CN1530444A - Gene related to blood sugar regulation, protein product and use thereof - Google Patents

Abstract

A novel gene pank 4 associated with blood sugar regulation, the protein product coded by it, and the application of said gene and protein in regulating blood sugar and treating diabetes are disclosed.

Description

The new gene that blood glucose regulation is relevant, its protein product and uses thereof

Technical field

The present invention relates to a kind of new gene relevant with blood glucose regulation, called after pank4 (claiming fang-1 again) the invention still further relates to the protein of described genes encoding and described gene and the purposes of protein in blood glucose regulation and treating diabetes.The preliminary discriminating of gene of the present invention is a kind of new Pantothen kinase gene.

Background technology

As everyone knows, type ii diabetes is a kind of disease of multifactorial inheritance.Think that at present the absorption that participates in insulin signaling pathway, glucose transport approach, glycogen route of synthesis, lipid acid all may be relevant with the generation of diabetes with protein involved molecule synthetic, the lipocyte differentiation pathway.Research onset diabetes mechanism, the research of illustrate molecular mechanism, identifying blood glucose regulation gene and function thereof all has crucial meaning to the prevention and the treatment of diabetes.Wherein, the insulin signaling pathway is the more field of studying at present, this approach is very complicated, comprises insulin receptor, IRS etc., and links together with Akt signal transduction path, MAPK signal transduction path, CAP/Cb1 signal transduction path etc.Insulin receptor belongs to receptor tyrosine kinase, comprises IGF-I and IRR.At least 9 insulin receptor kinase substrates have been identified at present, comprising 4 IRS IRS1, IRS2, IRS3 and IRS4.IRS (IRS) has high homology, but acts on different, additional mutually.The IRS-1 knock out mice shows as in utero slow with postnatal growth, insulin resistant, and glucose tolerates symptoms such as impaired.The IRS-2 knock out mice shows as at liver etc. and organizes insulin resistant and comprise a plurality of region growing defectives such as cerebral tissue, finally develops into type ii diabetes.

Identify that the diabetes tumor susceptibility gene is a large order, make slow progress at present that along with the end of human genome examining order, a lot of scientists are just using full genome scanning technology and seeking tumor susceptibility gene.But because diabetes are a kind of multigenic diseases, it is the generation that gene and gene interaction and gene and environmental interaction are just induced diabetes, some genes are related perhaps not strong with this disease separately, locatees clone's work and bring a lot of troubles so give.

At present, a lot of scientist's application of difference technique of display have been separated the gene and the disease gene of many differential expressions.Differential display technique is based on RT-PCR.With oligomerization (dT) is all mRNA in the primer reverse transcription cell, by different primers to resulting cDNA is carried out pcr amplification, wherein contain a kind of isotopic labeling dNTP, the PCR product separates in the polyacrylamide gel electrophoresis then, downcut the recovery differential fragment from gel after the radioautograph, after determining positive differential fragment, clone's full-length gene.The advantage that difference shows is fast simple, and this method does not need special instrument, and it is fairly simple to operate, and can obtain difference within a short period of time and show collection of illustrative plates; Repeatability is high, and about 90%-95% difference band can be repeated; The susceptibility height only needs the total RNA of 2ug be used for reverse transcription, reacts with 4 kinds of oligomerization (dT) primers and 20 AP primers pairing PCR, can cover mRNA more than 98% from statistics.Difference shows the research that is used for the medical science aspect in a large number.Liang etc. at first are used for this technology the research of tumour, and separated difference expression gene (Science 257:967-971,1992), and people such as Nishio Y are applied to diabetes study with this technology, they with bovine aortic smooth muscle cells after external different blood sugar concentrations are cultivated, the cDNA fragment of difference display separation to 3 differential expression (FASEB letter, 8:103-106,1994).But these researchs are at present used cultured cell in vitro mostly as research object, and cell can not represent them at the intravital normal physiological state of machine under condition of in vitro culture well.The inventor conducts a research from integral level rather than in the cultured cells level, carries out the mask work of blood glucose regulation genes involved, with the candidate gene as treating diabetes.The inventor adopts the SD rat, and through jugular vein right atrium intubation, glucose stimulates, and difference shows the gene of SD rat glucose stimulating group and non-stimulating group differential expression.At first obtaining the EST of differential expression, is that probe screening rat skeletal muscle cDNA obtains 1 full-length cDNA with these EST, further studies based on this, finds that new gene of the present invention is a kind of new Pantothen kinase gene.

Pantothen kinase is the enzyme of CoA building-up process the first step effect; it is with the pantothenic acid phosphorylation; then rapidly it is changed into CoA; CoA is a very important acyl carrier, and it mainly participates in tricarboxylic acid cycle, fatty acid metabolism and all kinds of intermediary metabolism reaction; part 4 ' among the CoA-PPA is an important precursor group in many enzyme systems; comprise acyl carrier protein, bacterium and Eukaryotic fatty acid synthetase, and citrate lyase.Pantothen kinase is considered to CoA synthetic rate-limiting enzyme in mammalian body.

Pantothen kinase studies show that in early days in isolating perfused hearts, Regular Insulin is considered to the agent of CoA synthetic strongly inhibited, reduces insulin concentration or remove inhibitor in the heart of diabetes sample, will make the synthetic enhancing of CoA.The same level that reduces Regular Insulin in the animal body of fasting or diabetes also can make the synthetic raising of CoA.In the heart that exsomatizes, the activity of Pank will and reduce along with glucose, pyruvic acid, lipid acid and Regular Insulin rising.And the activity of Pantothen kinase will be along with glycogen, hunger, high fat, first raises during anti-and diabetes in liver.

In the cardiac muscle, the synthetic control that is subjected to the first step in the approach of CoA is just from pantothenic acid to 4 '-phosphoric acid pantothenic acid.Glucose such as pyruvic acid, β-Qiang Dingsuan, Palmiticacid, low amount in the heart that exsomatizes all can suppress the reaction of Pantothen kinase.Insulin requirement suppresses the activity of Pantothen kinase under the situation that glucose exists.If do not have glucose or Palmiticacid to replace glucose, Regular Insulin will can not work, and this shows that Regular Insulin exercises its function by the conversion carbon cycle.Because the catalyzed reaction efficient of Pantothen kinase is subjected to the influence of external source substrate, when above-mentioned substrate is utilized, will increase so, so Pantothen kinase is that promotion is glycometabolic such as intermediate products such as tricarboxylic acid cycle or glycolysis-s.

In a word, above-mentioned data shows that Pantothen kinase may be the very crucial factor of glycemic control.Yet, up to the present also do not know the target site of Pantothen kinase gene in carbohydrate metabolism and insulin signaling pathway.

