CN113655225B - Application of protein and composition for identifying early pregnancy of Tibetan pigs - Google Patents

Abstract

The invention belongs to the technical field of biology, and particularly relates to an application of a protein, wherein the protein is at least one of the following proteins: a talin1 protein, wherein the sequence of the talin1 protein is shown as an amino acid sequence NO. 1; the sequence of the Profile protein is shown as amino acid sequence NO. 2; HGF activator protein, the sequence of which is shown in amino acid sequence NO. 3; the sequence of the Carbonic anhydride protein is shown as amino acid sequence NO. 4; the protein is used as a marker for identifying early pregnancy of Tibetan pigs. Another object of the present invention is to provide a composition of protein markers for early pregnancy identification of Tibetan pigs.

Description

Application of protein and composition for identifying early pregnancy of Tibetan pigs

Technical Field

The invention belongs to the technical field of biology, and particularly relates to application of a protein and a composition for identifying early pregnancy of Tibetan pigs.

Background

The production of live pigs is the core industry of animal husbandry, and the total pork consumption is always the first of animal products in the whole world; pork has no substitution in the status of meat food consumption of residents in China, and the pork production efficiency has a profound influence on the lives of the residents. The pregnancy diagnosis of the sow is taken as a key link in the reproduction process of the live pig, and the early and late diagnosis and the accuracy of the diagnosis influence the fertility rate of the sow, the lactation of the farrowing and the like to a great extent. The method has the advantages that the nonpregnant sows are detected in time, the arrangement and the complementary allocation of the nonpregnant sows are facilitated as early as possible, the nonpregnant period of the sows is shortened, the farrowing interval is shortened, the extra cost caused by ineffective feeding is reduced, the sows with low fecundity and sterility are eliminated in time, and the influence on the economic benefit is extremely obvious in the large-scale and high-efficiency breeding industry. Most of the current pregnancy diagnosis methods in production are ultrasonic diagnosis, hormone method, behavior observation and the like, which have advantages and disadvantages, the accuracy and efficiency depend on the experience of reproduction workers to a great extent, the identification time generally is 21-24 days after hybridization, the effects are uneven, and the identification time and the accuracy are all to be improved. The search for a sensitive and efficient novel pregnancy diagnosis method is still a leading-edge topic which has scientific guiding significance for pig production and has wide market application prospect.

Various kinds of biomolecules such as proteins, steroids, prostaglandins, and the like exist in liquids such as blood, urine, saliva, and the like, and these molecules play important functions in the animal body, and the synthesis thereof significantly changes with the physiological state of the animal; in addition, the liquids have the characteristics of convenient collection, sufficient sample analysis amount, low cost, strong stability, small stress on animals and the like, thereby having important biological significance in the aspects of livestock physiological health, disease monitoring and early diagnosis.

CN202110437164.0 discloses a protein biomarker for early pregnancy diagnosis of sows, which is characterized in that: the protein biomarker is one of the following protein markers: FLNC protein with sequence number <210> 1; ADIPOQ protein, the sequence number of which is <210> 2; an HP protein having sequence number <210> 3; TERF2 protein having sequence number <210> 4; the method for diagnosing the early pregnancy of the sow is characterized by comprising the following steps: the method comprises the following specific steps: firstly, blood sample collection: collecting peripheral blood of the sows 15 days after the mating; processing a blood sample: separating serum or plasma from the blood sample in the step (i): thirdly, detecting by using the kit: and (4) selecting an ELISA kit to detect the blood sample treated in the step (II).

CN202010525872.5 discloses an exosome miRNA marker for early pregnancy diagnosis of sows and application thereof, wherein exosome miRNA molecular markers comprise ssc-miR-192, ssc-miR-146a-5p and ssc-miR-149, and when serum exosome miRNA is singly used for diagnosis and differentiation of early pregnancy and non-pregnant sows, the areas under the ROC curve (AUC) are 0.863, 0.885 and 0.857 respectively; the combination AUC of ssc-miR-192 and ssc-miR-146a-5p is 0.921; the joint AUC of ssc-miR-192 and ssc-miR-149 is 0.902; the joint AUC of the ssc-miR-146a-5p and the ssc-miR-149 is 0.910, and when three miRNAs of the ssc-miR-192, the ssc-miR-146a-5p and the ssc-miR-149 are combined, the AUC is 0.959.

CN201911011796.X discloses an application of a serum exosome ssc-miR-92b-3p as a molecular marker for early pregnancy diagnosis of sows. The amino acid sequence of the serum exosome ssc-miR-92b-3p is shown in SEQ ID No. 1. The present invention identifies the changes in the levels of exosome mirnas in serum at the earliest day 9 of pregnancy and identifies the increased levels of ssc-miR-92b-3p expression during days 9 to 15 of pregnancy, which mirnas may serve as a novel biomarker for early pregnancy in pigs.

As can be seen, those skilled in the art have analyzed proteins in pig serum, secretions, and the like to detect pregnancy of sows as early as possible.

It should be noted that no corresponding identification index has been proposed by anyone for identifying early pregnancy of Tibetan pigs.

The technical problem that the present scheme will solve is: how to accurately identify early pregnancy of Tibetan pigs.

Disclosure of Invention

The invention aims to provide the application of the protein, the protein is used for identifying the early pregnancy of the Tibetan pigs, the identification can be carried out as early as the 12 th day of pregnancy, the technical blank of the identification of the early pregnancy of the Tibetan pigs is eliminated, and the scheme has high identification accuracy and early identification time.

Another object of the present invention is to provide a composition of protein markers for early pregnancy identification of Tibetan pigs.

The technical scheme of the invention is as follows:

use of a protein which is at least one of:

a talin1 protein, wherein the sequence of the talin1 protein is shown as an amino acid sequence NO. 1;

the sequence of the Profile protein is shown as amino acid sequence NO. 2;

HGF activator protein, the sequence of which is shown in amino acid sequence NO. 3;

the sequence of the Carbonic anhydride protein is shown as amino acid sequence NO. 4;

the protein is used as a marker for identifying early pregnancy of Tibetan pigs.

