CN101724033A - Human epididymal expression sperm binding protein HEL-28 as well as encoding gene and application thereof - Google Patents

Abstract

The invention belongs to the biotechnology and the medical field and discloses a new human epididymal specific expression sperm binding protein HEL-28 as well as preparation and application thereof. The invention discloses an amino acid sequence of the HEL-28 protein and provides a recombinant expression vector which carries a DNA sequence used for encoding the protein. The protein is located in the back region of the head of a mature sperm, and an RT-PCR result indicates that the protein has low expression level in an epididymis and various tissues of a humna body. A band of 187 KD is detected in the body part of the epididymis by using Western blot. The HEL-28 protein can be used as a target protein for developing an immunological contraceptive and clinically diagnosing and treating male infertility. A method for detecting a corresponding gene or protein based on development of the HEL-28 protein can be widely used for the research field of reproductive medicine.

Description

Human epididymis expression sperm binding protein HEL-28 and encoding gene thereof and application

Technical field

The invention belongs to biotechnology and medical field.Specifically, the present invention relates to a kind of new human epididymis expression sperm binding protein hereinafter to be referred as HEL-28 and encoding gene and application

Background technology

This century, the mankind's development was faced with the problem of two sternnesses: on the one hand, global population is estimated will reach 9,000,000,000 in the year two thousand fifty in rapid expansion; On the other hand, according to World Health Organization's investigation, about 15% Mr. and Mrs exist sterile problem, and the sickness rate of developed country's infertility soared at nearest 10 years, and European developed country can be up to 30%, and wherein male factor accounts for half.World Health Organization's prediction, along with the increase of paathogenic factors such as environmental pollution and sexually transmitted disease (STD), in this century, infertility will become the third-largest disease that is only second to tumour and cardiovascular and cerebrovascular disease.But male sterility does not cause enough attention of society and medical circle, and the progress of therefore relevant male reproductive health is slow, lags significantly behind other subject.The effective ways that lack molecular biology aspect Clinics and Practices infertility.Because the sperm development obstacle can not natural fertilization, needs the doctor to obtain the offspring by being the tube baby.Utilize this kind technology to obtain the offspring, not only expended valuable time and fund, and have a lot of unhealthful latencies, the natural combination of smart ovum, the advantage in the normal growing process of having lost selected hereditary offspring's chance.Brought heavy spirit and economical load for family and society.

The sharp increase of whole world population has caused resource to exhaust and environmental degradation, becomes the serious threat of human health and existence.Our times population surpasses 6,000,000,000, estimates that according to the expert maximum population's capacity that Chinese resource environment can support is 15-16 hundred million.At present can be also very limited for the human contraceptives of selecting, also differ far away from the standard of " efficient, safe, reversible ".Develop safe and effective, the advanced practical new technology that avoids conception and control birth, product innovation, requiring with the personalization of the contraception medication of satisfying different crowd, different levels is the general trend of technical development of avoiding conception and controling birth both at home and abroad in recent years.

Epididymis is the vitals of spermioteleosis, and the sperm that testis produces need could obtain to move, be fertilized, defend and keep biological functions such as normal embryo development and defence by interacting with epididymis tube chamber microenvironment.Kind of secretory protein and sperm interaction surplus in the of nearly 200 participate in the spermioteleosis process, but people knows little about it to these proteic functions in the epididymis.The research of the epididymal proteins relevant with spermioteleosis will help to promote the progress of male genetic medical science, for the diagnosis of male infertility and treatment and development of new contraceptive provide the candidate molecular target, might bring new thinking and breakthrough to the male contraception of epididymis link simultaneously.

Summary of the invention

First purpose of the present invention provides a kind of new human epididymis expression sperm binding protein HEL-28.

A kind of human epididymis expression sperm binding protein HEL-28, it have the aminoacid sequence of sequence 2 in the sequence table or with the amino acid residue sequence of sequence 2 through replacement, disappearance or the interpolation of one or several amino-acid residue and have identical active by sequence 2 deutero-protein or polypeptide with the amino acid residue sequence of sequence 2.

People's epididymis secretory protein called after of the present invention: HEL-28 is made up of 1663 amino-acid residues.HEL-1 albumen has 3 N glycosylation sites; 16 N-tetradecanoyl sites and 52 phosphorylation sites; comprise the protein kinase phosphorylation site that 1 cAMP relies on; 22 casein kinase i I phosphorylation sites and 26 protein kinase C phosphorylation sites, 3 Tyrosylprotein kinase phosphorylation sites.And, contain 2 Anaphylatoxin functional domains in the molecule, 1 Bipartite nuclearlocalization signal profile, 1 NTR functional domain.

Second purpose of the present invention provides the gene of coding human epididymal expression albumen HEL-28.It is one of following nucleotide sequences:

The dna sequence dna of sequence 1 in the sequence table, the GenBank number of registration is: EU794602;

The polynucleotide of SEQ ID No.2 protein sequence in the code sequence tabulation;

The dna sequence dna that limits with sequence in the sequence table 1 has 90% above homology, and coding identical function protein DNA sequence.

SEQID No.1 in the sequence table, full length cDNA sequence is 5091bp, is positioned on No. 19 karyomit(e)s of people, 1663 amino acid of encoding, proteins encoded is HEL-28, contains signal peptide, its open reading frame is 4992bp, is a complete reading frame.

A third aspect of the present invention provides and has adopted the genetic engineering technique preparation to have the method for human epididymis expression sperm binding protein HEL-28 active polypeptide, and it comprises the carrier that contains above-mentioned polynucleotide sequence, and the host cell that is transformed or transduce by this carrier.

A fourth aspect of the present invention provides and above-mentioned human epididymis expression sperm binding protein HEL-28 specificity bonded antibody, and the auxiliary diagnosis of the infertility of developing thus that is used for the epididymal function caused by abnormal.

A fifth aspect of the present invention, the method that whether has human epididymis expression sperm binding protein HEL-28 in the sample that detects is provided, it comprises: with the specific antibody acting in conjunction under certain condition of testing sample and secretory protein, observe whether form immune complex, have immune complex to form just to point out and have secretory protein in the sample.

A sixth aspect of the present invention provides the purposes of albumen of the present invention and its encoding sequence.Albumen for example of the present invention can be used as the molecular target of diagnosis infertility, or be used for the treatment of infertility as the active drug composition, or as the target molecule of development of new contraceptive, or be used to screening and promote the active agonist of human epididymis expression sperm binding protein HEL-28, or be used to the active antagonist of screening inhibition human epididymis expression sperm binding protein HEL-28, or be used to the peptide finger print identification.Encoding sequence or its fragment according to human epididymis expression sperm binding protein HEL-28 can design primer or probe and be used for PCR or nucleic acid hybridization, perhaps are used to prepare gene chip.Albumen of the present invention also can be used for pharmaceutical composition, as the effective active composition, is used to produce a kind of medicine that resists diseases such as cause pathogeny imcrobe infection and tumour with this.

A seventh aspect of the present invention provides a kind of pharmaceutical composition, and described composition comprises albumen of the present invention, nucleotide sequence, construction or cell, and pharmaceutically acceptable carrier.

Albumen of the present invention can be expressed at the epididymis internal specific, is positioned sperm head rear region, and it is relevant therefore may to discern, move, be fertilized with smart ovum.Because it is a kind of natural protein polypeptide, can predict and may have less side effect, other advantages of the present invention can be known from the following detailed description.

Description of drawings

The immunofluorescence location of Fig. 1 .HEL-28 albumen on people's sperm

Red fluorescence shows sperm nucleus; Green fluorescence shows the location of HEL-28 albumen on sperm;

The A group: positive control, A3 show that the HEL-75 protein binding is in whole sperm (article is delivered);

B group: negative control.

The C group: HEL-28 albumen location, C3. shows that the HEL-28 protein binding is in sperm head rear region.

Scale: 5mm.

Fig. 2 HEL-28 gene organization expression map

1 caput epididymidis, 2 bodies of epididymis, 3 cauda epididymidiss, 4 testis, 5 hearts, 6 livers, 7 spleens, 8 kidneys, 9 stomaches, 10 negative controls

The western blot result that Fig. 3 .HEL-28 albumen is expressed at epididymal fluid

M:Marker; 1: positive control, HEL-75 albumen; 2: negative control, mice serum; 3:HEL-28 albumen

Albumen of the present invention has comprised the variant form of the same or similar inhibition biological active of having of described polypeptide or function, these variant forms comprise (but being not limited to): the amino acid sequence with respect to native protein has several (to be generally 1-50, preferably 1-30,1-20 more preferably, best 1-10) amino acid whose disappearance, insertion and replacement. In addition, described disappearance or insertion (increase) also can occur in the C end and/or the N end (has in 20 usually, preferably be in 10, more preferably be 5 with interior amino acid deletions or increase), in the art, when replacing with the close or similar amino acid of performance, usually can not change amino acid whose function, it is known in the art that the amino acid whose preservative replacement table of functional similarity is provided. Following 5 groups contain the mutually amino acid of conservative substitution separately; Aliphatic series: glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I); Aromatics: phenylalanine (F), tyrosine (Y), tryptophan (W); Sulfur-bearing: methionine (M), cysteine (C); Alkalescence: arginine (R), lysine (K), histidine (H); Acid: aspartic acid (D), glutamine (Q). In addition, this term has also comprised fragment or the derivative of albumen, and condition is that this fragment or derivative have kept desirable protein biological activity.