Summary of the invention

The invention provides a kind of isolated nucleic acid molecule, it comprises a kind of proteinic polynucleotide that participate in the insulin signaling pathway of coding.Nucleic acid molecule of the present invention is a kind of new Pantothen kinase gene, and it is named as the pank4 gene, and it is from rat, and nucleotide sequence is shown in SEQID NO:1, and its amino acid sequence coded is shown in SEQ ID NO:2.The invention still further relates to and have the polynucleotide that have the nucleotide sequence of 90%, preferably at least 95%, 96%, 97%, 98% or 99% homogeny with the described nucleotide sequence of SEQ ID NO:1 at least, perhaps under stringent condition with the polynucleotide of the described nucleotide sequence hybridization of SEQ ID NO:1.Described stringent condition is well known to those skilled in the art, for example shows " molecular cloning laboratory manual " referring to people such as Sambrook.

The invention still further relates to the recombinant vectors that comprises isolated nucleic acid molecule of the present invention, comprise the host cell of this recombinant vectors, and they produce the proteic method of pank4 through recombinant technology to make such carrier and host cell and use.The present invention also relates to be specific to the proteic antibody of pank4.Those skilled in the art know various suitable expression vector and host cell, the carrier for expression of eukaryon pCDNA3.1 that the non-limitative example of expression vector for example uses in the embodiment of the invention, prokaryotic expression carrier pGEX-4T-3 (being gsh (Gst) carrier) etc.Host cell can be the conventional prokaryotic host cell that uses, yeast, mammalian cell, insect cell etc., the coli strain DH5a that non-limitative example for example uses in the embodiment of the invention, with coli strain BL21 (DE3), the normal myoblast strain of L-6TG SD rat etc.

Pank4 gene of the present invention can high expression level after blood sugar stimulates, and raises the expression of IRS IRS-1, and therefore pank4 gene of the present invention and albumen thereof can find the genomic medicine of effective lowering blood glucose as the potential target spot of treatment diabetes.

Description of drawings

Fig. 1 is the collection of illustrative plates that contains pank4 expression carrier pcDNA3.1-pank4 of the present invention.

Fig. 2 is the result that the RT-PCR of substrate 1 (IRS-1) analyzes.1 and 2 RT-PCR results that represent GADPH and IRS1 respectively wherein, swimming lane A and B represent respectively from control cells with the sample of pcDNA3.1-pank4 cells transfected.

Fig. 3 illustrates the Northern engram analysis of IRS-1, and wherein swimming lane 1 and 2 is represented respectively from control cells with the sample of pcDNA3.1-pank4 cells transfected.

Fig. 4 A-4L is the immunohistochemical methods detected result of pank4 tissue distribution.

Fig. 5 is the cellular localization result of Pank4.

Fig. 6 illustrates interior interaction of external and body of Pank4 and Pkm2.

Fig. 7 illustrates in the body of two structural domains of Pank4 and Pkm2 and interacts.

Fig. 8 illustrates the interactional laser co-focusing experimental result of Pank4 and Pkm2.

Fig. 9 illustrates Pank4 can raise the Pkm2 activity.

Embodiment

Pank4 gene of the present invention is to obtain by the mRNA differential display technique is applied to the integral animal level, is subjected to hyperglycemia to stimulate its expression to raise.NCBI analyzes, and the homology of Pank4 and people's Pantothen kinase is very high.Simultaneously, Pank4 albumen contains two conservative structural domains, is respectively KOG2201 (33aa-378aa) and KOG4584 (448aa-768aa).

The experiment of immunohistochemical methods, the result shows of the present invention, Pank4 extensively is distributed in each tissue (Fig. 4), simultaneously, and as can be seen, Pank4 is distributed in some more to be needed in high energy or the synthetic vigorous cell mass, such as, kidney, thyroid secretor (Fig. 4 D, I), some are being in the skeletal muscle and the cardiac muscle of contraction or diastole, and in some cell mass of vascular smooth muscle (Fig. 4 B, G, K), also has the islet cells interior (Fig. 4 C) of pancreas.These results show that Pank4 produces relevant with energy.

By the yeast two-hybrid experiment, found a gene relevant with Pank4, pyruvate kinase gene (Pkm2), an one new interaction protein also is a key enzyme in glycolysis-.Among the described later embodiment, further test (Fig6BC) and be total to positioning experiment (Fig8A-F) and verified the interaction of the two by pull-down (Fig6A), co-immunoprecipitation.The activity of Pkm2 is regulated and to the influence of IRS-1, Pank4 can be used to prepare the medicine of blood sugar regulation concentration from Pank4.

Below will further describe the present invention by embodiment.

The clone of embodiment 1 pank4 gene

1, the preparation rat blood sugar is regulated model automatically

10 of male SD rats (available from China Academy of TCM's Experimental Animal Center, in 3 months ages of mouse, body weight 200～250 grams are freely drunk water and ingested).Be divided into two groups, promptly experimental group and control group are respectively 5 for every group.Each is organized rat and all carries out external jugular vein one right atrium catheter intubation, and postoperative is given enough feed and water, and is standby.External jugular vein one right atrium catheter intubation method is summarized as follows: the etherization animal, cut skin at neck, peel off, appear external jugular vein, by drawing pin silicone tube is introduced the external jugular vein chamber, withdraw from and draw pin, silicone tube is inserted the right atrium, A/C, by the subcutaneous the other end of drawing silicone tube from back of the body neck, seal conduit, sew up the incision, animal is waken up, and the back is independent feeds.Animal is experimentizing under the waking state fully.The physiological saline 0.4ml that the injection of experimental group rat contains 400mmol/L glucose injects the physiological saline that 0.8ml contains 400mmol/L glucose again after 10 minutes.Control group is injected the physiological saline that does not contain glucose with amount respectively.After 30 minutes, the disconnected neck of animal is put to death, and under aseptic condition, every rat is got the about altogether 1g of bilateral quadriceps muscle of thigh, and is frozen immediately in liquid nitrogen.

2.mRNA difference display analysis

(GiBcoBRL USA), extracts the total RNA of two treated animal muscle tissues respectively according to TRIZOL Reagent test kit.Identify the integrity of RNA with 1.5% agarose gel electrophoresis.

Utilize total RNA of above-mentioned acquisition to carry out the synthetic of cDNA article one chain, synthetic according to the operation of reverse transcription test kit specification sheets (Superscript TM II RT, BRL).Total RNA and 2 μ l, 10 μ M anchor primers (1# 5 '-CGC GGA TCC TTTTTT TTT TTA-3 ' that 1 μ g DNase I handles; 2# 5 '-CGC GGA TCC TTT TTT TTT TTG-3 '; 3#5 '-CGC GGA TCC TTT TTT TTT TTC-3 ') mixes.70 ℃ of incubations of reaction solution 10 minutes, cooled on ice then.Add 2 μ l, 10 * the first chain synthesis reaction liquid, 2 μ l 25mMMgCl ₂, 1 μ l 10mM dNTP, 2 μ l 0.1M DTT.42 ℃ of incubations 5 minutes.Add 1 μ lSuperscript II, reaction solution is in 42 ℃ of incubations 50 minutes, then 70 ℃ 15 minutes, cooled on ice.Add 37 ℃ of 20 minutes degradation of rna of 1 μ l RNaseH at last.-20 ℃ of storages.

