CN1712532A - Pig tyraminase beta protein coding sequence - Google Patents

Abstract

Hog monoamine oxidase B protein coding sequence is characterized by having nucleotide sequence from 129-1691 bit in SEQ IDNO.1 and monoamine oxidase B protein coding sequence homology 92.73% with human, rat, ox and dog, having amino acid sequence polypeptide in SEQ ID No:1, or its conserved variant polypeptide, or its active fragment, or its active derivative, having monoamine oxidase B amino acid sequence homology 93.81% with human, rat, ox and dog, cloning according MAO-B gene sequence, sequencing, obtaining hog MOA-B cDNA sequence, and homologous comparing for different species CDS sequence of MAO-B gene. It can be used for relation of MAO-gene and hog stress sensitivity and human related stress diseases.

Description

Pig monoamine oxidase-B albumen coded sequence

Technical field

What the present invention relates to is a kind of protein coding sequence that is used for the biological gene engineering field, particularly the albumen coded sequence of pig monoamine oxidase-B (MAO-B).

Background technology

(monoamine oxidase B MAO-B) is a kind of flavo-enzyme that is combined on the mitochondrial outer membrane to monoamine oxidase-B, and this kind of enzyme is the key enzyme in several important monoamine neurotransmitter oxidative degradation processes.The monoamine material has important neural activity, vasoactive and the necessary function of some other vital movement, thereby monoamine oxidase-B plays an important role in the normal active adjusting of neuroendocrine system.Monoamine oxidase (MAO) is that nineteen twenty-eight Hare finds at first.1970, according to the specificity of its preference substrate and inhibitor, MAO divided A, B amphitypy.The preference substrate of MAO-B is phenylethylamine (PEA) and benzylamine, is suppressed by deprenyl specifically.

Research to MAO-B more concentrates on its inhibitor on the treatment of diseases such as dysthymia disorders, schizophrenia.Recent people MAO-B (Binda etc., 2002) crystalline structure is found, and told and striden film district, FAD land, substrate inhibition land,, efficiently inhibitor how single-minded for design, thus valid approach more provided for the treatment of relative disease.In modern Swine Production, when the change of environmental factorss such as weather, feed, wean and the stimulation of being transported, butchering etc., pig only can show and sting tail, fight, nervous, terrified uneasy, often yelp, do not eat sit up, symptoms such as maldigestion, diarrhoea, the pig that has is butchered the back and meat of poor quality (PSE meat) pale, soft, that seep water occurs.These symptoms of stress all can have a strong impact on production performance, bring tremendous loss to pig industry.The basis of stress reaction is to occur a series of neuroendocrine reaction disorders in the body.Because the vital role of MAO-B in regulating normal neuroendocrine system, its effect in the stress of baby pigs caused ablaction reaction should draw attention.

In analysis to the prior art document, 1988, the full-length cDNA of people liver MAO-B is cloned. and the amino acid sequence analysis of being derived by cDNA shows that the molecular mass of MAO-B is respectively 5810ku, and proved that MAO-A and MAO-B are by different genes encoding, be not by product with a kind of protein processing.The coding protein sequence of ox, mouse, rat, dog MAO-B is obtained in succession subsequently.Wherein the MAO-B albumen coded sequence of dog obtained in 2003, at Chie HASHIZUME, Masatoshi SUZUKI andKoji MASUDA, " Molecular Cloning of Canine Monoamine Oxidase Subtypes A (MAOA) and B (MAOB) cDNAs and Their Expression in the Brain " .J.Vet.Med.Sci..Vol.65:893-898. (2003), be Chie HASHIZUME, Masatoshi SUZUKI and KojiMASUDA etc. are published in Japan's " animal doctor's medical journal " (2003,65 volumes) report in " molecular cloning of dog MAO-A and MAO-B cDNA and the expression in the brain thereof " literary composition on, they obtained with the method for homologous clone dog MAO-B albumen coded sequence and compare analysis with its sequence with by the aminoacid sequence that sequence is derived with other Mammals.But do not find as yet that so far the clone obtains pig MAO-B gene coded protein sequence.

Summary of the invention

The objective of the invention is at the deficiencies in the prior art, a boar monoamine oxidase-B albumen coded sequence is provided.Make its can for the mankind stress relative disease research animal model is provided, the present invention is with reference to other Mammals MAO-B gene order, and utilize homologous clone and electronic cloning method, cloning and sequencing obtains the cDNA sequence of pig MOA-B, and the CDS sequence of different plant species MAO-B gene carried out homology relatively, be the relation research of studying MAO-B gene and pig stress sensitive from now on and the research and the treatment of human relative disease with going a long way greatly.