Nucleotide sequence of the present invention can use pcr amplification method, recombination method or artificial synthetic method to obtain usually. For the pcr amplification method, can be disclosed according to the present invention relevant nucleotide sequence, especially open reading frame sequence designs primer, with the prepared cDNA library of conventional method well known by persons skilled in the art as template, amplification and must be about sequence. This normally is cloned into carrier with it, changes cell over to again, separates then obtaining relevant sequence from the host cell after the propagation by conventional method.

Among the present invention, use conventional method well known by persons skilled in the art to comprise to encode the nucleotide sequence of albumen of the present invention to be inserted in the carrier, these methods include but not limited to the extracorporeal recombinant DNA technology, recombinant technique etc. in the body.

Above-mentioned carrier can be used for transforming or transfection is suitable protokaryon and eukaryotic, so that it can express coded albumen of the present invention, host cell can be prokaryotic, such as bacterial cell, or the eukaryotic such as low, such as yeast cells; Or higher eucaryotic cells, such as insect cell etc. Adoptable conversion, transfection method include, but are not limited to: calcium phosphate precipitation, microinjection, electroporation, liposome-mediated etc.

Albumen of the present invention if need, can utilize its physics, chemical separating by the whole bag of tricks and the purifying expression product with other characteristics at cell inner expression, and these methods are well-known to those skilled in the art. The example of these methods includes but not limited to: conventional renaturation processes, process the combination of (salt analysis method), centrifugal, the broken bacterium of infiltration, the broken bacterium of sound wave, super centrifugal, sieve chromatography, adsorption chromatography, ion-exchange chromatography, high performance liquid chroma-tography and other various chromatographic techniques and these methods with conventional protein precipitant.

The present invention comprises that also albumen or its fragment are had specific polyclonal antibody and monoclonal antibody, also comprises having immunocompetent antibody fragment or chimeric antibody, as has mouse-anti body binding specificity but still keep antibody from people's antibody moiety.

Antibody of the present invention can be prepared by the known various technology of those skilled in that art. For example the gene outcome of recombinant antibodies, purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody, and antibody of the present invention also can be monoclonal antibody. This type of monoclonal antibody can utilize hybridoma technology to prepare.

The inventor finds further that by research this protein localization is in a back zone of mature sperm (Fig. 1). RT-PCR result shows that this albumen is in epididymis and the various tissue expression amounts of human body very low (Fig. 2). The inventor at expression in escherichia coli people HEL-28 albumen, use its immune mouse, obtained its polyvalent antibody. The inventor detects a 187KD band (Fig. 3) with Western blot at the epididymis body.

Drug regimen of the present invention can be made various formulations according to various needs, usually pharmaceutical composition can be made injectable agent, for example liquid solution or suspension; Also can be made into before injection, be fit to allocate in solution or the suspension, the solid form of solution carrier, liposome is also included within the definition of pharmaceutically acceptable carrier, pharmaceutical composition of the present invention can by the doctor according to patient's kind, age, body weight and roughly the factor such as disease condition, administering mode determine the useful dosage of patient is used, in order to improve the medication effect, albumen of the present invention also can use jointly with other drug.

Below in conjunction with instantiation, further set forth the present invention, should understand these examples only is used for explanation the present invention and is not used in and limits the scope of the invention, unless description is arranged in addition, enforcement of the present invention will be adopted molecular biology, microbiology, recombinant DNA and immunologic routine techniques, and these all are known to those skilled in the art. These technology have complete description below in the document: Sambrook " molecular cloning experiment guide " second edition (1989) for example; " dna clone " I and II volume (D.N.Glover edits, 1985); " oligonucleotides is synthetic " (M.J.Gait edits, 1984); " nucleic acid hybridization " (B.D.Hames and S. J. Higgins edits. 1984); " protein purification; Principle and put into practice " the 2nd edition (Spring-Verlag, N.Y.), and " experiment immunization learn handbook I-IV volume (D.C.Weir and C.C.Blackwell edit 1986) or, can carry out according to the specification that the reagent manufacturer provides.

Embodiment

Embodiment 1. adopts genetic engineering technique to prepare human epididymal expression albumen HEL-28

Gene clone and sequential analysis

The large scale sequencing screening is carried out in people's epididymis cDNA library that this laboratory makes up, obtained expressed sequence tag (EST), utilize the Unigene database to carry out electronic cloning, obtain people HEL-28 full-length cDNA.Use Expasy Translate tool (http://us.expasy.orghttp: //us.expasy.org), InterProScan (http://www.ebi.ac.uk/Tools/http: //www.ebi.ac.uk/Tools/) and Protein Stakes-of-wood roteinblast (http://www.ncbi.nlm.nih.gov/BLAST/http: //www.ncbi.nlm.nih.gov/BLAST/) wait prediction HEL-28 peptide sequence and supposition function.SignalP (http://www.cbs.dtu.dkhttp: //www.cbs.dtu.dk), location in PSORTII and WoLFPSORT software analysis such as (http://psort.nibb.ac.jp) this proteic signal peptide of prediction and the born of the same parents.ProfileScan (http://myhits.isb-sib.ch/cgi-bin/PFSCANhttp: //myhits.isb-sib.ch/cgi-bin/PFSCAN) prediction N holds glycosylation and phosphorylation site.

To the people HEL-28 full-length cDNA that obtains, gene order according to information biology predictive coding HEL-28 mature peptide designs a pair of special primer: F01:5 '-TTGGTACCGACGACGACGACAAG GGACCCACCTCAGGTC-3 ', F02:5 '-GCGGCGAATTC GTTGGGGCACCCAAA-3 ', upstream and downstream primer two ends are introduced restriction endonuclease sites KpnI and EcoRI respectively.Primer is synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.The people's epididymis cDNA library that makes up with this laboratory is a template, pcr amplification HEL-28 gene fragment.The PCR reaction conditions: 94 ℃ of pre-sex change 10min, (94 ℃, 40s; 55 ℃, 45s; 70 ℃, 30s) 30 circulations, 70 ℃ are extended 10min, 4 ℃ of preservations.After reaction finished, 1.0% agarose gel electrophoresis detected and Separation and Recovery purpose fragment, inserted the evaluation of checking order among the cloning vector pMD-18T.

Reorganization HEL-28 protein expression and purifying

Order-checking identifies that back HEL-28 gene is cloned among the expression vector pET-32b (+) by KpnI and EcoRI site, and it is consistent to make itself and fusion tag read frame.Recombinant expression vector pET32b (+)-HEL-28 changes E.coli BL21 (DE3) competent cell over to, utilize bacterium colony PCR screening positive clone, engineering strain is at 1mM IPTG, after inducing 4h under 32 ℃ of conditions, the recombinant protein that the N end has His-tag carries out separation and purification by " two step nickel affinity chromatography methods " to reorganization HEL-28 albumen.Briefly, " the first step affinity chromatography " is used for purification of Recombinant Trx-HEL-28 fusion rotein.Fusion rotein recombinant enterokinase cracking then discharges fusion tag.At last, reorganization HEL-28 albumen is reclaimed in utilization " the second step affinity chromatography ".Recombinant protein behind the purifying carries out quantitatively preserving with lyophilize then with Bradford (Bradford 1976) method.

The preparation of embodiment 2. anti-HEL-28 polyclonal antiserums

How anti-with the preparation of reorganization HEL-28 protein immunization BALB/C mice.Briefly, every mouse was injected the complete freund adjuvant (CFA) of 50 μ g recombinant proteins and equivalent in the 1st day.Injected incomplete freund adjuvant (IFA) booster immunization of 25 μ g recombinant proteins and equivalent then at the 15th, 30 and 45 day.Get blood from eyeball on the 60th day, and analyzed tiring and specificity of antibody with ELASA and western blot behind the separation of serum.Elisa assay shows that antibody titer reaches 1: 10000.Westernblot shows that the natural HEL-28 that extracts to recombinant protein with from people's epididymal fluid has good specificity.

The research of embodiment 3. people HEL-28mRNA distribution expression patterns

In order to determine the expression pattern of HEL-28, adopt sxemiquantitative RT-PCR to analyze the HEL-28 gene, testis, the heart, liver, spleen, lung, kidney, the differential expression in the tissue such as stomach at people's caput epididymidis, body, tail.With TRIzol (Tiangen, Beijing, China) extracted total RNA, the total RNA of 1ug 20UAMV reversed transcriptive enzyme (Promega) and 0.3ugoligod _T18 (Promega) reverse transcription becomes cDNA.Then, 2ul synthetic cDNA utilizes gene specific primer to carry out pcr amplification.The 20ul reaction system contains: 2ul10 * PCR buffer (with MgCl ₂), 2ul dNTP Mix (10mmol/L), each draws ul (25umol/L) 1ul, 1ul Taq archaeal dna polymerase (2.5U/ul), 2ulcDNA template and 11ulddH ₂O.The program of PCR reaction is: 94 ℃, and 10min; 94 ℃, 1min; 54 ℃ of (to HEL-28)/49 ℃ (30s of β-actin); 72 ℃, 1min (35 circulation); 72 ℃ are extended 7min.The expression of β-actin is as confidential reference items.All pcr amplification products 1.5% agarose electrophoretic analysis.RT-PCR result shows that this albumen is very low at epididymis and the various tissue expression amounts of human body.