Carry out difference subsequently and show PCR, totally 10 of used random primers.Random primer designs voluntarily with reference to CloneTech mRNA difference visualizingre agent box, and 5 ' adds EcoR I restriction enzyme site.Wherein, primer sequence is: 1# 5 '-CGG AAT TCT CCA GTT CAG-3 '; 2#5 '-CGG AAT TCC AAG CGA GGT-3 '; 3# 5 '-CGG AAT TCC TTG TAGCCA-3 '; 4# 5 '-CGG AAT TCC TTT CTA GCC-3 '; 5# 5 '-CGG AATTCA CCT TGG ACA-3 '; 6# 5 ' CGG AAT TCA TGC TGG GGA 3 '; 7#5 ' CGG AAT TCT CGG TCA TAG 3 '; 8# 5 ' CGG AAT TCA TGC TGGTAG 3 '; 9# 5 ' CGG AAT TCG ATC TGA CTG 3 '; 10# 5 ' CGG AATTCA TGC TGT ATG 3 '.Add the above-mentioned cDNA of 20ng and 2.5 μ ci[α-32P in the PCR reaction system] Dctp, 0.5U Taq plus II polysaccharase (Sangon), 1 μ l, 10 * PCR reaction solution, 0.5 μ l, 25 μ M dNTP, 1 μ l, 2 μ M random primer and anchor primers, 4.35 μ l water make cumulative volume reach 10 μ l.Reaction conditions is: circulation (94 ℃ 5 minutes, 40 ℃ 5 minutes, 72 ℃ 5 minutes), two circulations (94 ℃ 30 seconds, 40 ℃ 40 seconds, 72 ℃ 5 minutes) 30 circulations then (and 94 ℃ 30 seconds, 52 ℃ 40 seconds, 72 ℃ 2 minutes).Of short duration centrifugal, collect reaction solution.

Above-mentioned PCR product electrophoresis on 6% denaturing polyacrylamide gel.To the X-ray exposure, obtain mRNA difference and show collection of illustrative plates.

3, the recovery of difference PCR product, again the amplification, purifying

The strict relative position of determining X-mating plate and gel.Show spectrum according to mRNA difference, cut required gel band with the cleaning scalpel.In the 0.5ml centrifuge tube with 100 μ lddH ₂O soaks gel, and 95 ℃ were heated centrifugal collection 20 minutes.Shift supernatant to another centrifuge tube, add 10 μ l3M NaAc (pH5.2), 5 μ l glycogens (10mg/ml), mixing.Add 450 μ l dehydrated alcohols, mixing.Put-70 ℃ freezing 60 minutes, 12000rpm is centrifugal 15 minutes afterwards.Abandon supernatant, with 4 ℃ of 75% washing with alcohol precipitation, air-dry.With 10 μ lddH ₂The O dissolving.PCR product with so obtaining is stored in-20 ℃.

Then carry out the amplification again of difference PCR product.In 0.5ml Eppendorf pipe, set up following reaction system: the PCR product of recovery (10ng/ μ l) 1 μ l, primer I (sequence 1# 5 '-CGCGGA TCC TTT TTT TTT TTA-3 ', or 2# 5 '-CGC GGA TCC TTT TTTTTT TTG-3 ', or 3# 5 '-CGC GGA TCC TTT TTT TTTTTC-3 ') (50pmol/ μ l) 1 μ l, primer I I (sequence 1# 5 '-CGG AAT TCT CCAGTT CAG-3 ' or 2# 5 '-CGG AAT TCC AAG CGA GGT-3 ', or 3#5 '-CGG AAT TCC TTG TAG CCA-3 ', or 4# 5 '-CGG AAT TCC TTTCTA GCC-3 ', or 5# 5 '-CGG AAT TCA CCT TGG ACA-3 ', or 6# 5 ' CGG AAT TCA TGC TGG GGA 3 ', or 7# 5 ' CGG AAT TCT CGG TCATAG 3 ', or 8# 5 ' CGG AAT TCA TGC TGG TAG 3 ', or 9# 5 ' CGGAAT TCG ATC TGA CTG 3 ', or 10# 5 ' CGG AAT TCA TGC TGT ATG3 ') (50pmol/ μ l) 1 μ l, 10 * PCR reaction buffer (15mMgCl ₂) 5 μ l, 4 * dNTP (each 5mM), 2 μ l, Taq polysaccharase (1u/ μ l) 2 μ l add deionized water to final volume 50 μ l.94 ℃ of sex change were carried out following cycling program 30 times after 2 minutes: 94 ℃ of sex change 40 seconds, and 55 ℃ of annealing 30 seconds, 72 ℃ were extended 40 seconds.After 30 loop ends, 72 ℃ are continued to extend 10 minutes, then the PCR product are separated with sepharose.

4, the slit hybridization analysis of difference cDNA

It is quantitative that above-mentioned PCR product is carried out agarose gel electrophoresis.Every kind of product is got 400ng and is handled through alkaline denaturation,, distinguishes in contrast with beta-actin on the Hybond-N nylon membrane with slit point film device (BioRad) point sample.2XSSC rinsing nylon membrane, airing.With UV-crosslinked method the PCR product is fixed on the film.

Adopt the single grappling base primers of above-mentioned mixing, preparation a- ³²The strand cDNA probe of P mark is used for slit hybridization and radioautograph.At first, (0.5%SDS), salmon sperm DNA is added 68 ℃ of prehybridization solutions to final concentration in 95 ℃ of sex change after 5 minutes be 100 μ g/ml for 5 * Denhart ' s solution, 6 * SSC above-mentioned nylon membrane to be placed the 15ml prehybridization solution.68 ℃ of prehybridizations 2 hours.Add above-mentioned mixing cDNA probe then, hybridized 20 hours for 68 ℃.It is as follows to wash the film condition: 500ml 2 * SSC, the of short duration rinsing nylon membrane of 0.1%SDS room temperature 5 minutes, repeat once; 250ml1 * SSC, 0.1%SDS room temperature were washed film 30 minutes, repeated once; 250ml 1 * SSC, 0.1%SDS washed film 15 minutes in 65 ℃, repeated once; Blot nylon membrane with filter paper, with the X-of Fuji mating plate compressing tablet, in-70 ℃ of exposures 3-4 days.Using Pharmacia GelScan XL (2.1 editions) gel scanning software wraps on the Pharmacia LKB Ultroscan XL scanner to each hybridization spot scanning, with the integrated value measurement gene order relative expression quantity at scanning peak.