The present invention is achieved by the following technical solutions, and pig MAO-B gene is that one section sequence with people's MAO-B is that probe carries out the nucleotide sequence that electronic cloning obtains complete proteins encoded among the present invention.After electronic cloning obtains complete coding protein sequence, on the sequence that obtains, design primer, and be that template is carried out reverse transcription PCR with the total RNA that from piglet colon, extracts, and cloning and sequencing, checking electronic cloning result has finally obtained pig MAO-B gene protein encoding sequence.The pig MAO-B gene protein encoding sequence that obtains is seen SEQ ID NO.1.The protein coding sequence that encoding sequence (CDS) in the pig MAO-B gene cDNA sequence is translated into by universal code is seen SEQ ID NO.1.

The pig MAO-B gene protein encoding sequence that the present invention obtained has among the SEQ ID NO.1 from the nucleotide sequence of 129-1691 position, and its same people, mouse, rat, ox, dog monoamine oxidase-B albumen coded sequence homology are 92.73%.

Described sequence encoding has polypeptide or its conservative property variation polypeptide or its active fragments of the aminoacid sequence shown in the SEQ ID NO.1, or its reactive derivative, its same people, mouse, rat, ox, dog monoamine oxidase-B amino acid sequence homology are 93.81%.

In the present invention, " pig MAO-B gene protein encoding sequence " refers to compare with the aminoacid sequence of SEQ ID NO.1, has 5-10 amino acid to be replaced by similar performance or close amino acid and forms polypeptide.These conservative property variation polypeptide are preferably replaced according to table 1 and are produced.

Table 1

Initial residue	Representational replacement	The preferred replacement
			??Ala(A)	?Val；Leu；Ile	??Val
??Arg(R)	?Lys；Gln；Asn	??Lys
			??Asn(N)	?Gln；His；Lys；Arg	??Gln
??Asp(D)	?Glu	??Glu
			??Cys(C)	?Ser	??Ser
??Gln(Q)	?Asn	??Asn
			??Glu(E)	?Asp	??Asp
??Gly(G)	?Pro；Ala	??Ala
			??His(H)	?Asn；Gln；Lys；Arg	??Arg
??Ile(I)	?Leu；Val；Met；Ala；Phe	??Leu
			??Leu(L)	?Ile；Val；Met；Ala；Phe	??Ile
??Lys(K)	?Arg；Gln；Asn	??Arg
			??Met(M)	?Leu；Phe；Ile	??Leu
??Phe(F)	?Leu；Val；Ile；Ala；Tyr	??Leu
			??Pro(P)	?Ala	??Ala
??Ser(S)	?Thr	??Thr
			??Thr(T)	?Ser	??Ser
??Trp(W)	?Tyr；Phe	??Tyr
			??Tyr(Y)	?Trp；Phe；Thr；Ser	??Phe
??Val(V)	?Ile；Leu；Met；Phe；Ala	??Leu

In SEQ ID NO.1, electronic cloning product length is 2559bp, wherein the 129-131 position is initiator codon ATG, the 1689-1691 position is terminator codon TGA, the 129-1691 position is coded protein zone (CDS), and the product of RT-PCR checking amplification and order-checking is for being the fragment of 1650bp from the 74-1763 bit length.At SEQ IDNO.1,520 amino acid of pig MAO-B genes encoding.

The present invention first from the tissue of pig the clone obtained the albumen coded sequence of MAO-B of pig, research MAO-B effect in the various stress reactions and then provide animal model to lay a good foundation for the research of human relative disease in Swine Production is provided.

Description of drawings

Fig. 1 pig MAO-B gene cDNA sequence clone process sketch.

Embodiment

Below in conjunction with specific embodiment, further set forth the present invention.These embodiment only are used to the present invention is described and are not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, for example the Sambrook equimolecular is cloned: laboratory manual (New York:Cold SpringHarbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

Embodiment 1

The clone of pig MAO-B gene cDNA sequence

MAO-B gene cDNA clone process is seen accompanying drawing 1.

Used experimental technique concrete operation method is as follows in clone's process:

1. electronic cloning

Search in the est database of pig with the BLASTn program that people MAO-B gene C DS nucleotides sequence is classified as among the information probe operation NCBI, obtain and the identical pig est sequence a series of different in size of probe height, then these sequences are carried out the assembly unit connection and obtain a long sequence, and then be that probe carries out BLASTn once more in the pig est database with this segment length's sequence that obtains respectively, obtain more est sequence, thereby sequence is extended to two ends, and the sequence that is obtained is the most at last spliced and is obtained complete sequence.

2. biological cloning checking

(1) separate tissue (Isolation)

Butcher 30 age in days DLY commodity piglets on the pig farm, get colon's sample, put into liquid nitrogen rapidly, be stored in ℃ refrigerator of laboratory-70 after freezing, prepare to extract total tissue RNA.