Embodiment 4. people HEL-28 albumen are in the immunofluorescence location of tissue

Get people's testis, epididymis tissue freezing section, carry out immunofluorescence dyeing, wherein one anti-is mouse-anti rHEL-28 how anti-(1: 400), and two anti-ly are FITC-mark sheep anti-mouse igg (1: 200), and nucleus dyes with PI.Sections all after the dyeing are used Laser Scanning Confocal Microscope (LeicaTCSSP2 AOBS) observations then with 80% glycerine mounting.Experiment is anti-as negative control with blood serum substituting before the mouse immune one simultaneously.

Embodiment 5. people's epididymal proteins extracts and Western blot

The total protein extract 20ug that will extract from people's epididymal fluid separates with 15%SDS-PAGE, and wet then forwarding to carried out western blot on the pvdf membrane.Mouse-anti people HEL-28 is how anti-to be one anti-(1: 5000), and two anti-ly are HRP-mark sheep anti-mouse igg (1: 500).The activity of horseradish peroxidase is analyzed Westernblot with enhancement type HRP-DAB substrate colouring reagents box and is shown anti-people HEL-28 resists the natural HEL-28 that extracts to recombinant protein with from people's epididymal fluid to have good specificity more.

Embodiment 6. people HEL-28 albumen are in the immunofluorescence location of sperm

Collect and coat on the slide glass of 1% gelatin bag quilt after sperm washs with PBS, dry naturally, fix 10 minutes with methyl alcohol then.The slide glass that contains sperm at room temperature sealed 1 hour with 3%BSA, added mouse-anti HEL-28 then and resisted (1: 200) more, and 4 ℃ are spent the night.Mice serum is done negative control before the immunity.With PBST (PBScontaining0.1%Tween-20) washing three times, add corresponding FITC-mark sheep anti-mouse igg two anti-(1: 200).Slide glass is used 80% glycerine mounting then with PBST washing three times.With Olympus BX-52 microscopic examination, the HEL-28 protein binding may and be moved, be fertilized relevant with smart ovum identification in sperm head back zone.

The anti-microbial activity of embodiment 7.HEL-28 recombinant protein

Utilization clone's colony-forming unit (CFU) is analyzed anti-microbial activity.Briefly, the E.coli XL-1blue of incubated overnight grows into 10mM PBS (pH 7.4) dilution of logarithmic phase (OD600=0.4-0.5) back.About 2 * 10 ⁶The CFU/ml bacterium is mixing with the HEL-28 of 12.5～100ug/ml, 37 ℃ of cultivations.15,30,60 and 120min after beginning to cultivate is respectively in sampling.Sample is done serial dilution with 10mMPBS (pH7.4), and each extent of dilution is got 100ul and is coated the LB agar plate, and 37 ℃ of overnight incubation are to forming single bacterium colony.Statistics bacterium colony number, anti-microbial activity represents that with the percentage ratio of bacteria living formula is: % survival=(albumen handle back survival clone number/handle clone's number of surviving in the back without albumen) * 100.Experiment replaces albumen to handle bacterium as negative control with 10mM PBS (pH 7.4).

Embodiment 8HEL-28 and sperm motility and relation research in vitro fertilization

Utilize HEL-28 to resist sealing normal people sperm more, carry out sperm motility and analysis in vitro fertilization then.Experiment is carried out according to the method that vitro life standard reagent box provides.Briefly, sperm uses SpermRinse-30 in 37 ℃, 5%CO earlier ₂Cultivate capacitation in 1 hour.Added mouse-anti rHEL-28 according to 1: 200 then and resist more, in 37 ℃, 6%CO ₂Cultivate and sealed in 2 hours.Sperm separated into two parts after the sealing, a part directly utilize computer aided system to analyze the mobility of sperm.Another part is with G-Fert washing 2 times, and to be adjusted to final concentration be 1.0 * 10 ⁷Cells/ml.Add the 100ul sample of sperm in the culture dish of 35mm, cover paraffin oil then and put into 37 ℃, 6%CO ₂With stand-by in the saturated humidity incubator.Handle the after ripening ovocyte through G-MOPS and add during preprepared fertilization drips, at 37 ℃, 6%CO ₂Observe the fertilization situation with cultivating in the saturated humidity incubator, write down the fetal development situation every day.More than all the experiment use simultaneously the immunity before mice serum as negative control.

Sequence table

＜110〉Li Jianyuan

＜120〉human epididymis expression sperm binding protein HEL-28 and encoding gene thereof and application

<160>2

<170>PatentIn?version?3.5

<210>1

<211>5091

<212>DNA

<213>Homo?sapiens

<220>

<221>CDS

<222>(33)..(5024)