6, the clone of difference cDNA and order-checking and cDNA library screening

According to the slit results of hybridization, selecting has the PCR product of notable difference to clone.Purified pcr product at first is according to the Wizard of Promega company ^TMThe operation of PCR DNA PurificationSystem test kit specification sheets is with the effect of 1.5% agarose gel electrophoresis purification Identification.Then, with the dna fragmentation behind the glass milk absorption method recovery purifying.All differences is expressed pGEM-T cloning vector and the order-checking acquisition est sequence that fragment all is cloned into Promega company.

3 up-regulated expression fragments and 3 downward modulations are expressed fragment all as probe hybridization screening library, and the sieve storehouse is the common method according to known probe sequence screening cDNA clone.

(Rat Skeletal Muscle 5 '-STRETCH PLUScDNA Library, RL3003b ClonTech) screen the work of cDNA total length with rat normal bone flesh library.6 EST (EST89, EST62, EST119, EST17, EST124, EST76) are labeled probe and screen with classical hybrid method, and the screening through three-wheel obtains EST89, EST62, EST124, EST76 mono-clonal respectively.The sequence that obtains with EST89 sieve storehouse is confirmed as the total length encoding gene after by analysis, called after pank4, its nucleotide sequence shown in SEQ ID NO:1, the about 2.3kb of its total length.

Embodiment 2 pank4 Construction of eukaryotic

Add an Xho1 restriction enzyme site with upstream primer 5 '-CAT CTC GAG AAA ATG GCG GAG TGC CGG-3 ', downstream primer 5 '-CA TGG ATC CTC GGCTGG GAC CTCGTA-3 ' adds a BamH I site, and total length pank4 gene is that template is carried out pcr amplification.The pcr amplification condition is: 94 ℃ of sex change 2 minutes, carry out following cycling program then 30 times: and 94 ℃ of sex change 40 seconds, 55 ℃ of annealing 60 seconds, 72 ℃ were extended 150 seconds.After 30 loop ends, 72 ℃ are continued to extend 10 minutes, then the PCR product are separated with sepharose, reclaim the PCR product with glass milk absorption method.The dna fragmentation that reclaims is quantitative through the electrophoresis electrophoresis, is connected to pGEM-T easy carrier.With two kinds of enzyme Xho1 and BamH1 the pank4 gene coding region is scaled off from pGEM-T easy carrier, electrophoresis reclaims this band, is connected with same pcDNA3.1 expression vector (Invitrogen) with XhoI and BamHI double digestion.The plasmid called after pcDNA3.1-pank4 (seeing shown in Figure 1) that obtains.

Embodiment 3 pank4 can raise the expression of IRS-1

This example selects for use the CBRH-7919 cell strain to study, this cell strain be the Wistar rat with the MCA-RH7777 that diethylnitrosamine brings out, derive from the Chinese Academy of Sciences of Shanghai Inst. of Cytobiology, Chinese Academy of Sciences cell bank.

The CBRH-7919 cell is transfection pCDNA3.1-pank4 and empty expression vector pCDNA3.1 control group respectively.After the transfection 36 hours, discard nutrient solution, extract RNA, quantitative RT-PCR.Quantitative RT-PCR with beta-actin in contrast.Choose the several key enzymes in the glucose metabolism respectively, glucokinase, glyceraldehyde 3-phosphate dehydro-genase, pyruvate kinase, isocitric acid dehydrogenase gene, insulin receptor, substrate 1, IRS 2, IRS 3, IRS 4, glucose transporter 4, glycogen synthetase, acetyl-CoA carboxylase, GSK1 designs primer, carries out quantitative RT-PCR and detects.The RT-PCR product is identified through electrophoresis and is found to have only substrate 1 (IRS-1) to express notable difference, as shown in Figure 2, further verifies RT-PCR result with the Northern trace, as shown in Figure 3.

The structure of embodiment 4 pank4 gene prokaryotic carriers

1, the construction of prokaryotic expression vector that contains the sequence of the proteic antigenic determinant of coding pank4

According to the sequence characteristic of pank4, determine the proteic antigenic determinant sequence of pank4 through software analysis, it is cloned into prokaryotic expression carrier pGEX-4T-3 (Pharmacia).For ease of construction expression, we add BamH I restriction enzyme site (a 5 ' primer: 5 '-ATC GGA TCC CTC CTA GTC AAC ATT GGC-3 ') at 5 ' end primer, (3 ' primer: 5 '-TGC TCG AGT CTG TCCAAT GTC ATT GCT-3 '), template is total length pank4 to add an Xho I restriction enzyme site at 3 ' end primer.The pcr amplification condition: 94 ℃ of sex change 2 minutes, carry out following cycling program then 30 times: 94 ℃ of sex change 40 seconds, 55 ℃ of annealing 60 seconds, 72 ℃ were extended 60 seconds.After 30 loop ends, 72 ℃ are continued to extend 10 minutes, then the PCR product are separated with sepharose, reclaim PCR product (about 300bp) with glass milk absorption method.The dna fragmentation that reclaims is quantitative through the electrophoresis electrophoresis.The dna fragmentation that reclaims is quantitative through the electrophoresis electrophoresis, is connected to pGEM-T easy carrier (Promega).With two kinds of enzyme Xho1 and BamH1 this fragment is scaled off from pGEM-T easy carrier, electrophoresis reclaims this band, is connected with same pGEX-4T-3 prokaryotic expression carrier (Pharmasia) with XhoI and BamHI double digestion.Called after pGEX-4T-3-f2.

2. the construction of prokaryotic expression vector that contains the pank4 full-length gene

The design primer, 5 ' end primer adds BamH I restriction enzyme site (a 5 ' primer: 5 '-AGGA TCC AAA ATG GCG GAG TGC CGG-3 '), (3 ' primer: 5 '-AC TCG AGT TGG GAC CTCGTA CTT GAA-3 '), template is total length pank4 to add an Xho I restriction enzyme site at 3 ' end primer.The pcr amplification condition: 94 ℃ of sex change 2 minutes, carry out following cycling program then 30 times: 94 ℃ of sex change 40 seconds, 62 ℃ of annealing 60 seconds, 72 ℃ were extended 2 minutes.After 30 loop ends, 72 ℃ are continued to extend 10 minutes, then the PCR product are separated with sepharose, reclaim PCR product (about 2.3kb) with glass milk absorption method.The dna fragmentation that reclaims is quantitative through the electrophoresis electrophoresis.The dna fragmentation that reclaims is quantitative through the electrophoresis electrophoresis, is connected to pGEM-T easy carrier.With two kinds of enzyme Xho1 and BamH1 this fragment is scaled off from pGEM-T easy carrier, electrophoresis reclaims this band, is connected with same pET-30a (+) prokaryotic expression carrier (Invitrogen) with XhoI and BamHI double digestion.Called after pET-30a (+)-pank4.