Amino acid sequence No.1 is as follows:

MVALSLKISIGNVVKTMQFEPSTMVYDACRIIRERIPEALAGPPSDFGLFLSDDDPKKGIWLEAGKALDYYMLRNGDTMEYRKKQRPLKIRMLDGTVKTVMVDDSKTVTDMLMTICARIGITNHDEYSLVREIMEEKKEEVTGTLRKDKTLLRDEKKMEKLKQKLHTDDELNWLDHGRTLREQGVEEHETLLLRRKFFYSDQNVDSRDPVQLNLLYVQARDDILNGSHPVSFDKACEFAGFQCQIQFGPHNEQKHKAGFLDLKDFLPKEYVKQKGERKIFQAHKNCGQMSEIEAKVRYVKLARSLKTYGVSFFLVKEKMKGKNKLVPRLLGITKECVMRVDEKTKEVIQEWNLTNIKRWAASPKSFTLDFGDYQDGYYSVQTTEGEQIAQLIAGYIDIILKKKKSKDHFGLEGDEESTMLEDSVSPKKSTVLQQQYNRVGKVEHGSVALPAIMRSGASGPENFQVGSMPPAQQQITSGQMHRGHMPPLTSAQQALTGTINSSMQAVQAAQATLDDFDTLPPLGQDAASKAWRKNKMDESKHEIHSQVDAITAGTASVVNLTAGDPAETDYTAVGCAVTTISSNLTEMSRGVKLLAALLEDEGGSGRPLLQAAKGLAGAVSELLRTAQPASAEPRQNLLQAAGNVGQASGELLQQIGESDTDPHFQICAPRGAGVRSPPDPPTDVLMQLAKAVASAAAALVLKAKSVAQRTEDSGLQTQVIAAATQCALSTSQLVACTKVVAPTISSPVCQEQLVEAGRLVAKAVEGCVSASQAATEDGQLLRGVGAAATAVTQALNELLQHVRAHATGAGPAGRYDQATDTILTVTENIFSSMGDAGEMVRQARILAQATSDLVNAIKADAEGESDLENSRKLLSAAKILADATAKMVEAAKGAAAHPDSEEQQQRLREAAEGLRMATNVAAQNAIKKKLVQRLEHAAKQAAASATQTIAAAQHAASTPKTTAGPQPLLVQSCKAVAEQIPLLVQGVRGSQAQPDSPSAQLALIAASQSFLQPGGKMVAAAKASVPTIQDQASAMQLSQCAKNLGTALAELRTAAQKAQEACGPLEMDSALSVVQNLERDLQEVKAAARDGKLKPLPGETMEKCAQDLGNSTKAVSSAIAQLLGEVAQGNENYAGIAAREVAGGLRSLAQAARGVAALTSDPAVQAIVLDTASDVLDKASSLIEEAKKAAGHPGDPESQQRLAQVAKAVTQALNRCVSCLPGQRDVDNALRAVGDASKRLLSDSLPPSTGTFQEAQSRLNEAAAGLNQAATELVQASRGTPQDLARASGRFGQDFSTFLEAGVEMASQAPSQEDRAQVVSNLKGISMSSSKLLLAAKALSTDPAAPNLKSQLAAAARAVTDSINQLITMCTQQAPGQKECDNALRELETVRELLENPVQPINDMSYFGCLDSVMENSKVLGEAMTGISQNAKNGNLPEFGEAIATASKALCGFTEAAAQAAYLVGVSDPNSQAGQQGLVEPTQFARANQAIQMACQSLGEPGCTQAQVLSAATIVAKHTSALCNSCRLASARTANPTAKRQFVQSAKEVANSTANLVKTIKALDGAFTEENRAQCRAATAPLLEAVDNLSAFASNPEFSSVPAQISPEGRAAMEPIVISAKTMLESAGGLIQTARALAVNPRDPPRWSVLAGHSRTVSDSIKKLITSMRDKAPGQLECETAIAALNSCLRDLDQASLAAVSQQLAPREGISQEALHTQMLTAVQEISHLIEPLASAARAEASQLGHKVSQMAQYFEPLTLAAVGAASKTLSHPQQMALLDQTKTLAESALQLLYTAKEAGGNPKQAAHTQEALEEAVQMMTEAVEDLTTTLNEAASAAGVVGGMVDSITQAINQLDEGPMGEPEGSFVDYQTTMVRTAKAIAVTVQEMVTKSNTSPEELGPLANQLTSDYGRLASEAKPAAVAAENEEIGAHIKHRVQELGHGCAALVTKAGALQCSPSDAYTKKELIECARRVSEKVSHVLAALQAGNRGTQACITAASAVSGIIADLDTTIMFATAGTLNREGAETFADHREGILKTAKVLVEDTKVLVQNAAGSQEKLAQAAQSSVATITRLADVVKLGAASLGAEDPETQVVLINAVKDVAKALGDLISATKAAAGKVGDDPAVWQLKNSAKVMVTNVTSLLKTVKAVEDEATKGTRALEATTEHIRQELAVFCSPEPPAKTSTPEDFIRMTKGITMATAKAVAAGNSCRQEDVIATANLSRRAIADMLRACKEAAFHPDVAPDVRLRALHYGRECANGYLELLDHVLLTLQKPSPELKQQLTGHSKRVAGSVTELIQAAEAMKGTEWVDPEDPTVIAENELLGAAAAIEAAAKKLEQLKPRAKPKEADESLNFEEQILEAAKSIAAATSALVKAASAAQRELVAQGKVGAIPANALDDGQWSQGLISAARMVAAATNNLCEAANAAVQGHASQEKLISSAKQVAASTAQLLVACKVKADQDSEAMKRLQAAGNAVKRASDNLVKAAQKAAAFEEPENETVVVKEKMVGGIAQIIAAQEEMLRKERELEEARKKLAQIRQQQYKFLPSELRDEH

amino acid sequence No.2 is as follows:

