Val Asn Phe Thr Gln His Thr Val

20 25

<210>208

<211>32

<212>PRT

＜213〉Bruce trypanosome (Trypanosoma brucei)

<400>208

Leu Arg Asn Ala Leu Arg Gly Arg Thr Leu Met Gly Arg Glu Val Ile

1 5 10 15

Leu Pro Ser Ser Tyr Val Ile Ala Cys Ala Ser Phe Thr Asn Lys Pro

20 25 30

<210>209

<211>32

<212>PRT

＜213〉Bruce trypanosome (Trypanosoma brucei)

<400>209

Val Ala Glu Ser Phe Cys Ile Trp Glu His Asp Lys Lys Pro Glu Arg

1 5 10 15

Ser Glu Met Val Thr Gln Trp Leu Arg Leu Ser Asn Asp Leu His Cys

20 25 30

<210>210

<211>24

<212>PRT

＜213〉discoideum net post bacterium (Dictyostelium discoideum)

<400>210

Leu Gln Arg Leu Pro Phe Ser Ile Gly Tyr Asn Gly Phe Ala Lys Val

1 5 10 15

Ser Asn Tyr Phe Gln Val Thr Asp

20

<210>211

<211>30

<212>PRT

＜213〉discoideum net post bacterium (Dictyostelium discoideum)

<400>211

Tyr Ser Ser Phe Arg Gly Ile Gln Leu Ile Gly Glu Lys Ile Lys Ile

1 5 10 15

Pro Asn Gly Phe Asp Gly Tyr Val Phe Arg Asp Glu Asp Glu

20 25 30

<210>212

<211>35

<212>PRT

＜213〉discoideum net post bacterium (Dictyostelium discoideum)

<400>212

Lys Phe Asn Glu Leu Thr Tyr Trp Asn Arg Glu Thr Val Pro Ser Asp

1 5 10 15

Phe Asp Lys Gln Ile Gln Ala Phe Lys Ser Leu Asn Ile Gln Ser Met

20 25 30

Val Asn Lys

35

<210>213

<211>24

<212>PRT

＜213〉ascomycetous yeast belongs to (Yarrowia lipolytica)

<400>213

Cys His Val Leu Pro Cys His Ile His Tyr Thr Gly Pro Ala Asn Val

1 5 10 15

Ala Glu Tyr Phe Thr Ile Glu Gly

20

<210>214

<211>31

<212>PRT

＜213〉ascomycetous yeast belongs to (Yarrowia lipolytica)

<400>214

Ser Thr His Phe Arg Gly Arg Lys Leu His Gly Val Glu Gln Asn Leu

1 5 10 15

Pro Glu Thr Tyr Arg Gly Tyr Arg Phe Thr Glu Gly Thr Asn Phe

20 25 30

<210>215

<211>14

<212>PRT

＜213〉ascomycetous yeast belongs to (Yarrowia lipolytica)

<400>215

Ser Phe Asp His Leu Val Ile Trp Gly His Glu Arg Val Pro

1 5 10

<210>216

<211>18

<212>PRT

＜213〉ascomycetous yeast belongs to (Yarrowia lipolytica)

<400>216

Gln Trp Ala Cys Gly Val Arg Asp Trp His Gln Val Ala Thr Ala Met

1 5 10 15

Asn Pro

<210>217

<211>25

<212>PRT

＜213〉kluyveromyces spp (Kluyveromyces waltii)

<400>217

Met His Leu Val Pro Cys Lys Ile Arg Val Ala Gly Pro Thr Ala Glu

1 5 10 15

Leu Thr Thr Asn Phe Asp Glu Asp Leu

20 25

<210>218

<211>31

<212>PRT

＜213〉kluyveromyces spp (Kluyveromyces waltii)

<400>218

Val Asn Tyr Ile Arg Gly Arg Lys Leu Val Gly Lys Ser Leu Ser Cys

1 5 10 15

Phe Gln Asp Cys Asp Ile Val Leu Leu Asp Gln Asp Cys Glu Ala

20 25 30

<210>219

<211>14

<212>PRT

＜213〉kluyveromyces spp (Kluyveromyces waltii)

<400>219

Arg Val Ser Ser Ile Val Asn Tyr Glu Arg Glu Gly Asn Glu

1 5 10

<210>220

<211>18

<212>PRT

＜213〉kluyveromyces spp (Kluyveromyces waltii)