(2) preparation work of extraction RNA

The NaOH soaked overnight of used in glass products 0.1M is washed repeatedly with tap water, uses distilled water flushing again 2 times, and 180 ℃ were toasted 4 hours.

Homogenizer, electrophoresis chamber be with 3% hydrogen peroxide dipping 20-30 minute, again with 0.1% DEPC water flushing.Because the DEPC of deactivation is not influential to the PCR reaction, available 0.5% SDS handles.

The DEPC water logging bubble of Tip head, EP effective 0.1% spends the night, and autoclaving makes the DEPC inactivation.

Distilled water and solution are handled with DEPC, and promptly every 100ml water or solution add 0.1ml DEPC liquid, and room temperature is placed and spent the night 30 minutes DEPC inactivations of autoclaving.

(3) total tissue RNA is extracted

Get 100mg frozen sample of organizing in liquid nitrogen and put into 1ml Trizol (trizalreagent, invitrogen BRL, homogenate in homogenate organ pipe USA).Centrifugal 10 minutes of 4 ℃ of 12000g.Draw supernatant liquor, 15-30 ℃ of incubation 5 minutes.Add the 0.2ml chloroform, cover lid is used hand concuss 15 seconds.15-30 ℃ incubation 2-3 minute.Centrifugal 15 minutes of 4 ℃ of 12000g.Draw supernatant liquor, add the 0.8ml Virahol, mix.15-30 ℃ of incubation 10 minutes, centrifugal 10 minutes of 4 ℃ of 12000g, centrifuge tube bottom white precipitate is RNA.Outwell supernatant, add 1ml 75% washing with alcohol RNA, centrifugal 10 minutes of 4 ℃ of 7500g.Packing among seasoning or the vacuum-drying RNA. water-soluble (RNase free) ,-70 ℃ of preservations.

(4) evaluation of total tissue RNA

By measuring rna content on the spectrophotometer and with the quality of agarose electrophoretic analysis RNA, concrete grammar is as follows: electrophoresis chamber RNA scavenging solution (100mM NaOH, 1mM EDTA) soaks 4-5 hour, wash repeatedly with the DEPC treated water again that to pour 1 * TBE into behind the several stand-by.Sepharose is prepared with 1 * TBE.The 10ul sample-loading buffer (2 * TBE, 13% phenanthrene can, 0.1% bromjophenol blue, 7M urea) in add the above total tissue RNA of 1ul, 65 ℃ of sex change were put into after 10 minutes frozen water 2-3 minute immediately.Point sample is electrophoresis in sepharose.

(5) design of primers and synthetic

Upstream and downstream primer (primer is to B1) is designed in CDS 5 ' upstream and 3 ' downstream respectively in the pig MAO-B sequence that electronic cloning obtains, with amplification total length CDS.Primer designs voluntarily with Primer Premier 5.0, and precious Bioisystech Co., Ltd is synthetic by Dalian.Primer sequence is as follows:

F：5’-CAGGGGCCGAGATCCAGACA-3’R：5’-CTCCTTCCCCAAACCCACA-3’

(6) RT-PCR amplification

Carry out reverse transcription by precious biological reverse transcription test kit (the BcaBEST RNA PCR Kit Ver.1.1) requirement in Dalian.Inverse transcription reaction liquid is formed: 10ul 2 * Bca 1st Buffer, 4ul 25Mm MgSO4,1ul dNTPMixture, 0.5ul Rnase Inhibitor (40U/ul), 1ul BcaBEST Polymerase (22U/ul), 1ul Oligo dT Primer, 1ul RNA Sample, 1.5ul Rnase Free dH2O totally is 20ul.The reverse transcription reaction condition: 65 ℃ 1 minute, 30 ℃ 5 minutes (in 15 minutes-30 minutes at the uniform velocity heat up), 65 ℃ 25 minutes, 98 ℃ 5 minutes, 5 ℃ 5 minutes.Pcr amplification condition: 97 ℃ of sex change 5 minutes; 95 ℃ 30 seconds → 55 ℃ 30 seconds → 72 ℃ 1 minute, so carry out 30 times the circulation; 72 ℃ were extended 4 ℃ of preservations 10 minutes.