MAGWNAYIDNLMADGTCQDAAIVGYKDSPSVWAAVPGKTFVNITPAEVGVLVGKDRSSFFVNGLTLGGQKCSVIRDSLLQDGEFTMDLRTKSTGGAPTFNITVTMTAKTLVLLMGKEGVHGGMINKKCYEMASHLRRSQY

amino acid sequence No.3 is as follows:

MGRWAWVPSPCPPPRLSLLLLLLLLVPRGAQPQAGRNQTEAPAQKATVTPGTPLIPVTSATLRPPATRAPKAEGPHGGGLTPLPRAAPSNSSAGGTVLTEAGQPCRFPFRYGGRMIHSCTSEGSAHRKWCATTHNYDRDRAWGYCVQASTPWEGPAALDPCASGPCLNGGSCSSTQDPESYHCTCPVDFAGKDCGSEKCFDESRYEYLEAGDRWARVHEGRVEQCECAGGQVRCQGTRHTACLSSPCLNGGTCHLIVATGTTVCACPPGHAGRLCNIVPAQSCFVGSGTEYRGVASTAASGLSCLAWNSDLLYQELHVDSVGAAALLGLGPHAYCRNPDKDERPWCYVVKDSALSWEYCRLAACESLARIQPLPPEVLLTLPEPTAAGPAGRQNCGKRHKKRTFLRPRIIGGSSSLPGSHPWLAAIYIGNNFCAGSLVHTCWVVSAAHCFANSLAGPWAGPPVASWAPRPQPLCPAGRHPWDALAPFCLSWAPARQADLASSRPVLIRLEKKGERCAVRSQFVQPICLPEPGSPFPAGHKCQIAGWGHQDENVSGYSSSLREALVPLVADHKCSSPEVYGADISPNMLCAGYFDCRSDACQGDSGGPLACEKNGVAHLYGIISWGDGCGRLNKPGVYTRVANYVDWINDRIWPSKRPADPS

amino acid sequence No.4 is as follows:

MTSPAWGYDGEYGPEHWSKVYPIANGNNQSPIDIKTSETKHDTSLKPISVSYNPATAKEIINVGHSFHVNFEDNDNRSVLKDGPLSESYRLLQFHFHWGKTDDYGSEHLVDGAKYSAELHIVHWNSAKYSSAAEAASHADGLAIIGVLVKVGQANPNLQKVLDALKGIKYKGKKAPFTNFDPSVLLPSSLDYWTYFGSLTHPPLHESVNWIILKENISISSDQLAQFRSLLSNAEGDKDVPIKHNNRPPQPLKGRIVKASF

in addition, the invention also provides a composition of the protein marker for identifying early pregnancy of the Tibetan pigs, which consists of carbon anhydrase protein and at least one of talin1 protein, profile protein and HGF activator protein;

a talin1 protein, wherein the sequence of the talin1 protein is shown as an amino acid sequence NO. 1;

the sequence of the Profile protein is shown as amino acid sequence NO. 2;

HGF activator protein, the sequence of which is shown in amino acid sequence NO. 3;

the sequence of the Carbonic anhydride protein is shown as amino acid sequence No. 4.

The invention has the following beneficial effects:

according to the scheme, a DIA proteomics method is utilized, proteins extracted from serum at different time points in 19 days in the early gestation period of the Tibetan pigs are subjected to enzymolysis, and then high performance liquid chromatography separation and mass spectrum detection are carried out on peptide fragments, so that 390 kinds of proteins are identified in total; screening differential expression protein to carry out PRM targeting quantitative verification.

The results show that compared with the control group, the expression levels of the A0a287AC34 (talin 1, TLN 1), A0A4X1UWL6 (Profilin, PFN 1), A0a286ZMW3 (HGF activator, HGFAC) proteins are significantly increased on day 16 of pregnancy, the expression levels of the A0a287AI92 (Carbonic anhydrase, CA 1) proteins are significantly increased on day 12 of pregnancy, and the relative expression trends of the four proteins at 4 time points in the early pregnancy are consistent with the expression trend in DIA, so that the A0a287AC34, A0A4X1UWL6, A0a ZMW3, and A0a AI 287 92 can be used as candidate marker proteins for early pregnancy diagnosis of tibetan pigs.

Drawings

FIG. 1A 0A287AC34 protein relative expression levels in the serum proteome (DIA, reference A) and PRM target quantitative validation (reference B) of Tibetan pigs at different times during early gestation;

FIG. 2A relative expression levels of 0A4X1UWL6 protein in serum proteome (DIA, reference A) and PRM target quantitative validation (reference B) at different times during early gestation in Tibetan pigs;

FIG. 3A relative expression levels of the 286ZMW3 protein in serum proteome (DIA, reference A) and PRM target quantitative validation (reference B) at different times during early gestation in Tibetan pigs;

FIG. 4A 0A287AI92 protein relative expression levels in different time serum proteomes (DIA, reference A) and PRM target quantitative validation (reference B) of early gestation in Tibetan pigs.

Detailed Description

The technical solutions of the present invention are further described in detail below with reference to the drawings and the detailed description, but the present invention is not limited thereto.

(1) Obtaining serum of Tibetan pigs at different time points in early gestation period

Selecting healthy female Tibetan pigs for testing, observing the oestrus rule, naturally mating after natural oestrus, counting as 0 day on the day of hybridization, collecting blood by puncture of anterior vena cava of neck on 5, 12, 16 and 19 days after hybridization, coagulating the blood sample for 1 hour at room temperature, and centrifuging at 2000 g for 10 minutes to separate serum. Serum samples were mixed with protease inhibitors (Sigma Aldrich) and stored at-80 ℃ until further analysis. Sows were slaughtered on day 35 post-partum and gestation was confirmed by the presence of embryos.

(2) Serum protein extraction and quality inspection

Total protein was extracted, quantitated using a Bradford protein quantitation kit, and the quality of protein extraction was checked by SDS-PAGE gel electrophoresis. Taking equivalent protein to carry out proteolysis, desalting by a C18 desalting column and freeze-drying.