<400>220

Glu Glu Met Glu Lys Phe Asn Glu Phe Leu Gln Leu Ser Glu Thr Ile

1 5 10 15

His Gly

<210>221

<211>51

<212>PRT

＜213〉Eremothecium (Eremothecium gossypii)

<400>221

Met Phe Leu Val Pro Cys Lys Val Arg Tyr Ser Gly Pro Thr Ala Glu

1 5 10 15

Phe Gln Ser Leu Asn His Ile Arg Gly Arg Lys Ile Val Gly Lys Asp

20 25 30

Ile Leu Ser Lys Phe Pro Asp Ser Asn Ala Tyr Leu Ala Arg Pro Asp

35 40 45

Asn Val Ala

50

<210>222

<211>13

<212>PRT

＜213〉Eremothecium (Eremothecium gossypii)

<400>222

Leu Asn Ala Ile Leu Asn Cys Glu Arg Asp Gly Asn Asp

1 5 10

<210>223

<211>18

<212>PRT

＜213〉Eremothecium (Eremothecium gossypii)

<400>223

Ser Glu Leu His Lys Phe His Glu Asn Leu Asp Leu Asn Asp Ala Ile

1 5 10 15

His Ala

<210>224

<211>25

<212>PRT

＜213〉yeast belong (Saccharomyces kluyveri)

<400>224

Ala His Phe Val Pro Cys Lys Val Arg Tyr Ser Gly Thr Thr Gln Glu

1 5 10 15

Phe Lys Asn Gln Phe Gln Leu Asp Gln

20 25

<210>225

<211>31

<212>PRT

＜213〉yeast belong (Saccharomyces kluyveri)

<400>225

Val Thr Tyr Ile Arg Gly Arg Lys Ile Val Gly Lys Glu Ile Thr Ala

1 5 10 15

Leu Glu Gly Cys Leu Ala Thr Leu Val Glu Glu Thr Thr Asn Pro

20 25 30

<210>226

<211>14

<212>PRT

＜213〉yeast belong (Saccharomyces kluyveri)

<400>226

Ser Leu Ser Lys Leu Val Asn Tyr Glu Arg Glu Gly Asn Glu

1 5 10

<210>227

<211>18

<212>PRT

＜213〉yeast belong (Saccharomyces kluyveri)

<400>227

Glu Glu Met Gly Lys Phe Gln Glu Phe Val Glu Leu Ala Asp Leu Ile

1 5 10 15

His Gly

<210>228

<211>25

<212>PRT

＜213〉kluyveromyces spp (Kluyveromyces lactis)

<400>228

Ala His Leu Val Pro Cys Lys Ile Arg Tyr Thr Gly Gln Thr Asp Glu

1 5 10 15

Leu Glu Glu Gln Phe Ile Met Asp Lys

20 25

<210>229

<211>30

<212>PRT

＜213〉kluyveromyces spp (Kluyveromyces lactis)

<400>229

Val Thr Tyr Ile Arg Gly Arg Lys Val Ile Gly Glu Gln Val Val Phe

1 5 10 15

Asp Asp Arg Thr Thr Tyr Ile Met Ser Thr Arg Glu Asp Asp

20 25 30

<210>230

<211>14

<212>PRT

＜213〉kluyveromyces spp (Kluyveromyces lactis)

<400>230

Lys Val Thr Glu Ile Phe Asn Tyr Glu Arg Glu Gly Asn Glu

1 5 10

<210>231

<211>18

<212>PRT

＜213〉kluyveromyces spp (Kluyveromyces lactis)

<400>231

Asp Glu Ile Ser Lys Phe Ile Glu Cys Arg Glu Leu Asp Ser Leu Ile

1 5 10 15

His Glu

<210>232

<211>25

<212>PRT

＜213〉Saccharomycodes (Saccharomyces castellii)

<400>232

Ala Ser Phe Val Pro Leu Lys Ile Arg Tyr Asn Gly Pro Thr Thr Glu

1 5 10 15

Phe Val Asn Asn Phe Lys Asp Asn Lys

20 25

<210>233

<211>32

<212>PRT

＜213〉Saccharomycodes (Saccharomyces castellii)