(7) clone and order-checking

The recovery of pcr amplified fragment.Fragment reclaims by the explanation of Shanghai China Shun a small amount of glue recovery test kit is undertaken, and operating process is as follows: as far as possible little cutting off contains the agar sugar of DNA, puts into the 1.5mlEP pipe.The ratio that adds 300ulS1 liquid in every 100mg agarose adds S1 liquid, 50 ℃ of water-baths 10 minutes.The agar liquid glucose that will dissolve moves into adsorption column, and centrifugal 1 minute of 10000g outwells liquid in the pipe.Add 500ul W1 liquid in adsorption column, centrifugal 15 seconds of 10000g outwells liquid in the pipe.In adsorption column, add 500ul W1 liquid, leave standstill 1 minute after, centrifugal 15 seconds of 10000g outwells liquid in the pipe.Centrifugal 1 minute.Adsorption column is put into a clean 1.5mlEP pipe, after adsorption film central authorities add 30ulT1 liquid, leave standstill 1 minute, centrifugal 1 minute of 10000g, liquid is the DNA of recovery in the EP pipe.

Reclaim the connection of fragment with the T carrier.Connect the test kit with TaKaRa pMD18-T Vector, operating process is as follows: the following ligation liquid of preparation in the EP pipe: pMD 18-T Vector (50ng/ul) 0.5ul, and DNA20-40ng, Solution 15ul adds dH ₂O to 10ul.30 minutes (also can spend the night) of 16 ℃ of reactions, product is used for the transformed competence colibacillus cell with above-mentioned reaction solution.

The conversion of plasmid.From-70 ℃ of refrigerators, take out the competent cell of preserving, ice bath hydrotropy.The 100ul competent cell adds 10ul and connects product, ice bath 30 minutes.In 42 ℃ of 90 seconds of water-bath heat stress, inserted immediately in the ice bath 2 minutes.Add 400ul liquid LB substratum, 37 ℃ brought back to life 50 minutes.Get 100ul and be laid on the LB flat board, flat board contains the ammonia benzyl mould Amp (100mg/ml) of 0.1% (V/V).37 ℃ constant temperature culture 10-15 hour.

The PCR that transforms bacterium colony identifies and cultivation.Transform single bacterium colony of cultivating with the marking pen mark, dip in the toothpick of sterilizing and get single bacterium colony.Dentiscalprum head is placed in to be added with in the PCR reaction solution EP pipe of (not containing template) rocks, make single bacterium colony serve as template.Increase with this segmental PCR condition of acquisition.The PCR product carries out agarose gel electrophoresis together with Marker, differentiates on the T carrier whether contain the purpose fragment.If obtain the purpose fragment; the choicest of available sterilization rifle is taken at the corresponding with it single bacterium colony on the flat board, puts into 40ml LB liquid nutrient medium, adds earlier the benzylpenicillin sodium (100mg/ml) of 0.1% (V/V) in liquid; 37 ℃ are spent the night and shake bacterium and cultivate, and are used for plasmid and reclaim.

The recovery of plasmid.Plasmid reclaims with Shanghai China Shun a small amount of plasmid extraction purification kit, and operation steps is as follows: will transform the bacterium liquid of cultivating and add in the 1.5mlEP pipe, 4000 rev/mins centrifugal, outwells liquid and obtain bacterial precipitation.It is centrifugal to add a bacterium liquid when bacterial precipitation is not enough again.Add 250ulP1 liquid in bacterial precipitation, vibration suspends.Add 250ulP2 liquid, gentleness shakes up, and room temperature left standstill 4 minutes.Add 350ulP3 liquid, gentleness shakes up.Centrifugal 10 minutes, supernatant liquor is carefully moved into adsorption column, centrifugal 15 seconds, outwell liquid.In adsorption column, add 500ulW1, centrifugal 15 seconds, outwell liquid.In adsorption column, add 500ulW1, left standstill 1 minute, centrifugal 15 seconds, outwell liquid.Centrifugal 1 minute.Adsorption column is put into a clean 1.5mlEP pipe, after adsorption film central authorities add 25-30ulT1 liquid, leave standstill 1 minute, centrifugal 1 minute.Liquid is the plasmid of recovery in the EP pipe.

The enzyme of plasmid is cut evaluation.Reclaim plasmid really for inserting the segmental recombinant plasmid of purpose in order to prove, adopt the double digestion method to identify. this research concrete operations are as follows: according to TaKaRa pMD18-T Vector figure, select restriction endonuclease Bam HI and Hind III. to guarantee not have in the purpose fragment point of contact of these two kinds of enzymes.Endonuclease reaction system composed as follows: Bam HI 1ul, Hin d III 1ul, 10 * K Buffer 2ul, DNA≤1ul adds dH2O to 20ul.30 ℃ were reacted 3 hours.Agarose gel electrophoresis detects.

The order-checking of recombinant plasmid.Get the 20ul recombinant plasmid in the EP pipe, seal film phonograph seal, the order-checking of intergrowth thing technology company.