<400>1

ctgtccctct?gaccctgcac?tgtcccagca?cc?atg?gga?ccc?acc?tca?ggt?ccc 53

Met?Gly?Pro?Thr?Ser?Gly?Pro

1 5

agc?ctg?ctg?ctc?ctg?cta?cta?acc?cac?ctc?ccc?ctg?gct?ctg?ggg?agt 101

Ser?Leu?Leu?Leu?Leu?Leu?Leu?Thr?His?Leu?Pro?Leu?Ala?Leu?Gly?Ser

10 15 20

ccc?atg?tac?tct?atc?atc?acc?ccc?aac?atc?ttg?cgg?ctg?gag?agc?gag 149

Pro?Met?Tyr?Ser?Ile?Ile?Thr?Pro?Asn?Ile?Leu?Arg?Leu?Glu?Ser?Glu

25 30 35

gag?acc?atg?gtg?ctg?gag?gcc?cac?gac?gcg?caa?ggg?gat?gtt?cca?gtc 197

Glu?Thr?Met?Val?Leu?Glu?Ala?His?Asp?Ala?Gln?Gly?Asp?Val?Pro?Val

40 45 50 55

act?gtt?act?gtc?cac?gac?ttc?cca?ggc?aaa?aaa?cta?gtg?ctg?tcc?agt 245

Thr?Val?Thr?Val?His?Asp?Phe?Pro?Gly?Lys?Lys?Leu?Val?Leu?Ser?Ser

60 65 70

gag?aag?act?gtg?ctg?acc?cct?gcc?acc?aac?cac?atg?ggc?aac?gtc?acc 293

Glu?Lys?Thr?Val?Leu?Thr?Pro?Ala?Thr?Asn?His?Met?Gly?Asn?Val?Thr

75 80 85

ttc?acg?atc?cca?gcc?aac?agg?gag?ttc?aag?tca?gaa?aag?ggg?cgc?aac 341

Phe?Thr?Ile?Pro?Ala?Asn?Arg?Glu?Phe?Lys?Ser?Glu?Lys?Gly?Arg?Asn

90 95 100

aag?ttc?gtg?acc?gtg?cag?gcc?acc?ttc?ggg?acc?caa?gtg?gtg?gag?aag 389

Lys?Phe?Val?Thr?Val?Gln?Ala?Thr?Phe?Gly?Thr?Gln?Val?Val?Glu?Lys

105 110 115

gtg?gtg?ctg?gtc?agc?ctg?cag?agc?ggg?tac?ctc?ttc?atc?cag?aca?gac 437

Val?Val?Leu?Val?Ser?Leu?Gln?Ser?Gly?Tyr?Leu?Phe?Ile?Gln?Thr?Asp

120 125 130 135

aag?acc?atc?tac?acc?cct?ggc?tcc?aca?gtt?ctc?tat?cgg?atc?ttc?acc 485

Lys?Thr?Ile?Tyr?Thr?Pro?Gly?Ser?Thr?Val?Leu?Tyr?Arg?Ile?Phe?Thr

140 145 150

gtc?aac?cac?aag?ctg?cta?ccc?gtg?ggc?cgg?acg?gtc?atg?gtc?aac?att 533

Val?Asn?His?Lys?Leu?Leu?Pro?Val?Gly?Arg?Thr?Val?Met?Val?Asn?Ile

155 160 165

gag?aac?ccg?gaa?ggc?atc?ccg?gtc?aag?cag?gac?tcc?ttg?tct?tct?cag 581

Glu?Asn?Pro?Glu?Gly?Ile?Pro?Val?Lys?Gln?Asp?Ser?Leu?Ser?Ser?Gln

170 175 180

aac?cag?ctt?ggc?gtc?ttg?ccc?ttg?tct?tgg?gac?att?ccg?gaa?ctc?gtc 629

Asn?Gln?Leu?Gly?Val?Leu?Pro?Leu?Ser?Trp?Asp?Ile?Pro?Glu?Leu?Val

185 190 195

aac?atg?ggc?cag?tgg?aag?atc?cga?gcc?tac?tat?gaa?aac?tca?cca?cag 677

Asn?Met?Gly?Gln?Trp?Lys?Ile?Arg?Ala?Tyr?Tyr?Glu?Asn?Ser?Pro?Gln

200 205 210 215

cag?gtc?ttc?tcc?act?gag?ttt?gag?gtg?aag?gag?tac?gtg?ctg?ccc?agt 725

Gln?Val?Phe?Ser?Thr?Glu?Phe?Glu?Val?Lys?Glu?Tyr?Val?Leu?Pro?Ser

220 225 230

ttc?gag?gtc?ata?gtg?gag?cct?aca?gag?aaa?ttc?tac?tac?atc?tat?aac 773

Phe?Glu?Val?Ile?Val?Glu?Pro?Thr?Glu?Lys?Phe?Tyr?Tyr?Ile?Tyr?Asn

235 240 245

gag?aag?ggc?ctg?gag?gtc?acc?atc?acc?gcc?agg?ttc?ctc?tac?ggg?aag 821

Glu?Lys?Gly?Leu?Glu?Val?Thr?Ile?Thr?Ala?Arg?Phe?Leu?Tyr?Gly?Lys

250 255 260

aaa?gtg?gag?gga?act?gcc?ttt?gtc?atc?ttc?ggg?atc?cag?gat?ggc?gaa 869

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

265 270 275

cag?agg?att?tcc?ctg?cct?gaa?tcc?ctc?aag?cgc?att?ccg?att?gag?gat 917

Gln?Arg?Ile?Ser?Leu?Pro?Glu?Ser?Leu?Lys?Arg?Ile?Pro?Ile?Glu?Asp

280 285 290 295

ggc?tcg?ggg?gag?gtt?gtg?ctg?agc?cga?aag?gta?ctg?ctg?gac?ggg?gtg 965

Gly?Ser?Gly?Glu?Val?Val?Leu?Ser?Arg?Lys?Val?Leu?Leu?Asp?Gly?Val

300 305 310

cag?aac?ccc?cga?gca?gaa?gac?ctg?gtg?ggg?aag?tct?ttg?tac?gtg?tct 1013

Gln?Asn?Pro?Arg?Ala?Glu?Asp?Leu?Val?Gly?Lys?Ser?Leu?Tyr?Val?Ser

315 320 325

gcc?acc?gtc?atc?ttg?cac?tca?ggc?agt?gac?atg?gtg?cag?gca?gag?cgc 1061

Ala?Thr?Val?Ile?Leu?His?Ser?Gly?Ser?Asp?Met?Val?Gln?Ala?Glu?Arg

330 335 340

agc?ggg?atc?ccc?atc?gtg?acc?tct?ccc?tac?cag?atc?cac?ttc?acc?aag 1109

Ser?Gly?Ile?Pro?Ile?Val?Thr?Ser?Pro?Tyr?Gln?Ile?His?Phe?Thr?Lys

345 350 355

aca?ccc?aag?tac?ttc?aaa?cca?gga?atg?ccc?ttt?gac?ctc?atg?gtg?ttc 1157

Thr?Pro?Lys?Tyr?Phe?Lys?Pro?Gly?Met?Pro?Phe?Asp?Leu?Met?Val?Phe

360 365 370 375

gtg?acg?aac?cct?gat?ggc?tct?cca?gcc?tac?cga?gtc?ccc?gtg?gca?gtc 1205

Val?Thr?Asn?Pro?Asp?Gly?Ser?Pro?Ala?Tyr?Arg?Val?Pro?Val?Ala?Val

380 385 390

cag?ggc?gag?gac?act?gtg?cag?tct?cta?acc?cag?gga?gat?ggc?gtg?gcc 1253

Gln?Gly?Glu?Asp?Thr?Val?Gln?Ser?Leu?Thr?Gln?Gly?Asp?Gly?Val?Ala

395 400 405

aaa?ctc?agc?atc?aac?aca?cac?ccc?agc?cag?aag?ccc?ttg?agc?atc?acg 1301

Lys?Leu?Ser?Ile?Asn?Thr?His?Pro?Ser?Gln?Lys?Pro?Leu?Ser?Ile?Thr

410 415 420

gtg?cgc?acg?aag?aag?cag?gag?ctc?tcg?gag?gca?gag?cag?gct?acc?agg 1349

Val?Arg?Thr?Lys?Lys?Gln?Glu?Leu?Ser?Glu?Ala?Glu?Gln?Ala?Thr?Arg

425 430 435

acc?atg?cag?gct?ctg?ccc?tac?agc?acc?gtg?ggc?aac?tcc?aac?aat?tac 1397

Thr?Met?Gln?Ala?Leu?Pro?Tyr?Ser?Thr?Val?Gly?Asn?Ser?Asn?Asn?Tyr

440 445 450 455

ctg?cat?ctc?tca?gtg?cta?cgt?aca?gag?ctc?aga?ccc?ggg?gag?acc?ctc 1445

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

460 465 470

aac?gtc?aac?ttc?ctc?ctg?cga?atg?gac?cgc?gcc?cac?gag?gcc?aag?atc 1493

Asn?Val?Asn?Phe?Leu?Leu?Arg?Met?Asp?Arg?Ala?His?Glu?Ala?Lys?Ile

475 480 485

cgc?tac?tac?acc?tac?ctg?atc?atg?aac?aag?ggc?agg?ctg?ttg?aag?gcg 1541

Arg?Tyr?Tyr?Thr?Tyr?Leu?Ile?Met?Asn?Lys?Gly?Arg?Leu?Leu?Lys?Ala

490 495 500

gga?cgc?cag?gtg?cga?gag?ccc?ggc?cag?gac?ctg?gtg?gtg?ctg?ccc?ctg 1589

Gly?Arg?Gln?Val?Arg?Glu?Pro?Gly?Gln?Asp?Leu?Val?Val?Leu?Pro?Leu

505 510 515

tcc?atc?acc?acc?gac?ttc?atc?cct?tcc?ttc?cgc?ctg?gtg?gcg?tac?tac 1637

Ser?Ile?Thr?Thr?Asp?Phe?Ile?Pro?Ser?Phe?Arg?Leu?Val?Ala?Tyr?Tyr

520 525 530 535

acg?ctg?atc?ggt?gcc?agc?ggc?cag?agg?gag?gtg?gtg?gcc?gac?tcc?gtg 1685

Thr?Leu?Ile?Gly?Ala?Ser?Gly?Gln?Arg?Glu?Val?Val?Ala?Asp?Ser?Val

540 545 550