<400>233

Thr Thr Phe Ile Arg Gly Arg Cys Leu His Gly Thr Pro Val Asn Lys

1 5 10 15

Tyr Phe Glu Ser Ala Ser Ala His Val Ile Gly Lys Ser Lys Gln Thr

20 25 30

<210>234

<211>14

<212>PRT

＜213〉Saccharomycodes (Saccharomyces castellii)

<400>234

Thr Val Asn Ser Ile Ile Asn Tyr Glu Arg Glu Gly Asn Thr

1 5 10

<210>235

<211>18

<212>PRT

＜213〉Saccharomycodes (Saccharomyces castellii)

<400>235

Glu Glu Leu Ala His Leu Lys Glu Phe Val Gln Leu Gln Asn His Ile

1 5 10 15

His Ser

<210>236

<211>42

<212>PRT

＜213〉Saccharomyces paradoxus (Saccharomyces paradoxus)

<400>236

Val Asn Phe Met Pro Phe Tyr Thr Glu Tyr Glu Gly Pro Thr Gly Glu

1 5 10 15

Phe Lys Asp Tyr Lys Phe Glu Asp Thr Val Tyr Phe Arg Gly Lys Glu

20 25 30

Leu Gln Arg Glu Lys Leu Val Thr Thr Ala

35 40

<210>237

<211>47

<212>PRT

＜213〉Saccharomyces paradoxus (Saccharomyces paradoxus)

<400>237

Phe Ser Asn Gly Ala Ile Phe Ser Gly Asn Thr Ile Ile Gly Lys Val

1 5 10 15

Val Ser Val Asn Asn Tyr Glu Arg Glu Gly Thr Asn Arg Asn Glu Leu

20 25 30

Ala Arg Leu Gln Glu Leu Ile Ser Leu Ile Asp Val Ile Asn Gln

35 40 45

<210>238

<211>42

<212>PRT

＜213〉S. cervisiae (Saccharomyces cerevisiae)

<400>238

Val Ser Phe Met Pro Phe Tyr Thr Glu Tyr Gln Gly Pro Thr Glu Glu

1 5 10 15

Phe Lys Asp Tyr Lys Phe Glu Asp Thr Ile Tyr Phe Arg Gly Lys Glu

20 25 30

Leu Lys Arg Glu Lys Ser Ala Thr Pro Ser

35 40

<210>239

<211>47

<212>PRT

＜213〉S. cervisiae (Saccharomyces cerevisiae)

<400>239

Phe Ser Asn Gly Ala Ile Leu Ser Gly Asn Thr Ile Thr Gly Lys Ile

1 5 10 15

Val Ser Val Asn Asn Tyr Glu Arg Glu Gly Thr Asp Arg Asn Glu Leu

20 25 30

Ala Arg Leu Gln Glu Leu Ile Ser Leu Ile Asp Val Ile Asn Gln

35 40 45

<210>240

<211>42

<212>PRT

＜213〉Saccharomycodes (Saccharomyces mikatae)

<400>240

Leu Asn Phe Met Pro Phe Tyr Thr Glu Tyr Gln Gly Pro Thr Lys Glu

1 5 10 15

Phe Lys Asp Tyr Lys Phe Glu Asp Thr Ile Tyr Phe Arg Gly Lys Glu

20 25 30

Leu Gln Arg Glu Lys Pro Ala Thr Thr Ser

35 40

<210>241

<211>47

<212>PRT

＜213〉Saccharomycodes (Saccharomyces mikatae)

<400>241

Leu Ser Asn Gly Ala Ile Leu Ser Gly Asn Ala Ile Thr Asp Arg Val

1 5 10 15

Val Ser Val Asn Asn Tyr Glu Arg Glu Gly Thr Asn Arg Asn Glu Leu

20 25 30

Ala Arg Leu Gln Glu Leu Met Ser Leu Ile Asp Leu Ile Asn Gln

35 40 45

<210>242

<211>42

<212>PRT

＜213〉Saccharomycodes (Saccharomyces kudriavzevii)

<400>242

Val Ser Phe Met Pro Phe Tyr Thr Glu Tyr Gln Gly Pro Thr Ala Glu

1 5 10 15

Leu Lys Asp Tyr Thr Phe Gly Asp Thr Ile Tyr Phe Arg Gly Lys Glu

20 25 30

Leu Gln Arg Glu Lys Leu Ala Thr Ala Thr

35 40

<210>243

<211>47

<212>PRT

＜213〉Saccharomycodes (Saccharomyces kudriavzevii)