(3) Construction of a library of de-abundant DDA spectra

And (3) eluting and separating the polypeptide fractions by using liquid chromatography, collecting and combining 4 fractions, and carrying out DDA mode liquid quality detection after collecting the fractions: eluting by using an EASY-nLCTM 1200 nano-upgrading UHPLC system, detecting by using a Q active (TM) HF-X mass spectrometer, carrying out Nanospray Flex (ESI) ion source, wherein the ion spray voltage is 2.5 kV, the mass spectrum adopts a Data Dependent Acquisition (DDA) mode, the full scanning range of the mass spectrum is m/z 350-1500, and the resolution of the primary mass spectrum is 60000 (200 m/z); and (3) selecting the parent ions with the ion intensity of TOP 40 in the full scan, fragmenting by using a high energy collision fragmentation (HCD) method, carrying out secondary mass spectrum detection, setting the resolution of the secondary mass spectrum as 15000 (200 m/z), and generating mass spectrum detection original data (. raw) for constructing a DDA spectrogram library of the high-abundance protein.

(4) DIA mode liquid quality detection

Using EASY-nLCTM 1200 nano upgrading UHPLC system, using Q active (TM) HF-X mass spectrometer and Nanospray Flex (ESI) ion source, setting ion spray voltage to be 2.5 kV, adopting non-data dependent acquisition mode (DIA) for mass spectrum, setting full scan range of mass spectrum to be m/z 350-1500 and setting primary mass spectrum resolution to be 60000 (200 m/z); secondary mass spectrometry detection was performed using HCD method fragmentation with secondary mass resolution set to 30000 (200 m/z), and scan window information generated mass spectrometry detection raw data (. raw).

(5) Data processing

The mass spectrum off-line data is imported into the Proteome discovery 2.2 software for suc scrofa UniProt database search, and the spectrum peptide and protein are quantified. The mass deviation tolerance ranges at the time of precursor ion library search and at the time of fragment ion library search in the analysis parameters were set to 10ppm and 0.02Da, respectively. The Spectrum Peptide (PSMs) with the reliability of more than 99 percent is reserved as credible PSMs, the protein at least containing one unique Peptide segment is credible protein, and the FDR (pulse discovery rate) is less than or equal to 1 percent. And (3) constructing a DIA spectrogram library by using the information of peptide fragment parent ions and corresponding secondary fragment ions in the identified result of the DDA mode as a spectrogram library template. Based on these identification results, information such as ion intensity, chromatographic peak shape, retention time, etc. of the spectrum library template is compared as features with the results of the DIA acquisition mode. When the relative protein quantitative analysis is carried out, the protein abundance of the other four early pregnancy groups is compared by taking the protein abundance of the mating 0 day as a reference, and meanwhile, the quantitative comparative analysis is carried out on different time points. The difference is obvious when Pvalue is less than or equal to 0.05. When the difference FC between the protein groups is more than or equal to 1.2, the protein groups are set as up-regulation and down-regulation.

(6) Differential validation of protein expression

Screening differential expression protein to perform protein PRM target verification. The mass spectrometer data in the preliminary experiment were analyzed using Skyline software to determine if selected peptides were available based on reproducibility and stability. In the formal test, mass spectrum off-machine data are analyzed by using Skyline software, and peak areas are corrected by using internal standard peptide fragments.

In the embodiment, a DIA proteomics method is utilized, proteins extracted from serum of Tibetan pigs at different time points within 19 days of early gestation are subjected to enzymolysis, and then peptide fragments are subjected to high performance liquid chromatography separation and mass spectrometry detection to identify 390 proteins in total; screening differential expression protein to carry out PRM targeting quantitative verification. The results show that compared with the control group, the expression levels of the A0a287AC34 (talin 1, TLN 1), A0A4X1UWL6 (Profilin, PFN 1), A0a286ZMW3 (HGF activator, HGFAC) proteins are significantly increased on day 16 of pregnancy, the expression levels of the A0a287AI92 (Carbonic anhydrase, CA 1) proteins are significantly increased on day 12 of pregnancy, and the relative expression trends of the four proteins at 4 time points in the early pregnancy are consistent with the expression trend in DIA, so that the A0a287AC34, A0A4X1UWL6, A0a ZMW3, and A0a AI 287 92 can be used as candidate marker proteins for early pregnancy diagnosis of tibetan pigs.

Preferably, in this embodiment, since the peak expression value of the A0a287AI92 (Carbonic anhydrase, CA 1) protein is significantly different from that of the other three proteins, it is more preferable that the A0a287AI92 (Carbonic anhydrase, CA 1) protein is combined with one or two or three of the other three proteins to identify the content of the relevant proteins of the tibetan pig in 12 days and 15-16 days, so that the early pregnancy of the tibetan pig can be more accurately determined.

The invention screens and identifies the early pregnancy diagnosis marker protein of the Tibetan pigs by the DIA proteomics technology and the PRM targeted quantitative verification technology, thereby achieving the purposes of improving the accuracy of early pregnancy diagnosis, advancing the diagnosis time, enriching the diagnosis method and improving the breeding production benefit of livestock.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

<110> animal science institute of academy of agricultural sciences of Guangdong province

Application of <120> protein and composition for identifying early pregnancy of Tibetan pigs

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2558

<212> PRT

<213> talin1 protein ()