tgg?gtg?gac?gtc?aag?gac?tcc?tgc?gtg?ggc?tcg?ctg?gtg?gta?aaa?agc 1733

Trp?Val?Asp?Val?Lys?Asp?Ser?Cys?Val?Gly?Ser?Leu?Val?Val?Lys?Ser

555 560 565

ggc?cag?tca?gaa?gac?cgg?cag?cct?gta?cct?ggg?cag?cag?atg?acc?ctg 1781

Gly?Gln?Ser?Glu?Asp?Arg?Gln?Pro?Val?Pro?Gly?Gln?Gln?Met?Thr?Leu

570 575 580

aag?ata?gag?ggt?gac?cac?ggg?gcc?cgg?gtg?gta?ctg?gtg?gcc?gtg?gac 1829

Lys?Ile?Glu?Gly?Asp?His?Gly?Ala?Arg?Val?Val?Leu?Val?Ala?Val?Asp

585 590 595

aag?ggc?gtg?ttc?gtg?ctg?aat?aag?aag?aac?aaa?ctg?aca?cag?agt?aag 1877

Lys?Gly?Val?Phe?Val?Leu?Asn?Lys?Lys?Asn?Lys?Leu?Thr?Gln?Ser?Lys

600 605 610 615

atc?tgg?gac?gtg?gtg?gag?aag?gca?gac?atc?ggc?tgc?acc?ccg?ggc?agt 1925

Ile?Trp?Asp?Val?Val?Glu?Lys?Ala?Asp?Ile?Gly?Cys?Thr?Pro?Gly?Ser

620 625 630

ggg?aag?gat?tac?gcc?ggt?gtc?ttc?tcc?gac?gca?ggg?ctg?acc?ttc?acg 1973

Gly?Lys?Asp?Tyr?Ala?Gly?Val?Phe?Ser?Asp?Ala?Gly?Leu?Thr?Phe?Thr

635 640 645

agc?agc?agt?ggc?cag?cag?acc?gcc?cag?agg?gca?gaa?ctt?cag?tgc?ccg 2021

Ser?Ser?Ser?Gly?Gln?Gln?Thr?Ala?Gln?Arg?Ala?Glu?Leu?Gln?Cys?Pro

650 655 660

cag?cca?gcc?gcc?cgc?cga?cgc?cgt?tcc?gtg?cag?ctc?acg?gag?aag?cga 2069

Gln?Pro?Ala?Ala?Arg?Arg?Arg?Arg?Ser?Val?Gln?Leu?Thr?Glu?Lys?Arg

665 670 675

atg?gac?aaa?gtc?ggc?aag?tac?ccc?aag?gag?ctg?cgc?aag?tgc?tgc?gag 2117

Met?Asp?Lys?Val?Gly?Lys?Tyr?Pro?Lys?Glu?Leu?Arg?Lys?Cys?Cys?Glu

680 685 690 695

gac?ggc?atg?cgg?gag?aac?ccc?atg?agg?ttc?tcg?tgc?cag?cgc?cgg?acc 2165

Asp?Gly?Met?Arg?Glu?Asn?Pro?Met?Arg?Phe?Ser?Cys?Gln?Arg?Arg?Thr

700 705 710

cgt?ttc?atc?tcc?ctg?ggc?gag?gcg?tgc?aag?aag?gtc?ttc?ctg?gac?tgc 2213

Arg?Phe?Ile?Ser?Leu?Gly?Glu?Ala?Cys?Lys?Lys?Val?Phe?Leu?Asp?Cys

715 720 725

tgc?aac?tac?atc?aca?gag?ctg?cgg?cgg?cag?cac?gcg?cgg?gcc?agc?cac 2261

Cys?Asn?Tyr?Ile?Thr?Glu?Leu?Arg?Arg?Gln?His?Ala?Arg?Ala?Ser?His

730 735 740

ctg?ggc?ctg?gcc?agg?agt?aac?ctg?gat?gag?gac?atc?att?gca?gaa?gag 2309

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

745 750 755

aac?atc?gtt?tcc?cga?agt?gag?ttc?cca?gag?agc?tgg?ctg?tgg?aac?gtt 2357

Asn?Ile?Val?Ser?Arg?Ser?Glu?Phe?Pro?Glu?Ser?Trp?Leu?Trp?Asn?Val

760 765 770 775

gag?gac?ttg?aaa?gag?cca?ccg?aaa?aat?gga?atc?tct?acg?aag?ctc?atg 2405

Glu?Asp?Leu?Lys?Glu?Pro?Pro?Lys?Asn?Gly?Ile?Ser?Thr?Lys?Leu?Met

780 785 790

aat?ata?ttt?ttg?aaa?gac?tcc?atc?acc?acg?tgg?gag?att?ctg?gct?gtc 2453

Asn?Ile?Phe?Leu?Lys?Asp?Ser?Ile?Thr?Thr?Trp?Glu?Ile?Leu?Ala?Val

795 800 805

agc?atg?tcg?gac?aag?aaa?ggg?atc?tgt?gtg?gca?gac?ccc?ttc?gag?gtc 2501

Ser?Met?Ser?Asp?Lys?Lys?Gly?Ile?Cys?Val?Ala?Asp?Pro?Phe?Glu?Val

810 815 820

aca?gta?atg?cag?gac?ttc?ttc?atc?gac?ctg?cgg?cta?ccc?tac?tct?gtt 2549

Thr?Val?Met?Gln?Asp?Phe?Phe?Ile?Asp?Leu?Arg?Leu?Pro?Tyr?Ser?Val

825 830 835

gtt?cga?aac?gag?cag?gtg?gaa?atc?cga?gcc?gtt?ctc?tac?aat?tac?cgg 2597

Val?Arg?Asn?Glu?Gln?Val?Glu?Ile?Arg?Ala?Val?Leu?Tyr?Asn?Tyr?Arg

840 845 850 855

cag?aac?caa?gag?ctc?aag?gtg?agg?gtg?gaa?cta?ctc?cac?aat?cca?gcc 2645

Gln?Asn?Gln?Glu?Leu?Lys?Val?Arg?Val?Glu?Leu?Leu?His?Asn?Pro?Ala

860 865 870

ttc?tgc?agc?ctg?gcc?acc?acc?aag?agg?cgt?cac?cag?cag?acc?gta?acc 2693

Phe?Cys?Ser?Leu?Ala?Thr?Thr?Lys?Arg?Arg?His?Gln?Gln?Thr?Val?Thr

875 880 885

atc?ccc?ccc?aag?tcc?tcg?ttg?tcc?gtt?cca?tat?gtc?atc?gtg?ccg?cta 2741

Ile?Pro?Pro?Lys?Ser?Ser?Leu?Ser?Val?Pro?Tyr?Val?Ile?Val?Pro?Leu

890 895 900

aag?acc?ggc?ctg?cag?gaa?gtg?gaa?gtc?aag?gct?gcc?gtc?tac?cat?cat 2789

Lys?Thr?Gly?Leu?Gln?Glu?Val?Glu?Val?Lys?Ala?Ala?Val?Tyr?His?His

905 910 915

ttc?atc?agt?gac?ggt?gtc?agg?aag?tcc?ctg?aag?gtc?gtg?ccg?gaa?gga 2837

Phe?Ile?Ser?Asp?Gly?Val?Arg?Lys?Ser?Leu?Lys?Val?Val?Pro?Glu?Gly

920 925 930 935

atc?aga?atg?aac?aaa?act?gtg?gct?gtt?cgc?acc?ctg?gat?cca?gaa?cgc 2885

Ile?Arg?Met?Asn?Lys?Thr?Val?Ala?Val?Arg?Thr?Leu?Asp?Pro?Glu?Arg

940 945 950

ctg?ggc?cgt?gaa?gga?gtg?cag?aaa?gag?gac?atc?cca?cct?gca?gac?ctc 2933

Leu?Gly?Arg?Glu?Gly?Val?Gln?Lys?Glu?Asp?Ile?Pro?Pro?Ala?Asp?Leu

955 960 965

agt?gac?caa?gtc?ccg?gac?acc?gag?tct?gag?acc?aga?att?ctc?ctg?caa 2981

Ser?Asp?Gln?Val?Pro?Asp?Thr?Glu?Ser?Glu?Thr?Arg?Ile?Leu?Leu?Gln

970 975 980

ggg?acc?cca?gtg?gcc?cag?atg?aca?gag?gat?gcc?gtc?gac?gcg?gaa?cgg 3029

Gly?Thr?Pro?Val?Ala?Gln?Met?Thr?Glu?Asp?Ala?Val?Asp?Ala?Glu?Arg

985 990 995

ctg?aag?cac?ctc?att?gtg?acc?ccc?tcg?ggc?tgc?ggg?gaa?cag?aac 3074

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

1000 1005 1010

atg?atc?ggc?atg?acg?ccc?acg?gtc?atc?gct?gtg?cat?tac?ctg?gat 3119

Met?Ile?Gly?Met?Thr?Pro?Thr?Val?Ile?Ala?Val?His?Tyr?Leu?Asp

1015 1020 1025

gaa?acg?gag?cag?tgg?gag?aag?ttc?ggc?cta?gag?aag?cgg?cag?ggg 3164

Glu?Thr?Glu?Gln?Trp?Glu?Lys?Phe?Gly?Leu?Glu?Lys?Arg?Gln?Gly

1030 1035 1040

gcc?ttg?gag?ctc?atc?aag?aag?ggg?tac?acc?cag?cag?ctg?gcc?ttc 3209

Ala?Leu?Glu?Leu?Ile?Lys?Lys?Gly?Tyr?Thr?Gln?Gln?Leu?Ala?Phe

1045 1050 1055

aga?caa?ccc?agc?tct?gcc?ttt?gcg?gcc?ttc?gtg?aaa?cgg?gca?ccc 3254

Arg?Gln?Pro?Ser?Ser?Ala?Phe?Ala?Ala?Phe?Val?Lys?Arg?Ala?Pro

1060 1065 1070

agc?acc?tgg?ctg?acc?gcc?tac?gtg?gtc?aag?gtc?ttc?tct?ctg?gct 3299

Ser?Thr?Trp?Leu?Thr?Ala?Tyr?Val?Val?Lys?Val?Phe?Ser?Leu?Ala

1075 1080 1085

gtc?aac?ctc?atc?gcc?atc?gac?tcc?caa?gtc?ctc?tgc?ggg?gct?gtt 3344

Val?Asn?Leu?Ile?Ala?Ile?Asp?Ser?Gln?Val?Leu?Cys?Gly?Ala?Val

1090 1095 1100

aaa?tgg?ctg?atc?ctg?gag?aag?cag?aag?ccc?gac?ggg?gtc?ttc?cag 3389