Embodiment 2

Pig MAO-B encoding sequence homology relatively

Search and obtain people, ox, mouse, dog MAO-B gene coded protein sequence on GenBank, the SEQ ID NO.1 sequence that its present invention who obtains with cloning and sequencing is obtained is carried out sequence homology relatively together on molecular biology software DNAman.Comparative result shows: species MAO-B gene C DS sequence homologies such as SEQ ID NO.1 sequence and people, ox, mouse, dog are respectively 92.73%, and amino acid sequence homology is respectively 93.81%.

Sequence that the present invention relates to and mark apportion are as follows:

The information of SEQ ID NO.1

＜110〉Shanghai Communications University

＜120〉pig MAO-B albumen coded sequence

<160>2

<210>1

<211>2559

<212>DNA

＜213〉pig (sus scrofa)

<220>

<221>CDS

<222>(129)...(1691)

<400>1

cgccctgggc?gggcaatata?gccgctcggc?ggaggcgcga?gcgcgcgggg?gcagcgcagc??60

gggcaggggc?cgagatccag?acacccgagc?agctggccac?cggggcggcc?cagagaaggg??120

cgagcacc?atg?agc?agc?aag?tgc?gac?gtg?gtc?gtg?gta?ggg?ggc?ggc?atc?tca?ggt?176

Met?Ser?Ser?Lys?Cys?Asp?Val?Val?Val?Val?Gly?Gly?Gly?Ile?Ser?Gly

15?10?15

atg?gca?gcg?gcc?aaa?ctt?ctg?cat?gac?tct?ggc?ctg?agt?gtg?att?gtt??224

Met?Ala?Ala?Ala?Lys?Leu?Leu?His?Asp?Ser?Gly?Leu?Ser?Val?Ile?Val

20?25?30

ctg?gaa?gcc?cgg?gac?cgc?gtg?gga?ggc?agg?act?tac?acc?gtc?agg?aac??272

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

35?40?45

caa?caa?gtt?aaa?tat?gtg?gac?ctt?gga?gga?tct?tat?gtt?ggg?cca?act??320

Gln?Gln?Val?Lys?Tyr?Val?Asp?Leu?Gly?Gly?Ser?Tyr?Val?Gly?Pro?Thr

50?55?60

cag?aat?cgc?atc?tta?aga?ttg?tcc?aag?gag?cta?gga?ttg?gag?acc?tac??368

Gln?Asn?Arg?Ile?Leu?Arg?Leu?Ser?Lys?Glu?Leu?Gly?Leu?Glu?Thr?Tyr

65?70?75?80

aaa?gtg?aat?gaa?gtg?gag?cgt?ctg?att?cac?tat?gta?aag?ggc?aaa?tcc??416

Lys?Val?Asn?Glu?Val?Glu?Arg?Leu?Ile?His?Tyr?Val?Lys?Gly?Lys?Ser

85?90?95

tac?ccc?ttc?agg?ggc?cca?tta?cca?cct?gtg?agg?aat?ccg?att?acc?ttc??464

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

100?105?110

cta?gat?ctt?aac?aac?ctt?tgg?agg?acg?gtg?gat?gac?atg?gga?cga?gag??512

Leu?Asp?Leu?Asn?Asn?Leu?Trp?Arg?Thr?Val?Asp?Asp?Met?Gly?Arg?Glu

115?120?125

att?ccc?agt?gat?gcc?cca?tgg?aag?gcg?ccc?ctt?gca?gaa?cag?tgg?gac??560

Ile?Pro?Ser?Asp?Ala?Pro?Trp?Lys?Ala?Pro?Leu?Ala?Glu?Gln?Trp?Asp

130?135?140