<400> 1

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

1 5 10 15

Met Gln Phe Glu Pro Ser Thr Met Val Tyr Asp Ala Cys Arg Ile Ile

20 25 30

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

35 40 45

Leu Phe Leu Ser Asp Asp Asp Pro Lys Lys Gly Ile Trp Leu Glu Ala

50 55 60

Gly Lys Ala Leu Asp Tyr Tyr Met Leu Arg Asn Gly Asp Thr Met Glu

65 70 75 80

Tyr Arg Lys Lys Gln Arg Pro Leu Lys Ile Arg Met Leu Asp Gly Thr

85 90 95

Val Lys Thr Val Met Val Asp Asp Ser Lys Thr Val Thr Asp Met Leu

100 105 110

Met Thr Ile Cys Ala Arg Ile Gly Ile Thr Asn His Asp Glu Tyr Ser

115 120 125

Leu Val Arg Glu Ile Met Glu Glu Lys Lys Glu Glu Val Thr Gly Thr

130 135 140

Leu Arg Lys Asp Lys Thr Leu Leu Arg Asp Glu Lys Lys Met Glu Lys

145 150 155 160

Leu Lys Gln Lys Leu His Thr Asp Asp Glu Leu Asn Trp Leu Asp His

165 170 175

Gly Arg Thr Leu Arg Glu Gln Gly Val Glu Glu His Glu Thr Leu Leu

180 185 190

Leu Arg Arg Lys Phe Phe Tyr Ser Asp Gln Asn Val Asp Ser Arg Asp

195 200 205

Pro Val Gln Leu Asn Leu Leu Tyr Val Gln Ala Arg Asp Asp Ile Leu

210 215 220

Asn Gly Ser His Pro Val Ser Phe Asp Lys Ala Cys Glu Phe Ala Gly

225 230 235 240

Phe Gln Cys Gln Ile Gln Phe Gly Pro His Asn Glu Gln Lys His Lys

245 250 255

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

260 265 270

Gln Lys Gly Glu Arg Lys Ile Phe Gln Ala His Lys Asn Cys Gly Gln

275 280 285

Met Ser Glu Ile Glu Ala Lys Val Arg Tyr Val Lys Leu Ala Arg Ser

290 295 300

Leu Lys Thr Tyr Gly Val Ser Phe Phe Leu Val Lys Glu Lys Met Lys

305 310 315 320

Gly Lys Asn Lys Leu Val Pro Arg Leu Leu Gly Ile Thr Lys Glu Cys

325 330 335

Val Met Arg Val Asp Glu Lys Thr Lys Glu Val Ile Gln Glu Trp Asn

340 345 350

Leu Thr Asn Ile Lys Arg Trp Ala Ala Ser Pro Lys Ser Phe Thr Leu

355 360 365

Asp Phe Gly Asp Tyr Gln Asp Gly Tyr Tyr Ser Val Gln Thr Thr Glu

370 375 380

Gly Glu Gln Ile Ala Gln Leu Ile Ala Gly Tyr Ile Asp Ile Ile Leu

385 390 395 400

Lys Lys Lys Lys Ser Lys Asp His Phe Gly Leu Glu Gly Asp Glu Glu

405 410 415

Ser Thr Met Leu Glu Asp Ser Val Ser Pro Lys Lys Ser Thr Val Leu

420 425 430

Gln Gln Gln Tyr Asn Arg Val Gly Lys Val Glu His Gly Ser Val Ala

435 440 445

Leu Pro Ala Ile Met Arg Ser Gly Ala Ser Gly Pro Glu Asn Phe Gln

450 455 460

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

465 470 475 480

His Arg Gly His Met Pro Pro Leu Thr Ser Ala Gln Gln Ala Leu Thr

485 490 495

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

500 505 510

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

515 520 525

Lys Ala Trp Arg Lys Asn Lys Met Asp Glu Ser Lys His Glu Ile His

530 535 540

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

545 550 555 560

Thr Ala Gly Asp Pro Ala Glu Thr Asp Tyr Thr Ala Val Gly Cys Ala

565 570 575

Val Thr Thr Ile Ser Ser Asn Leu Thr Glu Met Ser Arg Gly Val Lys

580 585 590

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

595 600 605

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

610 615 620

Thr Ala Gln Pro Ala Ser Ala Glu Pro Arg Gln Asn Leu Leu Gln Ala

625 630 635 640

Ala Gly Asn Val Gly Gln Ala Ser Gly Glu Leu Leu Gln Gln Ile Gly

645 650 655

Glu Ser Asp Thr Asp Pro His Phe Gln Ile Cys Ala Pro Arg Gly Ala

660 665 670

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

675 680 685

Ala Lys Ala Val Ala Ser Ala Ala Ala Ala Leu Val Leu Lys Ala Lys

690 695 700

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

705 710 715 720

Ala Ala Ala Thr Gln Cys Ala Leu Ser Thr Ser Gln Leu Val Ala Cys

725 730 735

Thr Lys Val Val Ala Pro Thr Ile Ser Ser Pro Val Cys Gln Glu Gln

740 745 750

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

755 760 765

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

770 775 780

Gly Ala Ala Ala Thr Ala Val Thr Gln Ala Leu Asn Glu Leu Leu Gln

785 790 795 800

His Val Arg Ala His Ala Thr Gly Ala Gly Pro Ala Gly Arg Tyr Asp

805 810 815

Gln Ala Thr Asp Thr Ile Leu Thr Val Thr Glu Asn Ile Phe Ser Ser

820 825 830

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

835 840 845

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

850 855 860

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

865 870 875 880

Ala Asp Ala Thr Ala Lys Met Val Glu Ala Ala Lys Gly Ala Ala Ala

885 890 895

His Pro Asp Ser Glu Glu Gln Gln Gln Arg Leu Arg Glu Ala Ala Glu

900 905 910

Gly Leu Arg Met Ala Thr Asn Val Ala Ala Gln Asn Ala Ile Lys Lys

915 920 925