<400>243

Leu Ser Asn Gly Ala Ile Leu Ser Gly Asn Thr Ile Asn Gly Arg Ile

1 5 10 15

Val Ala Val Asn Asn Tyr Glu Arg Glu Gly Ser Asn Arg Asn Glu Leu

20 25 30

Ala Arg Leu Gln Glu Leu Ile Ser Leu Thr Asn Val Ile Asn Gln

35 40 45

<210>244

<211>42

<212>PRT

＜213〉saccharomyces bayanus (Saccharomyces bayanus)

<400>244

Val Asn Phe Met Pro Cys Tyr Thr Glu Tyr Glu Gly Pro Thr Thr Glu

1 5 10 15

Phe Lys Asp Tyr Lys Phe Asp Asp Thr Ile Tyr Phe Arg Gly Lys Glu

20 25 30

Leu Gln Lys Glu Lys Pro Thr Ser Thr Ala

35 40

<210>245

<211>47

<212>PRT

＜213〉saccharomyces bayanus (Saccharomyces bayanus)

<400>245

Leu Ser Asn Gly Ala Ile Leu Ser Gly Asn Thr Val Thr Gly Lys Ile

1 5 10 15

Val Ala Val Asn Asn Tyr Glu Arg Glu Gly Thr Asp Arg Asn Glu Leu

20 25 30

Glu Arg Leu Gln Glu Leu Ile Tyr Ile Thr Asn Val Ile Asn Gln

35 40 45

<210>246

<211>90

<212>PRT

＜213〉Candida glabrata (Candida glabrata)

<400>246

Val Tyr Leu Val Pro Ala Lys Ile Glu Leu Val Gly Asn Ala Gly Val

1 5 10 15

Pro Glu Glu Ile Lys Phe Glu Glu Val Ala Gln Leu Arg Gly Lys Glu

20 25 30

Leu His Gly Glu Asp Leu Ile Gln His Leu Asp Gly Tyr His Gly Gln

35 40 45

Leu Val Lys Asp Gly Lys Ile Glu Glu Leu Ser Ser Val Val Asp Phe

50 55 60

Glu Arg Glu Glu Glu Ile Phe Lys Gln Glu Leu Asn Ala Phe Lys Glu

65 70 75 80

Thr Ala Asp Ile Leu Ser Ala Leu His Asn

85 90

Claims (61)

1, a kind of RNA enzyme H2 recombinant chou comprises:

I) a kind of SEQ ID No 1, nucleotide sequence or its homologous chromosomes encoded protein matter shown in the SEQ ID No 3, a kind of among the SEQ ID No 5;

Or ii) a kind of protein comprises SEQ ID No 2, SEQ ID No 4, or the aminoacid sequence shown in the SEQ ID No 6, or its homologous chromosomes.

2, RNA enzyme H2 recombinant chou according to claim 1 comprises the nucleotide sequence shown in a kind of SEQ ID No 1, or its homologous chromosomes encoded protein matter.

3, RNA enzyme H2 recombinant chou according to claim 1 comprises the nucleotide sequence shown in a kind of SEQ ID No 3, or its homologous chromosomes encoded protein matter.

4, RNA enzyme H2 recombinant chou according to claim 1 comprises the nucleotide sequence shown in a kind of SEQ ID No 5, or its homologous chromosomes encoded protein matter.

5, RNA enzyme H2 recombinant chou according to claim 1 comprises the aminoacid sequence shown in a kind of SEQ ID No 2, or its homologous chromosomes encoded protein matter.

6, RNA enzyme H2 recombinant chou according to claim 1 comprises the aminoacid sequence shown in a kind of SEQ ID No 4, or its homologous chromosomes encoded protein matter.

7, RNA enzyme H2 recombinant chou according to claim 1 comprises the aminoacid sequence shown in a kind of SEQ ID No 6, or its homologous chromosomes encoded protein matter.