The purifying of embodiment 5 pank4 proteantigen determinants and the preparation of antibody

Positive plasmid transformed into escherichia coli BL21 with the correct embodiment 4.1 in order-checking back obtains coats on the LB agar plate that contains kantlex, cultivates 10-16 hour for 37 ℃.Select about 5 single colony inoculations in the fresh LB pipe of the 5ml that contains kantlex, 37 ℃ violent wave and culture 10-16 hour.Get above-mentioned culture and be inoculated into two fresh LB pipes of 5ml that contain kantlex with 1/20 ratio, 37 ℃ violent wave and culture 30-60 minute, make bacterium liquid OD _600nmReach 0.4-0.6.Adding final concentration in an above-mentioned transfer tube is the IPTG of 1mmol/L, 37 ℃ violent wave and culture 3-4 hour, another pipe is not induced and is compared.Collect the about 1ml of nutrient solution with the 1.5ml centrifuge tube respectively, 12000rpm 20 seconds, abandons clean supernatant substratum.Preserve thalline.Bacterial sediment is with the resuspended mixing of 100ul 1 * sample buffer, and in 100 ℃ of water-baths 10 minutes, 12000rpm was centrifugal 2 minutes before the last sample of SDS-PAGE.Get 10-20ul and go up sample, carry out 12%SDS-PAGE.Prepare the 12%SDS-PAGE separation gel according to molecular cloning method, behind the mixing, it is poured into (the Mini-Cell glue system of Bio-Rad company) in the glue plate, room temperature was solidified 45 minutes.Preparation 5%SDS-PAGE concentrates glue.Electrophoresis at first carries out with 8V/cm, treats to be adjusted into 15V/cm after sample-loading buffer enters separation gel about 2 hours of electrophoresis.When treating that tetrabromophenol sulfonphthalein is about to plastic emitting, stop electrophoresis.0.25% coomassie brilliant blue R250 dye liquor dyed glue 2 hours, and it is high-visible to strip type of albumen to decolour, and the analyzing proteins expression of results is used after plasmid that expression amount is the highest and bacterial strain are preserved again.

The great expression of recombinant protein

Positive strain after identifying is chosen a single bacterium colony go into the fresh LB substratum of 100ml (containing kantlex).37 ℃ are acutely shaken overnight incubation.The bacterium liquid that will spend the night is inoculated into one according to 1/10 ratio and contains in the 500ml LB substratum (containing kantlex), and 37 ℃ are acutely shaken, and cultivate 1-1.5 hour, make the OD of bacterium liquid _600nmReach 0.4-0.6.Adding final concentration is 0.5mmol/L IPTG abduction delivering 3-4 hour.Collect nutrient solution, centrifugal 10 minutes of 5000rpm abandons supernatant, and PBS washes bacterial sediment.Resuspended according to 5ml/g with PBS, ice bath is work down, is work 10s according to parameter, cooling 20s, the condition of power 300W, ultrasonic disruption bacterium 20 times.Centrifugal 20 minutes of 12000rpm takes a morsel respectively and goes up cleer and peaceful precipitation, carries out SDS-PAGE and detects, and analyzing proteins is expression-form in born of the same parents.All the other are frozen standby in-70 ℃.

The purifying of recombinant protein

If expressed proteins mainly exists in the supernatant, directly add GSH-Sepharose absorption fusion rotein, if target protein mainly in inclusion body, then needs repurity behind the solubilization of inclusion bodies.The processing of GSH-Sepharose 4B matrix: shake the bottle (GSH-Sepharose 4B matrix is kept in the ethanol) that GSH-Sepharose4B is housed gently, resuspended matrix.Get 1.33mlGSH-Sepharose 4B matrix and transfer to suitable centrifuge tube.Centrifugal 5 minutes of 500g removes supernatant.The PBS that adds 4 ℃ of precoolings of 10ml, mixing cleans GSH-Sepharose4B matrix, residual ethanol is cleaned, otherwise can be disturbed later result.Centrifugal 5 minutes of 500g removes supernatant.Add 1ml 1XPBS, be used for balance matrix.

GSH-Sepharose 4B pillar purifying protein:

Expressing protein uses following method purifying in inclusion body:

Thalline is resuspended among the 20ml STE (10mM Tris.HCl pH8.0,100mM NaCl, 1mM EDTA).Add N,O-Diacetylmuramidase (10mg/ml) to final concentration 100ug/ml, mixing, ice bath 15 minutes.Add 1M DTT and 10%Sarkosyl and be respectively 5mM and 1.5% to concentration, on vibrator, mix 5s.Ultrasonic wave is broken bacterium, ultrasonic 12s, cooling 30s, power 300W, ultrasonic 15 times.4 ℃ centrifugal, 12000rpm, 20 minutes.Supernatant add 30%TritonX-100 to final concentration be thorough mixing 30 minutes on 3%, 4 ℃ of gyroscope.Add 200ulGSH-Sepharose 4B, thorough mixing is 30 minutes on the gyroscope.To go up liquid and be added on the polypropylene post, wash 3 times with the PBS of 10X column volume.With the centrifuge tube of resin transfer, add 200ul GST elution buffer (50mM Tris.HCl, 10mM gsh), room temperature effect 10 minutes to 1.5ml.Centrifugal 2 minutes of 4 ℃ of 1000rpm, supernatant is purifying protein.Repeat wash-out 2 times, then, forint-phenol law is measured protein concentration, and SDS-PAGE detects protein concentration, and-70 ℃ frozen stand-by.

The acquisition of antibody

The new zealand white rabbits that 4 2kg are heavy, two as experimental group, and two are contrast.Fundamental immunity for the first time: 1mg antigen (fusion rotein of purifying), add equal-volume Fu Shi Freund's complete adjuvant and mix, until forming even water-in-oil shape emulsion, adopt the back intracutaneous, the subcutaneous multi-point injection in the intensive place of lymphs such as oxter, about 20-30 point.Contrast 50mM Tris.HCl mixes with adjuvant.Fundamental immunity for the second time: 400ug antigen adds the equal-volume freund 's incomplete adjuvant and mixes, and is the same, multi-point injection.After one week of immunity, get blood ELISA from auricular vein and measure antibody titer.Booster immunization: 300ug antigen adds isopyknic freund 's incomplete adjuvant and mixes, multi-point injection.According to the result of ELISA mensuration antibody titer, per two all immunity once reach required titre (1: 32) up to antibody titer.From the carotid artery bloodletting, the blood of collection tilting 30 minutes in room temperature moves on to 4 ℃ of refrigerators and treats that serum separates out, and collects behind the serum 1000rpm centrifugal 10 minutes, and 0.5ml/ props up packing ,-70 ℃ of preservations.It is higher that the Western trace detects antibody purity.