Lys Leu Val Gln Arg Leu Glu His Ala Ala Lys Gln Ala Ala Ala Ser

930 935 940

Ala Thr Gln Thr Ile Ala Ala Ala Gln His Ala Ala Ser Thr Pro Lys

945 950 955 960

Thr Thr Ala Gly Pro Gln Pro Leu Leu Val Gln Ser Cys Lys Ala Val

965 970 975

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

980 985 990

Gln Pro Asp Ser Pro Ser Ala Gln Leu Ala Leu Ile Ala Ala Ser Gln

995 1000 1005

Ser Phe Leu Gln Pro Gly Gly Lys Met Val Ala Ala Ala Lys Ala Ser

1010 1015 1020

Val Pro Thr Ile Gln Asp Gln Ala Ser Ala Met Gln Leu Ser Gln Cys

1025 1030 1035 1040

Ala Lys Asn Leu Gly Thr Ala Leu Ala Glu Leu Arg Thr Ala Ala Gln

1045 1050 1055

Lys Ala Gln Glu Ala Cys Gly Pro Leu Glu Met Asp Ser Ala Leu Ser

1060 1065 1070

Val Val Gln Asn Leu Glu Arg Asp Leu Gln Glu Val Lys Ala Ala Ala

1075 1080 1085

Arg Asp Gly Lys Leu Lys Pro Leu Pro Gly Glu Thr Met Glu Lys Cys

1090 1095 1100

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

1105 1110 1115 1120

Gln Leu Leu Gly Glu Val Ala Gln Gly Asn Glu Asn Tyr Ala Gly Ile

1125 1130 1135

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

1140 1145 1150

Arg Gly Val Ala Ala Leu Thr Ser Asp Pro Ala Val Gln Ala Ile Val

1155 1160 1165

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

1170 1175 1180

Glu Ala Lys Lys Ala Ala Gly His Pro Gly Asp Pro Glu Ser Gln Gln

1185 1190 1195 1200

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

1205 1210 1215

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

1220 1225 1230

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

1235 1240 1245

Thr Gly Thr Phe Gln Glu Ala Gln Ser Arg Leu Asn Glu Ala Ala Ala

1250 1255 1260

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

1265 1270 1275 1280

Pro Gln Asp Leu Ala Arg Ala Ser Gly Arg Phe Gly Gln Asp Phe Ser

1285 1290 1295

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

1300 1305 1310

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

1315 1320 1325

Ser Ser Lys Leu Leu Leu Ala Ala Lys Ala Leu Ser Thr Asp Pro Ala

1330 1335 1340

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

1345 1350 1355 1360

Asp Ser Ile Asn Gln Leu Ile Thr Met Cys Thr Gln Gln Ala Pro Gly

1365 1370 1375

Gln Lys Glu Cys Asp Asn Ala Leu Arg Glu Leu Glu Thr Val Arg Glu

1380 1385 1390

Leu Leu Glu Asn Pro Val Gln Pro Ile Asn Asp Met Ser Tyr Phe Gly

1395 1400 1405

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

1410 1415 1420

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

1425 1430 1435 1440

Glu Ala Ile Ala Thr Ala Ser Lys Ala Leu Cys Gly Phe Thr Glu Ala

1445 1450 1455

Ala Ala Gln Ala Ala Tyr Leu Val Gly Val Ser Asp Pro Asn Ser Gln

1460 1465 1470

Ala Gly Gln Gln Gly Leu Val Glu Pro Thr Gln Phe Ala Arg Ala Asn

1475 1480 1485

Gln Ala Ile Gln Met Ala Cys Gln Ser Leu Gly Glu Pro Gly Cys Thr

1490 1495 1500

Gln Ala Gln Val Leu Ser Ala Ala Thr Ile Val Ala Lys His Thr Ser

1505 1510 1515 1520

Ala Leu Cys Asn Ser Cys Arg Leu Ala Ser Ala Arg Thr Ala Asn Pro

1525 1530 1535

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

1540 1545 1550

Thr Ala Asn Leu Val Lys Thr Ile Lys Ala Leu Asp Gly Ala Phe Thr

1555 1560 1565

Glu Glu Asn Arg Ala Gln Cys Arg Ala Ala Thr Ala Pro Leu Leu Glu

1570 1575 1580

Ala Val Asp Asn Leu Ser Ala Phe Ala Ser Asn Pro Glu Phe Ser Ser

1585 1590 1595 1600

Val Pro Ala Gln Ile Ser Pro Glu Gly Arg Ala Ala Met Glu Pro Ile

1605 1610 1615

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

1620 1625 1630

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

1635 1640 1645

Val Leu Ala Gly His Ser Arg Thr Val Ser Asp Ser Ile Lys Lys Leu

1650 1655 1660

Ile Thr Ser Met Arg Asp Lys Ala Pro Gly Gln Leu Glu Cys Glu Thr

1665 1670 1675 1680

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

1685 1690 1695

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

1700 1705 1710

Glu Ala Leu His Thr Gln Met Leu Thr Ala Val Gln Glu Ile Ser His

1715 1720 1725

Leu Ile Glu Pro Leu Ala Ser Ala Ala Arg Ala Glu Ala Ser Gln Leu

1730 1735 1740

Gly His Lys Val Ser Gln Met Ala Gln Tyr Phe Glu Pro Leu Thr Leu

1745 1750 1755 1760

Ala Ala Val Gly Ala Ala Ser Lys Thr Leu Ser His Pro Gln Gln Met

1765 1770 1775

Ala Leu Leu Asp Gln Thr Lys Thr Leu Ala Glu Ser Ala Leu Gln Leu

1780 1785 1790

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

1795 1800 1805

Thr Gln Glu Ala Leu Glu Glu Ala Val Gln Met Met Thr Glu Ala Val

1810 1815 1820