cag?atg?aca?atg?aag?gag?ctg?ttg?gac?aag?ctc?tgc?tgg?act?gaa?tct??608

Gln?Met?Thr?Met?Lys?Glu?Leu?Leu?Asp?Lys?Leu?Cys?Trp?Thr?Glu?Ser

145?150?155?160

tcg?aag?cag?ctg?gcc?acc?ctt?ttt?gtg?aac?ctg?tgt?gtc?acc?gcg?gag??656

Ser?Lys?Gln?Leu?Ala?Thr?Leu?Phe?Val?Asn?Leu?Cys?Val?Thr?Ala?Glu

165?170?175

acc?cat?gag?gtc?tct?gct?ctc?tgg?ttc?ctg?tgg?tat?gtg?aag?cag?tgt??704

Thr?His?Glu?Val?Ser?Ala?Leu?Trp?Phe?Leu?Trp?Tyr?Val?Lys?Gln?Cys

180?185?190

gga?ggc?acc?acc?agg?atc?atc?tca?aca?act?aac?gga?ggg?cag?gag?agg??752

Gly?Gly?Thr?Thr?Arg?Ile?Ile?Ser?Thr?Thr?Asn?Gly?Gly?Gln?Glu?Arg

195?200?205

aaa?ttt?gtg?ggc?gga?tct?ggt?caa?gtg?acc?gag?cgg?ata?aag?gac?ctc??800

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

210?215?220

ctt?gga?gac?cga?gcg?aag?ctg?gag?agg?cct?gtg?gtc?cac?att?gac?cag??848

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

225?230?235?240

aca?gga?gaa?aat?gtc?ctc?gtg?gag?acc?cta?aac?cac?gag?gtg?tac?gag??896

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

245?250?255

gct?aag?tat?gtg?att?agc?gcc?att?cct?cct?gtc?ctg?ggc?atg?aag?att??944

Ala?Lys?Tyr?Val?Ile?Ser?Ala?Ile?Pro?Pro?Val?Leu?Gly?Met?Lys?Ile

260?265?270

cat?ttc?agt?ccc?cct?ctg?cca?atg?atg?aga?aac?cag?ctc?atc?act?cgt??992

His?Phe?Ser?Pro?Pro?Leu?Pro?Met?Met?Arg?Asn?Gln?Leu?Ile?Thr?Arg

275?280?285

gta?cct?ctg?ggc?tct?gtc?atc?aag?tgt?ata?gtt?tat?tac?aaa?gag?ccc??1040

Val?Pro?Leu?Gly?Ser?Val?Ile?Lys?Cys?Ile?Val?Tyr?Tyr?Lys?Glu?Pro

290?295?300

ttc?tgg?agg?cat?aag?gat?tac?tgt?gga?agc?atg?att?att?gaa?gga?gag??1088

Phe?Trp?Arg?His?Lys?Asp?Tyr?Cys?Gly?Ser?Met?Ile?Ile?Glu?Gly?Glu

305?310?315?320

gaa?gct?cca?atc?gcc?tac?acg?ttg?gat?gat?tcc?aag?cct?gat?ggc?agc??1136

Glu?Ala?Pro?Ile?Ala?Tyr?Thr?Leu?Asp?Asp?Ser?Lys?Pro?Asp?Gly?Ser

325?330?335

tgt?gcc?gcc?atc?ata?gga?ttt?atc?ctt?gcc?cac?aaa?gcc?aga?aaa?ctg??1184

Cys?Ala?Ala?Ile?Ile?Gly?Phe?Ile?Leu?Ala?His?Lys?Ala?Arg?Lys?Leu

340?345?350

gcc?cgt?ctt?acc?aaa?gaa?gaa?agg?ctg?gag?aaa?ctt?tgc?gac?ctc?tat??1232

Ala?Arg?Leu?Thr?Lys?Glu?Glu?Arg?Leu?Glu?Lys?Leu?Cys?Asp?Leu?Tyr

355?360?365

gca?aaa?gtt?ctg?ggt?tca?aaa?gaa?gct?ttg?aac?ccc?gtg?cac?tat?gaa??1280

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

370?375?380

gag?aag?aac?tgg?tgc?gag?gag?cag?tac?tcg?gcg?ggc?tgc?tac?acg?acc??1328