PET-30a (+)-pank4 is also by carrying out abduction delivering with quadrat method.

Embodiment 6 pank4 tissue distribution test experience

Utilize the method for immunohistochemical methods to carry out the location that pank4 distributes, animal material is 300 grammes per square metre SD rats, and it is anaesthetized back PBS damping fluid perfusion, the interference of fully removing blood, take out each organ rapidly, fixing in 4% Paraformaldehyde 96 (PBS joins), then paraffin embedding, cut into slices, take off cured, fixing, hatched 15 minutes with 3% hydrogen peroxide, distilled water flushing, PBS soaked 5 minutes, and the pank4 antibody that makes with embodiment 5 diluted 4 ℃ of overnight incubation by 1: 200.Inferior daily PBS gives a baby a bath on the third day after its birth inferior, each 5 minutes, drip versatility two anti-(goat-anti rabbit) (Beijing Zhong Shan Bioisystech Co., Ltd), hatched 40 minutes for 37 ℃, PBS gives a baby a bath on the third day after its birth inferior, each 5 minutes, DAB (Beijing Zhong Shan Bioisystech Co., Ltd) colour developing, distilled water flushing, Hematorylin dyestuff were redyed 3 minutes, dehydration, mounting.Microscopically is observed, 10 *, 20 *, 40 * take a picture.Shown in Fig. 4 A-4L.Among the figure, A is a liver, and pank4 is distributed more widely in new splitted cell.B is a skeletal muscle tissue, and pank4 distributes more in certain cluster cell, illustrates that it is relevant with contraction, thereby illustrates that also it is perhaps relevant with energy metabolism.C is a pancreatic tissue, can conclude obviously that it distributes in certain class cell that pancreas is led extensively.D is a renal tissue, and visible pank4 has high the distribution among the figure near uriniferous tubules.E is a testis, is distributed in matrix as the arrow indication.F is a lung, is distributed in matrix as the arrow indication.G is a blood vessel, and is similar with skeletal muscle tissue, and pank4 distributes more in certain cluster cell.H is a small intestine, distributes in basal cell.I is a Tiroidina, is distributed in secretory duct as the arrow indication.J is a brain, distributes in neuronal cell.K is a cardiac muscle, and similar with skeletal muscle tissue, pank4 distributes more in certain cluster cell.L is a spleen, and pank4 does not have obvious characteristics to distribute.

The location of embodiment 7 Pank4 in cell

PEGFP-N1 carrier (Clonetech company) contains promotor and the enhanser of CMV, with the full gene of Pank4 is that the template pcr amplification obtains the Pank4 full-length clone to pEGFP-N1, process is as follows: upstream primer is: P1 (5 '-GAA CTC GAG AAA ATG GCG GAGTGC CGG-3 '), contain the Xho1 restriction enzyme site, downstream primer is: P2 (5 '-CAT GGATCC TCG GCT GGG ACC TCG TA-3 '), contain the BamH1 site.The PCR product is cut and is connected into same enzyme by the Xho1-BamH1 enzyme and cuts among the carrier pEGFP-N1 that obtains.

L-6TG cell (Shanghai cell institute of the Chinese Academy of Sciences) is cultivated in the RPM1640 substratum (10% heat-inactivated foetal calf serum), cell is inoculated on six orifice plates that are coated with cover glass with 50% ratio, treat after 24 hours that cell length was by 80% o'clock, with Lipofectamine2000 with plasmid pPank4-EGFP-N1 transfectional cell, pEGFP-N1 in contrast, after 24 hours, in OLYMPUS FV-500 confocal laser scanning microscopy observations.

HEK293 cell and Hela cell (available from Shanghai cell institute of the Chinese Academy of Sciences) are cultivated in the MEM substratum (10% heat-inactivated foetal calf serum), with the pPank4-EGFP-N1 transfectional cell, pEGFP-N1 in contrast, transfection method is the same.Then in OLYMPUSFV-500 microscopic examination result.

In addition, the Skeletal Muscle Cell of SD rat is incubated at 37 ℃ of the Eagle substratum (DMEM) (10% heat-inactivated foetal calf serum) of Dulbeccos improvement, 5%CO with Explant culture former generation ₂Treat that cell is fixed in 4% Paraformaldehyde 96 (preparing) after forming monolayer cell on the cover glass in PBS, after saturatingization of 0.1%TritonX-100 (in PBS, preparing), with 37 ℃ of sealings of 3%BSA (in PBS, preparing), mark an anti-half an hour (antibody that SD P of Rats ank4 protein immunization rabbit obtains) for 37 ℃, PBS washes cell three times, then the two anti-half an hour approximately of goat antirabbit of room temperature mark coupling FITC, the fixed cap slide is on slide glass, in OLYMPUSFV-500 microscopic examination result.

Plasmid pEGFP-Pank4 and pEGFP-N1 transfection found that green fluorescent protein is positioned full cell (Fig. 5 A, B, C) in L-6TG, HEK293, the HeLa clone, and EGFP-Pank4 is positioned (Fig. 5 E, F, G) in the tenuigenin.

SD rat skeletal muscle cultured cells of former generation is after fixing, and is anti-as one with Pank4 antibody, and the goat anti-rabbit antibody of coupling FITC anti-ly carries out immunofluorescence experiment as two, and the result shows that Pank4 albumen is positioned (Fig. 5 D, H) in the tenuigenin.

Embodiment 8 pank4 genes and proteic further evaluation

The yeast two-hybrid experiment

Utilize MATCHMAKER Library construction ﹠amp; Screening System (CLONTECH) carries out the yeast two-hybrid experiment, SD P of Rats ank4 full length gene is cloned in the pGBKT7 carrier (Clonetech): upstream primer: P1 (5 '-A GAA TTC AAAATG GCG GAG TGC CGG-3 ') contains the EcoRI site, downstream primer: P2 (5 '-AGGA TCC TGG GAC CTC GTA CTT GAA-3 ') contains the BamHI site, the PCR product is connected among the T-vector, be connected into after cutting with the EcoRI-BamH1 enzyme then among the carrier pGBKT7 that same enzyme cuts, recombinant plasmid pPank4-GBKT7 is transformed among the yeast strain Y187 after self activation and toxicity detect, Pank4 as the hybridization of bait protein and SD rat skeletal muscle cDNA library (SD rat skeletal muscle cDNA library be the cDNA segment with pGADT7-Rec as carrier, and be transformed among the yeast strain AH109), after 24-26 hour, after the observation zygote forms, collect yeast, and it is laid on trp ^-, leu ^-, his ^-, ade ^-On+50mM 3-AT the flat board, cultivated about 10 days for 30 ℃, the method for pressing CLONTECHMATCHMAKER library construction and screening reagent box is measured the activity of beta-galactosidase enzymes, screening positive clone.