8, a kind of RNA enzyme H2 recombinant chou comprises at least a:

I) the RNA enzyme H2B of the nucleotide sequence shown in a kind of SEQ ID No 1 or its homologous chromosomes coding perhaps contains the RNA enzyme H2B of the aminoacid sequence shown in the SEQID No 2 or its homologous chromosomes;

Or the RNA enzyme H2C of the nucleotide sequence shown in ii) a kind of SEQ ID No 3 or its homologous chromosomes coding, perhaps contain the RNA enzyme H2C of the aminoacid sequence shown in the SEQ ID No 4 or its homologous chromosomes;

Or the RNA enzyme H2A of the nucleotide sequence shown in iii) a kind of SEQ ID No 3 or its homologous chromosomes coding, perhaps contain the RNA enzyme H2A of the aminoacid sequence shown in the SEQ ID No 6 or its homologous chromosomes;

9, RNA enzyme H2 recombinant chou according to claim 8 comprises component i), ii) or iii) at least two kinds.

10, RNA enzyme H2 recombinant chou according to claim 8 comprises each component i), ii) or iii).

11, a kind of polynucleotide comprise as SEQ ID No 1,3 or 5 or the listed nucleotide sequence of its homologous chromosomes.

12, a kind of protein of the polynucleotide encoding by claim 11.

13, a kind of protein comprises as SEQ ID No 2,4 or 6 or the listed aminoacid sequence of its homologous chromosomes.

14, the proteinic polynucleotide of a kind of coding claim 13.

15, a kind of carrier comprises the polynucleotide of claim 1 or claim 14.

16, a kind of carrier transformed host cells as claimed in claim 15.

17, a kind of LCL (LCL) is expressed a kind of RNA enzyme H2 of sudden change.

18, the clone of claim 17, described RNA enzyme H2 has the sudden change A177T of RNA enzyme H2B.

19, the clone of claim 17, described RNA enzyme H2 has the sudden change G37S of RNA enzyme H2A.

20, a kind of host cell of claim 16 is a kind of ES clone.

21, a kind of for the effective antibody of arbitrary protein in claim 12 and 13.

22, the antibody of claim 21 and the protein special combination that contains the protein or the polynucleotide encoding shown in the SEQ ID No.1 of aminoacid sequence shown in the SEQ ID No.2.

23, the antibody of claim 21 and the protein special combination that contains the protein or the polynucleotide encoding shown in the SEQ ID No.3 of aminoacid sequence shown in the SEQ ID No.4.

24, the antibody of claim 21 combines with the protein specific of the polynucleotide encoding shown in protein that contains aminoacid sequence shown in the SEQ ID No.6 or the SEQ ID No.5.

25, any antibody of claim 21 to 24 is polyclonal antibody.

26, any antibody of claim 21 to 24 is monoclonal antibody.

27, each described antibody of claim 21 to 26 is humanized antibody.

28, each described antibody of claim 21 to 27 is applied to treatment.

29, a kind of activator or inactivator or its component that detects RNA enzyme H2, or the method for RNA enzyme H2 substrate specificity conditioning agent, described detection method comprises:

I) with RNA enzyme H2 recombinant chou or its component contact detection material, described component is RNA enzyme H2B, RNA enzyme H2C, RNA enzyme H2A or its any combination;

Ii) estimate RNA enzyme H2 or the activity of its component in sample, alternatively, continuity for some time;

And iii) determine the effect of the detection material of RNA enzyme H2 or its composition activity.

30, detection method according to claim 29 is carried out described step I i),, estimate the activity of RNA enzyme H2 at conventional time point therebetween through after a while.

31, claim 29 and 30 each described detection methods, described RNA enzyme H2 mixture is presented on step I) in.

32, detection method according to claim 31, described RNA enzyme H2 mixture is presented in the cell.

33, claim 29 and 30 each described detection methods, described RNA enzyme H2A, RNA enzyme H2B, a kind of step I that is presented among the RNA enzyme H2C) in.

34, claim 29 and 33 each described detection methods, described RNA enzyme H2 or its component are by at step I i) in the oligomeric nucleotide evaluation of importing mark.

35, a kind of activator or inactivator or its component that detects RNA enzyme H2, or the method for RNA enzyme H2 substrate specificity conditioning agent, described detection method comprises:

I) with mutant rna enzyme H2 or its sudden change component contact detection material, described component is RNA enzyme H2B, RNA enzyme H2C, RNA enzyme H2A or its any combination;

Ii) estimate RNA enzyme H2 or the activity of its component in sample, alternatively, continuity for some time;

And iii) determine the effect of the detection material of RNA enzyme H2 or its composition activity.

36, detection method according to claim 35 is carried out described step I ii) therebetween, through after a while, estimates the activity of RNA enzyme H2 at conventional time point.