Glu?Lys?Asn?Trp?Cys?Glu?Glu?Gln?Tyr?Ser?Ala?Gly?Cys?Tyr?Thr?Thr

385?390?395?400

tac?ttc?ccc?cct?ggg?atc?atg?act?cag?tat?gga?agg?gtt?cta?cgc?cag??1376

Tyr?Phe?Pro?Pro?Gly?Ile?Met?Thr?Gln?Tyr?Gly?Arg?Val?Leu?Arg?Gln

405?410?415

cca?gtc?ggc?agg?att?tat?ttc?gcc?ggc?acg?gag?act?gcc?acg?cac?tgg??1424

Pro?Val?Gly?Arg?Ile?Tyr?Phe?Ala?Gly?Thr?Glu?Thr?Ala?Thr?His?Trp

420?425?430

agt?ggc?tac?atg?gag?ggg?gcc?gtg?gag?gcc?gga?gag?aga?gcg?gcc?cga??1472

Ser?Gly?Tyr?Met?Glu?Gly?Ala?Val?Glu?Ala?Gly?Glu?Arg?Ala?Ala?Arg

435?440?445

gag?atc?ctg?cat?gcc?atg?gga?aag?atc?cca?gaa?gat?gaa?atc?tgg?cag??1520

Glu?Ile?Leu?His?Ala?Met?Gly?Lys?Ile?Pro?Glu?Asp?Glu?11e?Trp?Gln

450?455?460

tct?gaa?cca?gag?tcc?gtg?gat?gtg?cct?gcg?aag?ccc?att?acc?acg?acc??1568

Ser?Glu?Pro?G1u?Ser?Val?Asp?Val?Pro?A1a?Lys?Pro?Ile?Thr?Thr?Thr

165?470?475?480

ttc?ttg?gag?aga?cac?ttg?ccc?tcg?gtg?ccc?ggc?ctg?ctg?agg?ctg?att??1616

Phe?Leu?Glu?Arg?His?Leu?Pro?Ser?Val?Pro?Gly?Leu?Leu?Arg?Leu?Ile

485?490?495

gga?ttg?acc?gcc?atc?ttt?tca?gcc?act?gct?ctc?ggc?tac?ctg?gcc?cac??1664

Gly?Leu?Thr?Ala?Ile?Phe?Ser?Ala?Thr?Ala?Leu?Gly?Tyr?Leu?Ala?His

500?505?510

aaa?agg?ggg?cta?ctc?gtg?cgg?gtc?tga?agggagggcc?tctgtaacca??1711

Lys?Arg?Gly?Leu?Leu?Val?Arg?Val

515?520

caccctctca?ccctcctctc?attctgtgtg?gtgtgtgggt?ttggggaagg?agttgtaata??1771

acgttgcatg?aagacccaaa?gaatgtagac?taaggtggga?gcgtgagatg?aggccgactt??1831

ccggtcgcag?gaaaccccgt?ctctcattct?ccattttgat?tcccgcggag?tatctcttcc??1891

gtcgcttagc?gctctgtctc?tctcgttccc?aggtttactg?gcccgcgaat?ctttatagtc??1951

gttaaacagg?cttgttaaag?gtccttgctc?tcctacaata?ttcccttgcc?atgcacaaaa??2011

atctcctttt?ttccccaccc?tgtggcttta?tgcttaccct?tccgcttggc?tgtcgtgtca??2071

tcatcttcca?agttctgtgc?attggtcctt?agacttctgt?gttgttacag?ttgcaagacc??2131

ttagagacca?cctcgtcctt?ttttctattt?tagagttgag?ccaactgaag?ctgagggaga??2191

tggaatgtaa?ttgcccagtg?tccacaataa?gccgctggta?cttggggccc?taggtcatgg??2251

gtcccttgct?tttcccaccc?cccaggatga?atttcccaat?cactttcctc?cactccctgc??2311

tgtggttgtt?ggtggtgtcc?ccttgcgtgg?tttgctctgt?gctaaattgt?tttggtttat??2371

tcagatgcga?ctctttccct?ttctatctcg?tgtactgtgt?cttcagctca?ttttaatttg??2431

gggggtagga?ggttgggtaa?gaatcttgtc?ttttcttcca?tttgtgttct?ccattgtatc??2491

tggatacaaa?ggttggtata?catttgggta?attaaaaata?aagttgattg?aaaaaaaaaa??2551

aaaaaaa??2559

<210>2

<211>520

<212>PRT

＜213〉pig (sus scrofa)