The Pull-down experiment

In order to carry out the pull-down experiment, in Escherichia coli BL21 (DE3), express warm albumen of GST-Pank4 and GST albumen respectively.The method purifying that GST and GST-Pank4 albumen instruct (Amersham Pharmacia Biotech) to provide by the gst gene emerging system.In experiment, 0.5 μ g GST and GST-Pank4 albumen are incorporated into 20 μ l respectively with lysis buffer (20mM Tris-HCl, 4 ℃ of pH8.0 at, 1mM EDTA, 200mM NaCl, the 10mM 2 mercapto ethanol, 25 μ g/ml PMSF, 0.7 μ g/ml pepstatin, 1 μ g/ml leupeptin, 2 μ g/ml aprotinin, 0.5%Nonidet P-40) gsh-Sepharose that balance is good ^TMOn the 4B pearl (Amersham Pharmacia Biotech), use each 1ml lysis buffer rinsing 3 times then.

The dna sequence dna (Pkm2) of the yeast positive colony that screens is cloned into carrier for expression of eukaryon pCDNA6v5hisB-FIAG (Invitrogen) by reading sign indicating number, the HEK293 cell inoculation is to the flat board of 50mm, recombinant plasmid pCDNA6v5hisB-FLAG-Pkm2 is with Lipofectamine2000 reagent (Invitrogen, USA) transfection is in the Hek293 cell, collecting cell after 48 hours, the lysis buffer lysing cell, under 4 ℃ of conditions with cell lysate and be combined GST albumen or the proteic gsh-Sepharose of GST-Pank4 ^TMThe 4B pearl is in conjunction with spending the night, then with lysis buffer rinsing gsh-Sepharose ^TM4B pearl 3 times, the proteic pearl that is combined adds 30 μ l2 * SDS damping fluid, detects as an anti-Western trace with anti-FLAG.

The co-immunoprecipitation experiment

Recombinant plasmid pcDNA6v5hisB-FLAG-Pkm2 and pcDNA6v5hisB-HA-pank4 are with Lipofectamine2000 reagent (Invitrogen, USA) cotransfection is in the Hek293 cell, collecting cell after 48 hours, EBC lysis buffer (50mM Tris-Cl, pH8.0,120mM NaCl, 0.5%Nonidet P-40,5 μ g/mlleupeptin, 10 μ g/ml aprotinin, 50 μ g/ml PMSF, 0.2mM sodiumorthovanadate, 100mM NaF) lysing cell, 1, centrifugal 10 minutes of 2000rpm, supernatant adding anti-HA monoclonal antibody (Sigma) or anti-FLAG monoclonal antibody (Sigma) added Protein G sepharose 4 Fast Flow (Amersham Pharmacia) in conjunction with 1 hour then in conjunction with 1 hour, supernatant is inhaled and is gone, NETN damping fluid (20mMTris-Cl, pH8.0,1mM EDTA, 900mM NaCl, 0.5%Nonidet P-40) rinsing sepharose pearl is used NETN damping fluid (20mM Tris-Cl, pH8.0 more than 5 times again, 1mM EDTA, 100mM NaCl, 0.5%Nonidet P-40) rinsing is 1 time, detects as an anti-western trace with anti-FLAG MoAB or anti-HA MoAB.

KOG2201 and the KOG4584 of Pank4 are cloned into carrier pcDNA6v5hisB-HA by amino acid whose order, carry out co-immunoprecipitation test, method such as preceding with Pkm2.

Laser is positioning experiment altogether

HEK293 and the rewinding of HeLa cell inoculation have on six orifice plates of cover glass, treat that cell length was by 80% o'clock, the method of liposome is with in pEGFP-N1-pank4 and thepcDNA6v5hisB-FLAG-Pkm2 cotransfection to two kind of the cell, after 24 hours, cell 4% Paraformaldehyde 96 (PBS joins), after saturatingization of 0.1%TritonX-100 (PBS joins), 37 ℃ of sealings of 3%BSA 30 minutes, hatched 30 minutes for anti-FLAG MoAB37 ℃, PBS rinsing 3 times, the fluorescein that Anti-mouse IgG (H+L) puts together (TRITC, Sigma) the room temperature mark is 3 minutes, and green fluorescence and red immunofluorescence are by OLYMPUS FV-500 microscopic examination and analyze the two and express overlapping situation.

KOG2201 and the KOG4584 of Pank4 are cloned into carrier pEGFP-N1 by amino acid whose order, carry out common positioning experiment with Pkm2, method such as preceding.

Pyruvate kinase is active to be detected

L-6TG cell and HEK293T cell (available from Shanghai cell institute of the Chinese Academy of Sciences) are with pcDNA6v5hisB-HA-pank4 and pcDNA6v5hisB-FLAG-Pkm2 transfection, and 25 ℃ of activity of carrying out pyruvate kinase detect then.Cell ice bath at first, the PBS of ice precooling washes twice, the lysis buffer lysing cell, 15000g is centrifugal 20 minutes then.Supernatant is measured protein concentration with the BCA method, and enzyme is lived and reacted as follows: 50mM Tris-Cl, pH7.6,100mM KCl, 5mM MgSO ₄, 2mM ADP (Sigma), 0.2 or 2mM phosphoenolpyruvate (PEP) (Sigma), 0.25mM NADH and 2U serum lactic dehydrogenase (Sigma), the photoabsorption under the 340nm is measured in reaction after half an hour.

The result

Pkm2 and Pank4 interact

In order to seek interactional albumen with Pank4, with the AD library of Pank4 albumen as bait protein screening SD rat skeletal muscle, isolate ten positive colonies, through verifying in this hybridization, sequencing, BLAST analyzes and finds that they are respectively: beta-hydroxysteroiddehydrogenase isomerase type II, Rattus norvegicus calcium channel beta1 subunit, Rattus norvegicus glycogen phosphorylase, Rattus norvegicuspyruvate kinase (Pkm2), Rattus norvegicus glyceraldehyde-3-phosphatedehydrogenase., but find to have only Pkm2 and Pank4 direct interaction through above-mentioned pull-down experimental verification.

Whether Pank4 combines with Pkm2 by GST pull-down experimental verification, at first construction expression Pank4-GST fusion rotein and GST albumen, change the HEK293 cell with recombinant plasmid pcDNA6v5hisB-FLAG-Pkm2 and obtain product of cell lysis, with combine the proteic glutathione s epharose of GST albumen and Pank4-GST pearl and combine, carried out the western trace by protein bound pearl and detect, the anti-FLAG monoclonal antibody is anti-as one.The result shows, Pkm2 albumen only with the Pank4-GST protein binding, and not with GST protein binding (Fig. 6 A).