Glu Asp Leu Thr Thr Thr Leu Asn Glu Ala Ala Ser Ala Ala Gly Val

1825 1830 1835 1840

Val Gly Gly Met Val Asp Ser Ile Thr Gln Ala Ile Asn Gln Leu Asp

1845 1850 1855

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

1860 1865 1870

Thr Met Val Arg Thr Ala Lys Ala Ile Ala Val Thr Val Gln Glu Met

1875 1880 1885

Val Thr Lys Ser Asn Thr Ser Pro Glu Glu Leu Gly Pro Leu Ala Asn

1890 1895 1900

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

1905 1910 1915 1920

Ala Val Ala Ala Glu Asn Glu Glu Ile Gly Ala His Ile Lys His Arg

1925 1930 1935

Val Gln Glu Leu Gly His Gly Cys Ala Ala Leu Val Thr Lys Ala Gly

1940 1945 1950

Ala Leu Gln Cys Ser Pro Ser Asp Ala Tyr Thr Lys Lys Glu Leu Ile

1955 1960 1965

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

1970 1975 1980

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

1985 1990 1995 2000

Ala Val Ser Gly Ile Ile Ala Asp Leu Asp Thr Thr Ile Met Phe Ala

2005 2010 2015

Thr Ala Gly Thr Leu Asn Arg Glu Gly Ala Glu Thr Phe Ala Asp His

2020 2025 2030

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

2035 2040 2045

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

2050 2055 2060

Ala Gln Ser Ser Val Ala Thr Ile Thr Arg Leu Ala Asp Val Val Lys

2065 2070 2075 2080

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

2085 2090 2095

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

2100 2105 2110

Ser Ala Thr Lys Ala Ala Ala Gly Lys Val Gly Asp Asp Pro Ala Val

2115 2120 2125

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

2130 2135 2140

Leu Leu Lys Thr Val Lys Ala Val Glu Asp Glu Ala Thr Lys Gly Thr

2145 2150 2155 2160

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

2165 2170 2175

Phe Cys Ser Pro Glu Pro Pro Ala Lys Thr Ser Thr Pro Glu Asp Phe

2180 2185 2190

Ile Arg Met Thr Lys Gly Ile Thr Met Ala Thr Ala Lys Ala Val Ala

2195 2200 2205

Ala Gly Asn Ser Cys Arg Gln Glu Asp Val Ile Ala Thr Ala Asn Leu

2210 2215 2220

Ser Arg Arg Ala Ile Ala Asp Met Leu Arg Ala Cys Lys Glu Ala Ala

2225 2230 2235 2240

Phe His Pro Asp Val Ala Pro Asp Val Arg Leu Arg Ala Leu His Tyr

2245 2250 2255

Gly Arg Glu Cys Ala Asn Gly Tyr Leu Glu Leu Leu Asp His Val Leu

2260 2265 2270

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

2275 2280 2285

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

2290 2295 2300

Glu Ala Met Lys Gly Thr Glu Trp Val Asp Pro Glu Asp Pro Thr Val

2305 2310 2315 2320

Ile Ala Glu Asn Glu Leu Leu Gly Ala Ala Ala Ala Ile Glu Ala Ala

2325 2330 2335

Ala Lys Lys Leu Glu Gln Leu Lys Pro Arg Ala Lys Pro Lys Glu Ala

2340 2345 2350

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

2355 2360 2365

Ile Ala Ala Ala Thr Ser Ala Leu Val Lys Ala Ala Ser Ala Ala Gln

2370 2375 2380

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

2385 2390 2395 2400

Leu Asp Asp Gly Gln Trp Ser Gln Gly Leu Ile Ser Ala Ala Arg Met

2405 2410 2415

Val Ala Ala Ala Thr Asn Asn Leu Cys Glu Ala Ala Asn Ala Ala Val

2420 2425 2430

Gln Gly His Ala Ser Gln Glu Lys Leu Ile Ser Ser Ala Lys Gln Val

2435 2440 2445

Ala Ala Ser Thr Ala Gln Leu Leu Val Ala Cys Lys Val Lys Ala Asp

2450 2455 2460

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

2465 2470 2475 2480

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

2485 2490 2495

Phe Glu Glu Pro Glu Asn Glu Thr Val Val Val Lys Glu Lys Met Val

2500 2505 2510

Gly Gly Ile Ala Gln Ile Ile Ala Ala Gln Glu Glu Met Leu Arg Lys

2515 2520 2525

Glu Arg Glu Leu Glu Glu Ala Arg Lys Lys Leu Ala Gln Ile Arg Gln

2530 2535 2540

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

2545 2550 2555

<210> 2

<211> 140

<212> PRT

<213> Profile protein ()

<400> 2

Met Ala Gly Trp Asn Ala Tyr Ile Asp Asn Leu Met Ala Asp Gly Thr

1 5 10 15

Cys Gln Asp Ala Ala Ile Val Gly Tyr Lys Asp Ser Pro Ser Val Trp

20 25 30

Ala Ala Val Pro Gly Lys Thr Phe Val Asn Ile Thr Pro Ala Glu Val

35 40 45

Gly Val Leu Val Gly Lys Asp Arg Ser Ser Phe Phe Val Asn Gly Leu

50 55 60

Thr Leu Gly Gly Gln Lys Cys Ser Val Ile Arg Asp Ser Leu Leu Gln

65 70 75 80

Asp Gly Glu Phe Thr Met Asp Leu Arg Thr Lys Ser Thr Gly Gly Ala

85 90 95

Pro Thr Phe Asn Ile Thr Val Thr Met Thr Ala Lys Thr Leu Val Leu

100 105 110

Leu Met Gly Lys Glu Gly Val His Gly Gly Met Ile Asn Lys Lys Cys

115 120 125

Tyr Glu Met Ala Ser His Leu Arg Arg Ser Gln Tyr

130 135 140

<210> 3

<211> 661

<212> PRT

<213> HGF activator protein ()