Lys?Trp?Leu?Ile?Leu?Glu?Lys?Gln?Lys?Pro?Asp?Gly?Val?Phe?Gln

1105 1110 1115

gag?gat?gcg?ccc?gtg?ata?cac?caa?gaa?atg?att?ggt?gga?tta?cgg 3434

Glu?Asp?Ala?Pro?Val?Ile?His?Gln?Glu?Met?Ile?Gly?Gly?Leu?Arg

1120 1125 1130

aac?aac?aac?gag?aaa?gac?atg?gcc?ctc?acg?gcc?ttt?gtt?ctc?atc 3479

Asn?Asn?Asn?Glu?Lys?Asp?Met?Ala?Leu?Thr?Ala?Phe?Val?Leu?Ile

1135 1140 1145

tcg?ctg?cag?gag?gct?aaa?gat?att?tgc?gag?gag?cag?gtc?aac?agc 3524

Ser?Leu?Gln?Glu?Ala?Lys?Asp?Ile?Cys?Glu?Glu?Gln?Val?Asn?Ser

1150 1155 1160

ctg?cca?ggc?agc?atc?act?aaa?gca?gga?gac?ttc?ctt?gaa?gcc?aac 3569

Leu?Pro?Gly?Ser?Ile?Thr?Lys?Ala?Gly?Asp?Phe?Leu?Glu?Ala?Asn

1165 1170 1175

tac?atg?aac?cta?cag?aga?tcc?tac?act?gtg?gcc?att?gct?ggc?tat 3614

Tyr?Met?Asn?Leu?Gln?Arg?Ser?Tyr?Thr?Val?Ala?Ile?Ala?Gly?Tyr

1180 1185 1190

gct?ctg?gcc?cag?atg?ggc?agg?ctg?aag?ggg?cct?ctt?ctt?aac?aaa 3659

Ala?Leu?Ala?Gln?Met?Gly?Arg?Leu?Lys?Gly?Pro?Leu?Leu?Asn?Lys

1195 1200 1205

ttt?ctg?acc?aca?gcc?aaa?gat?aag?aac?cgc?tgg?gag?gac?cct?ggt 3704

Phe?Leu?Thr?Thr?Ala?Lys?Asp?Lys?Asn?Arg?Trp?Glu?Asp?Pro?Gly

1210 1215 1220

aag?cag?ctc?tac?aac?gtg?gag?gcc?aca?tcc?tat?gcc?ctc?ttg?gcc 3749

Lys?Gln?Leu?Tyr?Asn?Val?Glu?Ala?Thr?Ser?Tyr?Ala?Leu?Leu?Ala

1225 1230 1235

cta?ctg?cag?cta?aaa?gac?ttt?gac?ttt?gtg?cct?ccc?gtc?gtg?cgt 3794

Leu?Leu?Gln?Leu?Lys?Asp?Phe?Asp?Phe?Val?Pro?Pro?Val?Val?Arg

1240 1245 1250

tgg?ctc?aat?gaa?cag?aga?tac?tac?ggt?ggt?ggc?tat?ggc?tct?acc 3839

Trp?Leu?Asn?Glu?Gln?Arg?Tyr?Tyr?Gly?Gly?Gly?Tyr?Gly?Ser?Thr

1255 1260 1265

cag?gcc?acc?ttc?atg?gtg?ttc?caa?gcc?ttg?gct?caa?tac?caa?aag 3884

Gln?Ala?Thr?Phe?Met?Val?Phe?Gln?Ala?Leu?Ala?Gln?Tyr?Gln?Lys

1270 1275 1280

gac?gcc?cct?gac?cac?cag?gaa?ctg?aac?ctt?gat?gtg?tcc?ctc?caa 3929

Asp?Ala?Pro?Asp?His?Gln?Glu?Leu?Asn?Leu?Asp?Val?Ser?Leu?Gln

1285 1290 1295

ctg?ccc?agc?cgc?agc?tcc?aag?atc?acc?cac?cgt?atc?cac?tgg?gaa 3974

Leu?Pro?Ser?Arg?Ser?Ser?Lys?Ile?Thr?His?Arg?Ile?His?Trp?Glu

1300 1305 1310

tct?gcc?agc?ctc?ctg?cga?tca?gaa?gag?acc?aag?gaa?aat?gag?ggt 4019

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

1315 1320 1325

ttc?aca?gtc?aca?gct?gaa?gga?aaa?ggc?caa?ggc?acc?ttg?tcg?gtg 4064

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

1330 1335 1340

gtg?aca?atg?tac?cat?gct?aag?gcc?aaa?gat?caa?ctc?acc?tgt?aat 4109

Val?Thr?Met?Tyr?His?Ala?Lys?Ala?Lys?Asp?Gln?Leu?Thr?Cys?Asn

1345 1350 1355

aaa?ttc?gac?ctc?aag?gtc?acc?ata?aaa?cca?gca?ccg?gaa?aca?gaa 4154

Lys?Phe?Asp?Leu?Lys?Val?Thr?Ile?Lys?Pro?Ala?Pro?Glu?Thr?Glu

1360 1365 1370

aag?agg?cct?cag?gat?gcc?aag?aac?act?atg?atc?ctt?gag?atc?tgt 4199

Lys?Arg?Pro?Gln?Asp?Ala?Lys?Asn?Thr?Met?Ile?Leu?Glu?Ile?Cys

1375 1380 1385

acc?agg?tac?cgg?gga?gac?cag?gat?gcc?act?atg?tct?ata?ttg?gac 4244

Thr?Arg?Tyr?Arg?Gly?Asp?Gln?Asp?Ala?Thr?Met?Ser?Ile?Leu?Asp

1390 1395 1400

ata?tcc?atg?atg?act?ggc?ttt?gct?cca?gac?aca?gat?gac?ctg?aag 4289

Ile?Ser?Met?Met?Thr?Gly?Phe?Ala?Pro?Asp?Thr?Asp?Asp?Leu?Lys

1405 1410 1415

cag?ctg?gcc?aat?ggt?gtt?gac?aga?tac?atc?tcc?aag?tat?gag?ctg 4334

Gln?Leu?Ala?Asn?Gly?Val?Asp?Arg?Tyr?Ile?Ser?Lys?Tyr?Glu?Leu

1420 1425 1430

gac?aaa?gcc?ttc?tcc?gat?agg?aac?acc?ctc?atc?atc?tac?ctg?gac 4379

Asp?Lys?Ala?Phe?Ser?Asp?Arg?Asn?Thr?Leu?Ile?Ile?Tyr?Leu?Asp

1435 1440 1445

aag?gtc?tca?cac?tct?gag?gat?gac?tgt?cta?gct?ttc?aaa?gtt?cac 4424

Lys?Val?Ser?His?Ser?Glu?Asp?Asp?Cys?Leu?Ala?Phe?Lys?Val?His

1450 1455 1460

caa?tac?ttt?aat?gta?gag?ctt?atc?cag?cct?gga?gca?gtc?aag?gtc 4469

Gln?Tyr?Phe?Asn?Val?Glu?Leu?Ile?Gln?Pro?Gly?Ala?Val?Lys?Val

1465 1470 1475

tac?gcc?tat?tac?aac?ctg?gag?gaa?agc?tgt?acc?cgg?ttc?tac?cat 4514

Tyr?Ala?Tyr?Tyr?Asn?Leu?Glu?Glu?Ser?Cys?Thr?Arg?Phe?Tyr?His

1480 1485 1490

ccg?gaa?aag?gag?gat?gga?aag?ctg?aac?aag?ctc?tgc?cgt?gat?gaa 4559

Pro?Glu?Lys?Glu?Asp?Gly?Lys?Leu?Asn?Lys?Leu?Cys?Arg?Asp?Glu

1495 1500 1505

ctg?tgc?cgc?tgt?gct?gag?gag?aat?tgc?ttc?ata?caa?aag?tcg?gat 4604

Leu?Cys?Arg?Cys?Ala?Glu?Glu?Asn?Cys?Phe?Ile?Gln?Lys?Ser?Asp

1510 1515 1520

gac?aag?gtc?acc?ctg?gaa?gaa?cgg?ctg?gac?aag?gcc?tgt?gag?cca 4649

Asp?Lys?Val?Thr?Leu?Glu?Glu?Arg?Leu?Asp?Lys?Ala?Cys?Glu?Pro

1525 1530 1535

gga?gtg?gac?tat?gtg?tac?aag?acc?cga?ctg?gtc?aag?gtt?cag?ctg 4694

Gly?Val?Asp?Tyr?Val?Tyr?Lys?Thr?Arg?Leu?Val?Lys?Val?Gln?Leu

1540 1545 1550

tcc?aat?gac?ttt?gac?gag?tac?atc?atg?gcc?att?gag?cag?acc?atc 4739

Ser?Asn?Asp?Phe?Asp?Glu?Tyr?Ile?Met?Ala?Ile?Glu?Gln?Thr?Ile

1555 1560 1565

aag?tca?ggc?tcg?gat?gag?gtg?cag?gtt?gga?cag?cag?cgc?acg?ttc 4784

Lys?Ser?Gly?Ser?Asp?Glu?Val?Gln?Val?Gly?Gln?Gln?Arg?Thr?Phe

1570 1575 1580

atc?agc?ccc?atc?aag?tgc?aga?gaa?gcc?ctg?aag?ctg?gag?gag?aag 4829

Ile?Ser?Pro?Ile?Lys?Cys?Arg?Glu?Ala?Leu?Lys?Leu?Glu?Glu?Lys

1585 1590 1595

aaa?cac?tac?ctc?atg?tgg?ggt?ctc?tcc?tcc?gat?ttc?tgg?gga?gag 4874

Lys?His?Tyr?Leu?Met?Trp?Gly?Leu?Ser?Ser?Asp?Phe?Trp?Gly?Glu

1600 1605 1610

aag?ccc?aac?ctc?agc?tac?atc?atc?ggg?aag?gac?act?tgg?gtg?gag 4919

Lys?Pro?Asn?Leu?Ser?Tyr?Ile?Ile?Gly?Lys?Asp?Thr?Trp?Val?Glu

1615 1620 1625

cac?tgg?ccc?gag?gag?gac?gaa?tgc?caa?gac?gaa?gag?aac?cag?aaa 4964

His?Trp?Pro?Glu?Glu?Asp?Glu?Cys?Gln?Asp?Glu?Glu?Asn?Gln?Lys

1630 1635 1640

caa?tgc?cag?gac?ctc?ggc?gcc?ttc?acc?gag?agc?atg?gtt?gtc?ttt 5009