37, claim 35 and 36 each described detection methods, described RNA enzyme H2 is a recombinant chou.

38, claim 35 and 36 each described detection methods, described RNA enzyme H2 expresses from lymphoblastoid cell lines.

39, claim 35 and 36 each described detection methods, described RNA enzyme H2 expresses from non-human transgenic animal.

40, claim 35 and 36 each described detection methods, described RNA enzyme H2 mixture is presented in the cell.

41, a kind of activator or inactivator or its component that detects RNA enzyme H2, or the method for RNA enzyme H2 substrate specificity conditioning agent, described detection method comprises:

I) with mutant rna enzyme H2 or its sudden change component contact detection material, described component is RNA enzyme H2B, RNA enzyme H2C, RNA enzyme H2A or its any combination;

Ii) estimate RNA enzyme H2 or the activity of its component in sample, alternatively, continuity for some time;

And iii) determine the effect of the detection material of RNA enzyme H2 or its composition activity.

42, according to the detection method of claim 41, described step I through after a while, is estimated the activity of RNA enzyme H2 ii) therebetween at conventional time point.

43, according to claim 41 and 42 each described detection methods, described RNA enzyme H2 expresses from LCL.

44, according to claim 41 and 42 each described detection methods, described RNA enzyme H2 expresses from inhuman transgenic animal.

45, according to claim 41 and 42 each described detection methods, described RNA enzyme H2 mixture is presented in the cell.

46, a kind of detection RNA enzyme H2B, RNA enzyme H2C, or the method for suddenling change in the RNA enzyme H2A gene, in patient's genome, described detection method comprises:

I) cause the molten born of the same parents of the white cell of taking from patient; With

Ii) assess the RNA enzyme H2 activity of dissolved white cell.

47, a kind of assistance diagnosis SLE, AGS, autoimmune disease, to the infected by microbes enhanced sensitivity, or the method for its genetic risk, described method comprises the RNA enzyme H2A with patient, RNA enzyme H2B, or RNA enzyme H2C gene type, and with result and wild type gene comparison.

48, according to the method for claim 47, described infected by microbes is a virus infection.

49, a kind of production has encoding mutant RNA enzyme H2A, the method for the transgenic nonhuman animal of RNA enzyme H2B or RNA enzyme H2C, and described method comprises:

A) will comprise an encoding mutant RNA enzyme H2A, the recombination group of RNA enzyme H2B or RNA enzyme H2C polynucleotide sequence group imports non-human zygote or non-human embryonic stem cell;

B) produce a transgenic nonhuman animal from described non-human zygote or non-human embryonic stem cell; And

C) produce a kind of have encoding mutant RNA enzyme H2A, the genomic transgenic nonhuman animal of RNA enzyme H2B or RNA enzyme H2C.

50, according to the method for claim 49, described transgenic animal are rodents.

51, according to the method for claim 50, described transgenic animal are mouse or rat.

52, a kind of non-human transgenic animal has from RNA enzyme H2A, the mutator gene of RNA enzyme H2B or RNA enzyme H2C.

53,, has cell expressing RNA enzyme H2A, RNA enzyme H2B or a RNA enzyme H2C according to the transgenic animal of claim 52.

54, are rodents according to claim 52 or 53 each described transgenic animal.

55, according to the method for claim 54, described transgenic animal are mouse or rat.

56, use as each described transgenic animal of claim 52 to 53 as AGS SLE, the model of autoimmune disease or infected by microbes.

57, a kind ofly be used to identify whether patient suffers from AGS, SLE, autoimmune disease or to the infected by microbes enhanced sensitivity, or the diagnostic kit of tendency or its risk, described test kit contains in conjunction with the RNA enzyme H2A that is fit to primer, the primer of RNA enzyme H2B or RNA enzyme H2C.

58, according to the diagnostic kit of claim 57, described infected by microbes is a virus infection.

59, a kind of evaluation has the detection method of inflammation promotor action medium, and described detection method contains:

I) with RNA enzyme H2 or its component contact detection material, described component is RNA enzyme H2B, RNA enzyme H2C, RNA enzyme H2A or its any combination;

Ii) estimate RNA enzyme H2 or the activity of its component in sample, alternatively, continuity for some time.

60, according to the detection method of claim 59, described medium is an anti-inflammatory agent.

61, according to the detection method of claim 59, described medium is short scorching agent.

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