<400> 3

Met Gly Arg Trp Ala Trp Val Pro Ser Pro Cys Pro Pro Pro Arg Leu

1 5 10 15

Ser Leu Leu Leu Leu Leu Leu Leu Leu Val Pro Arg Gly Ala Gln Pro

20 25 30

Gln Ala Gly Arg Asn Gln Thr Glu Ala Pro Ala Gln Lys Ala Thr Val

35 40 45

Thr Pro Gly Thr Pro Leu Ile Pro Val Thr Ser Ala Thr Leu Arg Pro

50 55 60

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

65 70 75 80

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

85 90 95

Val Leu Thr Glu Ala Gly Gln Pro Cys Arg Phe Pro Phe Arg Tyr Gly

100 105 110

Gly Arg Met Ile His Ser Cys Thr Ser Glu Gly Ser Ala His Arg Lys

115 120 125

Trp Cys Ala Thr Thr His Asn Tyr Asp Arg Asp Arg Ala Trp Gly Tyr

130 135 140

Cys Val Gln Ala Ser Thr Pro Trp Glu Gly Pro Ala Ala Leu Asp Pro

145 150 155 160

Cys Ala Ser Gly Pro Cys Leu Asn Gly Gly Ser Cys Ser Ser Thr Gln

165 170 175

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

180 185 190

Asp Cys Gly Ser Glu Lys Cys Phe Asp Glu Ser Arg Tyr Glu Tyr Leu

195 200 205

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

210 215 220

Cys Glu Cys Ala Gly Gly Gln Val Arg Cys Gln Gly Thr Arg His Thr

225 230 235 240

Ala Cys Leu Ser Ser Pro Cys Leu Asn Gly Gly Thr Cys His Leu Ile

245 250 255

Val Ala Thr Gly Thr Thr Val Cys Ala Cys Pro Pro Gly His Ala Gly

260 265 270

Arg Leu Cys Asn Ile Val Pro Ala Gln Ser Cys Phe Val Gly Ser Gly

275 280 285

Thr Glu Tyr Arg Gly Val Ala Ser Thr Ala Ala Ser Gly Leu Ser Cys

290 295 300

Leu Ala Trp Asn Ser Asp Leu Leu Tyr Gln Glu Leu His Val Asp Ser

305 310 315 320

Val Gly Ala Ala Ala Leu Leu Gly Leu Gly Pro His Ala Tyr Cys Arg

325 330 335

Asn Pro Asp Lys Asp Glu Arg Pro Trp Cys Tyr Val Val Lys Asp Ser

340 345 350

Ala Leu Ser Trp Glu Tyr Cys Arg Leu Ala Ala Cys Glu Ser Leu Ala

355 360 365

Arg Ile Gln Pro Leu Pro Pro Glu Val Leu Leu Thr Leu Pro Glu Pro

370 375 380

Thr Ala Ala Gly Pro Ala Gly Arg Gln Asn Cys Gly Lys Arg His Lys

385 390 395 400

Lys Arg Thr Phe Leu Arg Pro Arg Ile Ile Gly Gly Ser Ser Ser Leu

405 410 415

Pro Gly Ser His Pro Trp Leu Ala Ala Ile Tyr Ile Gly Asn Asn Phe

420 425 430

Cys Ala Gly Ser Leu Val His Thr Cys Trp Val Val Ser Ala Ala His

435 440 445

Cys Phe Ala Asn Ser Leu Ala Gly Pro Trp Ala Gly Pro Pro Val Ala

450 455 460

Ser Trp Ala Pro Arg Pro Gln Pro Leu Cys Pro Ala Gly Arg His Pro

465 470 475 480

Trp Asp Ala Leu Ala Pro Phe Cys Leu Ser Trp Ala Pro Ala Arg Gln

485 490 495

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

500 505 510

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

515 520 525

Pro Glu Pro Gly Ser Pro Phe Pro Ala Gly His Lys Cys Gln Ile Ala

530 535 540

Gly Trp Gly His Gln Asp Glu Asn Val Ser Gly Tyr Ser Ser Ser Leu

545 550 555 560

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

565 570 575

Glu Val Tyr Gly Ala Asp Ile Ser Pro Asn Met Leu Cys Ala Gly Tyr

580 585 590

Phe Asp Cys Arg Ser Asp Ala Cys Gln Gly Asp Ser Gly Gly Pro Leu

595 600 605

Ala Cys Glu Lys Asn Gly Val Ala His Leu Tyr Gly Ile Ile Ser Trp

610 615 620

Gly Asp Gly Cys Gly Arg Leu Asn Lys Pro Gly Val Tyr Thr Arg Val

625 630 635 640

Ala Asn Tyr Val Asp Trp Ile Asn Asp Arg Ile Trp Pro Ser Lys Arg

645 650 655

Pro Ala Asp Pro Ser

660

<210> 4

<211> 261

<212> PRT

<213> Carbonic anhydrase protein ()

<400> 4

Met Thr Ser Pro Ala Trp Gly Tyr Asp Gly Glu Tyr Gly Pro Glu His

1 5 10 15

Trp Ser Lys Val Tyr Pro Ile Ala Asn Gly Asn Asn Gln Ser Pro Ile

20 25 30

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

35 40 45

Ser Val Ser Tyr Asn Pro Ala Thr Ala Lys Glu Ile Ile Asn Val Gly

50 55 60

His Ser Phe His Val Asn Phe Glu Asp Asn Asp Asn Arg Ser Val Leu

65 70 75 80

Lys Asp Gly Pro Leu Ser Glu Ser Tyr Arg Leu Leu Gln Phe His Phe

85 90 95

His Trp Gly Lys Thr Asp Asp Tyr Gly Ser Glu His Leu Val Asp Gly

100 105 110

Ala Lys Tyr Ser Ala Glu Leu His Ile Val His Trp Asn Ser Ala Lys

115 120 125

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

130 135 140

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

145 150 155 160

Val Leu Asp Ala Leu Lys Gly Ile Lys Tyr Lys Gly Lys Lys Ala Pro

165 170 175

Phe Thr Asn Phe Asp Pro Ser Val Leu Leu Pro Ser Ser Leu Asp Tyr

180 185 190

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

195 200 205

Asn Trp Ile Ile Leu Lys Glu Asn Ile Ser Ile Ser Ser Asp Gln Leu

210 215 220

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

225 230 235 240

Pro Ile Lys His Asn Asn Arg Pro Pro Gln Pro Leu Lys Gly Arg Ile

245 250 255

Val Lys Ala Ser Phe

260

Claims (1)

1. The use of the protein is characterized in that the protein is the combination of any one of talin1 protein, Profilin protein and HGF activator protein and Carbonic anhydrase protein:

a talin1 protein, wherein the sequence of the talin1 protein is shown as an amino acid sequence NO. 1;

the sequence of the Profile protein is shown as amino acid sequence NO. 2;

HGF activator protein, the sequence of which is shown in amino acid sequence NO. 3;

the sequence of the Carbonic anhydride protein is shown as amino acid sequence NO. 4;

the protein is used as a marker for identifying early pregnancy of Tibetan pigs.

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