Gln?Cys?Gln?Asp?Leu?Gly?Ala?Phe?Thr?Glu?Ser?Met?Val?Val?Phe

1645 1650 1655

ggg?tgc?ccc?aac?tga?ccacaccccc?attcccccac?tccagataaa?gcttcagtta 5064

Gly?Cys?Pro?Asn

1660

taaaaaaaaa?aaaaaaaaaa?aaaaaaa 5091

<210>2

<211>1663

<212>PRT

<213>Homo?sapiens

<400>2

Met?Gly?Pro?Thr?Ser?Gly?Pro?Ser?Leu?Leu?Leu?Leu?Leu?Leu?Thr?His

1 5 10 15

Leu?Pro?Leu?Ala?Leu?Gly?Ser?Pro?Met?Tyr?Ser?Ile?Ile?Thr?Pro?Asn

20 25 30

Ile?Leu?Arg?Leu?Glu?Ser?Glu?Glu?Thr?Met?Val?Leu?Glu?Ala?His?Asp

35 40 45

Ala?Gln?Gly?Asp?Val?Pro?Val?Thr?Val?Thr?Val?His?Asp?Phe?Pro?Gly

50 55 60

Lys?Lys?Leu?Val?Leu?Ser?Ser?Glu?Lys?Thr?Val?Leu?Thr?Pro?Ala?Thr

65 70 75 80

Asn?His?Met?Gly?Asn?Val?Thr?Phe?Thr?Ile?Pro?Ala?Asn?Arg?Glu?Phe

85 90 95

Lys?Ser?Glu?Lys?Gly?Arg?Asn?Lys?Phe?Val?Thr?Val?Gln?Ala?Thr?Phe

100 105 110

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

115 120 125

Tyr?Leu?Phe?Ile?Gln?Thr?Asp?Lys?Thr?Ile?Tyr?Thr?Pro?Gly?Ser?Thr

130 135 140

Val?Leu?Tyr?Arg?Ile?Phe?Thr?Val?Asn?His?Lys?Leu?Leu?Pro?Val?Gly

145 150 155 160

Arg?Thr?Val?Met?Val?Asn?Ile?Glu?Asn?Pro?Glu?Gly?Ile?Pro?Val?Lys

165 170 175

Gln?Asp?Ser?Leu?Ser?Ser?Gln?Asn?Gln?Leu?Gly?Val?Leu?Pro?Leu?Ser

180 185 190

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

195 200 205

Tyr?Tyr?Glu?Asn?Ser?Pro?Gln?Gln?Val?Phe?Ser?Thr?Glu?Phe?Glu?Val

210 215 220

Lys?Glu?Tyr?Val?Leu?Pro?Ser?Phe?Glu?Val?Ile?Val?Glu?Pro?Thr?Glu

225 230 235 240

Lys?Phe?Tyr?Tyr?Ile?Tyr?Asn?Glu?Lys?Gly?Leu?Glu?Val?Thr?Ile?Thr

245 250 255

Ala?Arg?Phe?Leu?Tyr?Gly?Lys?Lys?Val?Glu?Gly?Thr?Ala?Phe?Val?Ile

260 265 270

Phe?Gly?Ile?Gln?Asp?Gly?Glu?Gln?Arg?Ile?Ser?Leu?Pro?Glu?Ser?Leu

275 280 285

Lys?Arg?Ile?Pro?Ile?Glu?Asp?Gly?Ser?Gly?Glu?Val?Val?Leu?Ser?Arg

290 295 300

Lys?Val?Leu?Leu?Asp?Gly?Val?Gln?Asn?Pro?Arg?Ala?Glu?Asp?Leu?Val

305 310 315 320

Gly?Lys?Ser?Leu?Tyr?Val?Ser?Ala?Thr?Val?Ile?Leu?His?Ser?Gly?Ser

325 330 335

Asp?Met?Val?Gln?Ala?Glu?Arg?Ser?Gly?Ile?Pro?Ile?Val?Thr?Ser?Pro

340 345 350

Tyr?Gln?Ile?His?Phe?Thr?Lys?Thr?Pro?Lys?Tyr?Phe?Lys?Pro?Gly?Met

355 360 365

Pro?Phe?Asp?Leu?Met?Val?Phe?Val?Thr?Asn?Pro?Asp?Gly?Ser?Pro?Ala

370 375 380

Tyr?Arg?Val?Pro?Val?Ala?Val?Gln?Gly?Glu?Asp?Thr?Val?Gln?Ser?Leu

385 390 395 400

Thr?Gln?Gly?Asp?Gly?Val?Ala?Lys?Leu?Ser?Ile?Asn?Thr?His?Pro?Ser

405 410 415

Gln?Lys?Pro?Leu?Ser?Ile?Thr?Val?Arg?Thr?Lys?Lys?Gln?Glu?Leu?Ser

420 425 430

Glu?Ala?Glu?Gln?Ala?Thr?Arg?Thr?Met?Gln?Ala?Leu?Pro?Tyr?Ser?Thr

435 440 445

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

450 455 460

Leu?Arg?Pro?Gly?Glu?Thr?Leu?Asn?Val?Asn?Phe?Leu?Leu?Arg?Met?Asp

465 470 475 480

Arg?Ala?His?Glu?Ala?Lys?Ile?Arg?Tyr?Tyr?Thr?Tyr?Leu?Ile?Met?Asn

485 490 495

Lys?Gly?Arg?Leu?Leu?Lys?Ala?Gly?Arg?Gln?Val?Arg?Glu?Pro?Gly?Gln

500 505 510

Asp?Leu?Val?Val?Leu?Pro?Leu?Ser?Ile?Thr?Thr?Asp?Phe?Ile?Pro?Ser

515 520 525

Phe?Arg?Leu?Val?Ala?Tyr?Tyr?Thr?Leu?Ile?Gly?Ala?Ser?Gly?Gln?Arg

530 535 540

Glu?Val?Val?Ala?Asp?Ser?Val?Trp?Val?Asp?Val?Lys?Asp?Ser?Cys?Val

545 550 555 560

Gly?Ser?Leu?Val?Val?Lys?Ser?Gly?Gln?Ser?Glu?Asp?Arg?Gln?Pro?Val

565 570 575

Pro?Gly?Gln?Gln?Met?Thr?Leu?Lys?Ile?Glu?Gly?Asp?His?Gly?Ala?Arg

580 585 590

Val?Val?Leu?Val?Ala?Val?Asp?Lys?Gly?Val?Phe?Val?Leu?Asn?Lys?Lys

595 600 605

Asn?Lys?Leu?Thr?Gln?Ser?Lys?Ile?Trp?Asp?Val?Val?Glu?Lys?Ala?Asp

610 615 620

Ile?Gly?Cys?Thr?Pro?Gly?Ser?Gly?Lys?Asp?Tyr?Ala?Gly?Val?Phe?Ser

625 630 635 640

Asp?Ala?Gly?Leu?Thr?Phe?Thr?Ser?Ser?Ser?Gly?Gln?Gln?Thr?Ala?Gln

645 650 655

Arg?Ala?Glu?Leu?Gln?Cys?Pro?Gln?Pro?Ala?Ala?Arg?Arg?Arg?Arg?Ser

660 665 670

Val?Gln?Leu?Thr?Glu?Lys?Arg?Met?Asp?Lys?Val?Gly?Lys?Tyr?Pro?Lys

675 680 685

Glu?Leu?Arg?Lys?Cys?Cys?Glu?Asp?Gly?Met?Arg?Glu?Asn?Pro?Met?Arg

690 695 700

Phe?Ser?Cys?Gln?Arg?Arg?Thr?Arg?Phe?Ile?Ser?Leu?Gly?Glu?Ala?Cys

705 710 715 720

Lys?Lys?Val?Phe?Leu?Asp?Cys?Cys?Asn?Tyr?Ile?Thr?Glu?Leu?Arg?Arg

725 730 735

Gln?His?Ala?Arg?Ala?Ser?His?Leu?Gly?Leu?Ala?Arg?Ser?Asn?Leu?Asp

740 745 750

Glu?Asp?Ile?Ile?Ala?Glu?Glu?Asn?Ile?Val?Ser?Arg?Ser?Glu?Phe?Pro

755 760 765

Glu?Ser?Trp?Leu?Trp?Asn?Val?Glu?Asp?Leu?Lys?Glu?Pro?Pro?Lys?Asn

770 775 780

Gly?Ile?Ser?Thr?Lys?Leu?Met?Asn?Ile?Phe?Leu?Lys?Asp?Ser?Ile?Thr

785 790 795 800

Thr?Trp?Glu?Ile?Leu?Ala?Val?Ser?Met?Ser?Asp?Lys?Lys?Gly?Ile?Cys

805 810 815

Val?Ala?Asp?Pro?Phe?Glu?Val?Thr?Val?Met?Gln?Asp?Phe?Phe?Ile?Asp

820 825 830

Leu?Arg?Leu?Pro?Tyr?Ser?Val?Val?Arg?Asn?Glu?Gln?Val?Glu?Ile?Arg

835 840 845

Ala?Val?Leu?Tyr?Asn?Tyr?Arg?Gln?Asn?Gln?Glu?Leu?Lys?Val?Arg?Val

850 855 860

Glu?Leu?Leu?His?Asn?Pro?Ala?Phe?Cys?Ser?Leu?Ala?Thr?Thr?Lys?Arg

865 870 875 880

Arg?His?Gln?Gln?Thr?Val?Thr?Ile?Pro?Pro?Lys?Ser?Ser?Leu?Ser?Val

885 890 895

Pro?Tyr?Val?Ile?Val?Pro?Leu?Lys?Thr?Gly?Leu?Gln?Glu?Val?Glu?Val

900 905 910

Lys?Ala?Ala?Val?Tyr?His?His?Phe?Ile?Ser?Asp?Gly?Val?Arg?Lys?Ser

915 920 925

Leu?Lys?Val?Val?Pro?Glu?Gly?Ile?Arg?Met?Asn?Lys?Thr?Val?Ala?Val

930 935 940

Arg?Thr?Leu?Asp?Pro?Glu?Arg?Leu?Gly?Arg?Glu?Gly?Val?Gln?Lys?Glu

945 950 955 960

Asp?Ile?Pro?Pro?Ala?Asp?Leu?Ser?Asp?Gln?Val?Pro?Asp?Thr?Glu?Ser

965 970 975

Glu?Thr?Arg?Ile?Leu?Leu?Gln?Gly?Thr?Pro?Val?Ala?Gln?Met?Thr?Glu

980 985 990