<400>2

Met?Ser?Ser?Lys?Cys?Asp?Val?Val?Val?Val?Gly?Gly?Gly?Ile?Ser?Gly

1?5?10?15

Met?Ala?Ala?Ala?Lys?Leu?Leu?His?Asp?Ser?Gly?Leu?Ser?Val?Ile?Val

20?25?30

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

35?40?45

Gln?Gln?Val?Lys?Tyr?Val?Asp?Leu?Gly?Gly?Ser?Tyr?Val?Gly?Pro?Thr

50?55?60

Gln?Asn?Arg?Ile?Leu?Arg?Leu?Ser?Lys?Glu?Leu?Gly?Leu?Glu?Thr?Tyr

65?70?75?80

Lys?Val?Asn?Glu?Val?Glu?Arg?Leu?Ile?His?Tyr?Val?Lys?Gly?Lys?Ser

85?90?95

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

100?105?110

Leu?Asp?Leu?Asn?Asn?Leu?Trp?Arg?Thr?Val?Asp?Asp?Met?Gly?Arg?Glu

115?120?125

Ile?Pro?Ser?Asp?Ala?Pro?Trp?Lys?Ala?Pro?Leu?Ala?Glu?Gln?Trp?Asp

130?135?140

Gln?Met?Thr?Met?Lys?Glu?Leu?Leu?Asp?Lys?Leu?Cys?Trp?Thr?Glu?Ser

145?150?155?160

Ser?Lys?Gln?Leu?Ala?Thr?Leu?Phe?Val?Asn?Leu?Cys?Val?Thr?Ala?Glu

165?170?175

Thr?His?Glu?Val?Ser?Ala?Leu?Trp?Phe?Leu?Trp?Tyr?Val?Lys?Gln?Cys

180?185?190

Gly?Gly?Thr?Thr?Arg?Ile?Ile?Ser?Thr?Thr?Asn?Gly?Gly?Gln?Glu?Arg

195?200?205

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

210?215?220

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

225?230?235?240

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

245?250?255

Ala?Lys?Tyr?Val?Ile?Ser?Ala?Ile?Pro?Pro?Val?Leu?Gly?Met?Lys?Ile

260?265?270

His?Phe?Ser?Pro?Pro?Leu?Pro?Met?Met?Arg?Asn?Gln?Leu?Ile?Thr?Arg

275?280?285

Val?Pro?Leu?Gly?Ser?Val?Ile?Lys?Cys?Ile?Val?Tyr?Tyr?Lys?Glu?Pro

290?295?300

Phe?Trp?Arg?His?Lys?Asp?Tyr?Cys?Gly?Ser?Met?Ile?Ile?Glu?Gly?Glu

305?310?315?320

Glu?Ala?Pro?Ile?Ala?Tyr?Thr?Leu?Asp?Asp?Ser?Lys?Pro?Asp?Gly?Ser

325?330?335

Cys?Ala?Ala?Ile?Ile?Gly?Phe?Ile?Leu?Ala?His?Lys?Ala?Arg?Lys?Leu

340?345?350

Ala?Arg?Leu?Thr?Lys?Glu?Glu?Arg?Leu?Glu?Lys?Leu?Cys?Asp?Leu?Tyr

355?360?365

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

370?375?380

Glu?Lys?Asn?Trp?Cys?Glu?Glu?Gln?Tyr?Ser?Ala?Gly?Cys?Tyr?Thr?Thr

385?390?395?400

Tyr?Phe?Pro?Pro?Gly?Ile?Met?Thr?Gln?Tyr?Gly?Arg?Val?Leu?Arg?Gln

405?410?415

Pro?Val?Gly?Arg?Ile?Tyr?Phe?Ala?Gly?Thr?Glu?Thr?Ala?Thr?His?Trp

420?425?430

Ser?Gly?Tyr?Met?Glu?Gly?Ala?Val?Glu?Ala?Gly?Glu?Arg?Ala?Ala?Arg

435?440?445

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

450?455?460

Ser?Glu?Pro?Glu?Ser?Val?Asp?Val?Pro?Ala?Lys?Pro?Ile?Thr?Thr?Thr

165?470?475?480

Phe?Leu?Glu?Arg?His?Leu?Pro?Ser?Val?Pro?Gly?Leu?Leu?Arg?Leu?Ile

485?490?495

Gly?Leu?Thr?Ala?Ile?Phe?Ser?Ala?Thr?Ala?Leu?Gly?Tyr?Leu?Ala?His

500?505?510

Lys?Arg?Gly?Leu?Leu?Val?Arg?Val

515?520

Claims (5)

1, a boar monoamine oxidase-B albumen coded sequence, it is characterized in that, the pig MAO-B gene protein encoding sequence that is obtained has among the SEQ ID NO.1 from the nucleotide sequence of 129-1691 position, and its same people, mouse, rat, ox, dog monoamine oxidase-B albumen coded sequence homology are 92.73%.

2, pig monoamine oxidase-B albumen coded sequence according to claim 1, it is characterized in that, the pig MAO-B gene protein encoding sequence of described acquisition has polypeptide or its conservative property variation polypeptide or its active fragments of the aminoacid sequence shown in the SEQ ID NO.1, or its reactive derivative, its same people, mouse, rat, ox, dog monoamine oxidase-B amino acid sequence homology are 93.81%.

3, according to claim 1 or 2 described pig monoamine oxidase-B albumen coded sequences, it is characterized in that, described pig MAO-B gene protein encoding sequence, finger is compared with the aminoacid sequence of SEQ ID NO.1, has 5-10 amino acid to be replaced by similar performance or close amino acid and forms polypeptide.

4, according to claim 1 or 2 or 3 described pig monoamine oxidase-B albumen coded sequences, it is characterized in that, described SEQ ID NO.1, therein, electronic cloning product length is 2559bp, and wherein the 129-131 position is initiator codon ATG, and the 1689-1691 position is terminator codon TGA, the 129-1691 position is coded protein zone (CDS), and the product of RT-PCR checking amplification and order-checking is for being the fragment of 1650bp from the 74-1763 bit length.

5, according to claim 1 or 2 or 3 described pig monoamine oxidase-B albumen coded sequences, it is characterized in that, described SEQ ID NO.1, therein, 520 amino acid of pig MAO-B genes encoding.

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