Asp?Ala?Val?Asp?Ala?Glu?Arg?Leu?Lys?His?Leu?Ile?Val?Thr?Pro?Ser

995 1000 1005

Gly?Cys?Gly?Glu?Gln?Asn?Met?Ile?Gly?Met?Thr?Pro?Thr?Val?Ile

1010 1015 1020

Ala?Val?His?Tyr?Leu?Asp?Glu?Thr?Glu?Gln?Trp?Glu?Lys?Phe?Gly

1025 1030 1035

Leu?Glu?Lys?Arg?Gln?Gly?Ala?Leu?Glu?Leu?Ile?Lys?Lys?Gly?Tyr

1040 1045 1050

Thr?Gln?Gln?Leu?Ala?Phe?Arg?Gln?Pro?Ser?Ser?Ala?Phe?Ala?Ala

1055 1060 1065

Phe?Val?Lys?Arg?Ala?Pro?Ser?Thr?Trp?Leu?Thr?Ala?Tyr?Val?Val

1070 1075 1080

Lys?Val?Phe?Ser?Leu?Ala?Val?Asn?Leu?Ile?Ala?Ile?Asp?Ser?Gln

1085 1090 1095

Val?Leu?Cys?Gly?Ala?Val?Lys?Trp?Leu?Ile?Leu?Glu?Lys?Gln?Lys

1100 1105 1110

Pro?Asp?Gly?Val?Phe?Gln?Glu?Asp?Ala?Pro?Val?Ile?His?Gln?Glu

1115 1120 1125

Met?Ile?Gly?Gly?Leu?Arg?Asn?Asn?Asn?Glu?Lys?Asp?Met?Ala?Leu

1130 1135 1140

Thr?Ala?Phe?Val?Leu?Ile?Ser?Leu?Gln?Glu?Ala?Lys?Asp?Ile?Cys

1145 1150 1155

Glu?Glu?Gln?Val?Asn?Ser?Leu?Pro?Gly?Ser?Ile?Thr?Lys?Ala?Gly

1160 1165 1170

Asp?Phe?Leu?Glu?Ala?Asn?Tyr?Met?Asn?Leu?Gln?Arg?Ser?Tyr?Thr

1175 1180 1185

Val?Ala?Ile?Ala?Gly?Tyr?Ala?Leu?Ala?Gln?Met?Gly?Arg?Leu?Lys

1190 1195 1200

Gly?Pro?Leu?Leu?Asn?Lys?Phe?Leu?Thr?Thr?Ala?Lys?Asp?Lys?Asn

1205 1210 1215

Arg?Trp?Glu?Asp?Pro?Gly?Lys?Gln?Leu?Tyr?Asn?Val?Glu?Ala?Thr

1220 1225 1230

Ser?Tyr?Ala?Leu?Leu?Ala?Leu?Leu?Gln?Leu?Lys?Asp?Phe?Asp?Phe

1235 1240 1245

Val?Pro?Pro?Val?Val?Arg?Trp?Leu?Asn?Glu?Gln?Arg?Tyr?Tyr?Gly

1250 1255 1260

Gly?Gly?Tyr?Gly?Ser?Thr?Gln?Ala?Thr?Phe?Met?Val?Phe?Gln?Ala

1265 1270 1275

Leu?Ala?Gln?Tyr?Gln?Lys?Asp?Ala?Pro?Asp?His?Gln?Glu?Leu?Asn

1280 1285 1290

Leu?Asp?Val?Ser?Leu?Gln?Leu?Pro?Ser?Arg?Ser?Ser?Lys?Ile?Thr

1295 1300 1305

His?Arg?Ile?His?Trp?Glu?Ser?Ala?Ser?Leu?Leu?Arg?Ser?Glu?Glu

1310 1315 1320

Thr?Lys?Glu?Asn?Glu?Gly?Phe?Thr?Val?Thr?Ala?Glu?Gly?Lys?Gly

1325 1330 1335

Gln?Gly?Thr?Leu?Ser?Val?Val?Thr?Met?Tyr?His?Ala?Lys?Ala?Lys

1340 1345 1350

Asp?Gln?Leu?Thr?Cys?Asn?Lys?Phe?Asp?Leu?Lys?Val?Thr?Ile?Lys

1355 1360 1365

Pro?Ala?Pro?Glu?Thr?Glu?Lys?Arg?Pro?Gln?Asp?Ala?Lys?Asn?Thr

1370 1375 1380

Met?Ile?Leu?Glu?Ile?Cys?Thr?Arg?Tyr?Arg?Gly?Asp?Gln?Asp?Ala

1385 1390 1395

Thr?Met?Ser?Ile?Leu?Asp?Ile?Ser?Met?Met?Thr?Gly?Phe?Ala?Pro

1400 1405 1410

Asp?Thr?Asp?Asp?Leu?Lys?Gln?Leu?Ala?Asn?Gly?Val?Asp?Arg?Tyr

1415 1420 1425

Ile?Ser?Lys?Tyr?Glu?Leu?Asp?Lys?Ala?Phe?Ser?Asp?Arg?Asn?Thr

1430 1435 1440

Leu?Ile?Ile?Tyr?Leu?Asp?Lys?Val?Ser?His?Ser?Glu?Asp?Asp?Cys

1445 1450 1455

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

1460 1465 1470

Pro?Gly?Ala?Val?Lys?Val?Tyr?Ala?Tyr?Tyr?Asn?Leu?Glu?Glu?Ser

1475 1480 1485

Cys?Thr?Arg?Phe?Tyr?His?Pro?Glu?Lys?Glu?Asp?Gly?Lys?Leu?Asn

1490 1495 1500

Lys?Leu?Cys?Arg?Asp?Glu?Leu?Cys?Arg?Cys?Ala?Glu?Glu?Asn?Cys

1505 1510 1515

Phe?Ile?Gln?Lys?Ser?Asp?Asp?Lys?Val?Thr?Leu?Glu?Glu?Arg?Leu

1520 1525 1530

Asp?Lys?Ala?Cys?Glu?Pro?Gly?Val?Asp?Tyr?Val?Tyr?Lys?Thr?Arg

1535 1540 1545

Leu?Val?Lys?Val?Gln?Leu?Ser?Asn?Asp?Phe?Asp?Glu?Tyr?Ile?Met

1550 1555 1560

Ala?Ile?Glu?Gln?Thr?Ile?Lys?Ser?Gly?Ser?Asp?Glu?Val?Gln?Val

1565 1570 1575

Gly?Gln?Gln?Arg?Thr?Phe?Ile?Ser?ProIle?Lys?Cys?Arg?Glu?Ala

1580 1585 1590

Leu?Lys?Leu?Glu?Glu?Lys?Lys?His?Tyr?Leu?Met?Trp?Gly?Leu?Ser

1595 1600 1605

Ser?Asp?Phe?Trp?Gly?Glu?Lys?Pro?Asn?Leu?Ser?Tyr?Ile?Ile?Gly

1610 1615 1620

Lys?Asp?Thr?Trp?Val?Glu?His?Trp?Pro?Glu?Glu?Asp?Glu?Cys?Gln

1625 1630 1635

Asp?Glu?Glu?Asn?Gln?Lys?Gln?Cys?Gln?Asp?Leu?Gly?Ala?Phe?Thr

1640 1645 1650

Glu?Ser?Met?Val?Val?Phe?Gly?Cys?Pro?Asn

1655 1660

Claims (10)

1. human epididymis expression sperm binding protein HEL-28, it is characterized in that, it have the aminoacid sequence of sequence 2 in the sequence table or with the aminoacid sequence of sequence 2 through replacement, disappearance or the interpolation of one or several amino-acid residue and have with the identical active sequence 2 deutero-protein of amino acid residue sequence of sequence 2 with by chemical process synthetic protein.

2. the encoding gene of a human epididymis expression sperm binding protein HEL-28, it is one of following nucleotide sequences:

The nucleotide sequence of sequence 1 in the sequence table;

The dna sequence dna that limits with sequence in the sequence table 1 has 90% above homology, and the proteic dna sequence dna of coding identical function.

3. the eucaryon and the prokaryotic expression carrier that contain the described gene of claim 2.

4. the clone that contains the described gene of claim 2.

5. be the reagent of activeconstituents at the prepared various antibody of the described protein of claim 1 and with this antibody.

6. the pharmaceutical composition that provides at the described protein of claim 1, described composition comprises albumen of the present invention, nucleotide sequence, construction or cell, and pharmaceutically acceptable carrier.

7. the application of albumen according to claim 1 in the infertility diagnosis.

8. the application of albumen according to claim 1 in the infertility treatment.

9. the application of gene according to claim 2 in the new contraceptive of exploitation.

10. the application of albumen according to claim 1 in the new microbiotic of preparation.

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