Behind recombinant plasmid HA-Pank4 and FLAG-Pkm2 and HA-Pank4 and the FLAG difference cotransfection HEK293 cell, obtain cell lysate, proteinG pearl to the anti-HA monoclonal antibody immunity after coprecipitated carries out the western trace with anti-FLAG MoAb and detects, and is that the proteinG pearl after coprecipitated carries out western trace detected result (Fig. 6 B, C) with the anti-HA monoclonal antibody to the anti-FLAG monoclonal antibody immunity in its corresponding experiment.

We utilize recombinant plasmid pEGFP-Pank4 and pFLAG-Pkm2 cotransfection HEK293 and HeLa cell to investigate Pank4 and the proteic location of Pkm2, and Pank4 albumen can be determined by the location of green fluorescent protein in HEK293 cell and HeLa cell.The proteic location of Pkm2 can be carried out immunofluorescence by anti-FLAG antibody and be determined (Fig. 8 A-F).

Two structural domains of KOG2201 and KOG4584 all interact with Pkm2

KOG2201 and KOG4584 are cloned into carrier pcDNA6v5hisB-HA and Pkm2 respectively and are carried out the co-immunoprecipitation experiment, and the result shows, the two all with Pkm2 interaction (Fig. 7 A, B).The laser co-focusing experiment shows that also two structural domains are respectively to be total to localized (Fig. 8 G-Q) with Pkm2.

Pank4 can activate Pkm2

At first, plasmid pcDNA6v5hisB-HA-Pank4 and pcDNA6v5hisB-HA transfection HEK293T cell extract total RNA, carry out reverse transcription-PCR reaction, and from picture 9 as can be seen, the expression amount of Pkm2 does not have considerable change in control group and experimental group.Whether influence the activity of Pkm2 in order to analyze Pank4, we are with pcDNA6v5hisB-FLAG-Pkm2 and pcDNA6v5hisB-FLAG-Pank4, KOG2201, KOG4584 and independent carrier pcDNA6v5hisB-FLAG-Pkm2 and pcDNA6v5hisB-FLAG-Pkm transfection HEK293T cell, carrying out kinase activity detects, the result shows (Fig. 9), the KOG2201 structural domain is very big for the activity influence of Pkm2, can strengthen the activity of Pkm2 to a great extent, perhaps, this is the machine-processed place that Pank4 activates Pkm2, but KOG4584 is not clearly to the activity influence of Pkm2.

Sequence table

＜110〉Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences

＜120〉the new gene that blood glucose regulation is relevant, its protein product and uses thereof

<130>I20021195cb

<160>2

<170>PatentIn?version?3.1

<210>1

<211>2322

<212>DNA

＜213〉rat

<220>

<221>CDS

<222>(1)..(2322)

<223>

<400>1

atg?gcg?gag?tgc?cgg?gcg?agt?ggc?ggc?ggg?agc?ggc?ggg?gac?agt?ctg 48

Met?Ala?Glu?Cys?Arg?Ala?Ser?Gly?Gly?Gly?Ser?Gly?Gly?Asp?Ser?Leu

1 5 10 15

gac?aag?agc?atc?acg?ctg?ccc?ccc?gac?gag?atc?ttc?cga?aac?ctg?gag 96

Asp?Lys?Ser?Ile?Thr?Leu?Pro?Pro?Asp?Glu?Ile?Phe?Arg?Asn?Leu?Glu

20 25 30

aac?gcc?aag?cgc?ttc?gcc?att?gat?ata?ggt?gga?tca?ctg?acc?aag?ttg 144

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

35 40 45

gca?tac?tat?tcc?aca?gta?cag?cac?aaa?gtg?gcc?aaa?gtg?agg?tct?ttt 192

Ala?Tyr?Tyr?Ser?Thr?Val?Gln?His?Lys?Val?Ala?Lys?Val?Arg?Ser?Phe

50 55 60

gac?cac?cca?gga?aag?gac?gca?gaa?cag?gac?cat?gag?ccg?ccc?tat?gag 240

Asp?His?Pro?Gly?Lys?Asp?Ala?Glu?Gln?Asp?His?Glu?Pro?Pro?Tyr?Glu

65 70 75 80

atc?tca?gtt?cag?gag?gag?atc?aca?gct?cgt?ctg?cat?ttc?atc?aag?ttt 288

Ile?Ser?Val?Gln?Glu?Glu?Ile?Thr?Ala?Arg?Leu?His?Phe?Ile?Lys?Phe

85 90 95

gag?aac?acc?tac?atg?gaa?gcc?tgc?ctg?gac?ttt?atc?aga?gac?cac?ctt 336

Glu?Asn?Thr?Tyr?Met?Glu?Ala?Cys?Leu?Asp?Phe?Ile?Arg?Asp?His?Leu

100 105 110

gtc?aac?act?gag?acc?aag?gtc?atc?cag?gcc?aca?ggg?ggt?gga?gcc?tac 384

Val?Asn?Thr?Glu?Thr?Lys?Val?Ile?Gln?Ala?Thr?Gly?Gly?Gly?Ala?Tyr

115 120 125

aag?ttc?aag?gac?ctc?atc?gag?gag?aag?ctg?cgt?ctg?aag?gtg?gac?aaa 432

Lys?Phe?Lys?Asp?Leu?Ile?Glu?Glu?Lys?Leu?Arg?Leu?Lys?Val?Asp?Lys

130 135 140

gag?gat?gtg?atg?acc?tgc?ttg?att?aag?ggg?tgc?aac?ttc?gtg?ctg?aag 480

Glu?Asp?Val?Met?Thr?Cys?Leu?Ile?Lys?Gly?Cys?Asn?Phe?Val?Leu?Lys

145 150 155 160

aac?atc?cca?cat?gag?gcc?ttc?atg?tac?cag?aaa?gac?tca?gac?cca?gag 528

Asn?Ile?Pro?His?Glu?Ala?Phe?Met?Tyr?Gln?Lys?Asp?Ser?Asp?Pro?Glu

165 170 175

ttt?cgg?ttt?cag?aca?aat?cac?ccg?aac?atc?ttc?ccc?tac?ctc?cta?gtc 576

Phe?Arg?Phe?Gln?Thr?Asn?His?Pro?Asn?Ile?Phe?Pro?Tyr?Leu?Leu?Val

180 185 190

aac?att?ggc?tct?gga?gtc?tcc?atc?gtg?aag?gtg?gag?aca?gag?gac?cgg 624

Asn?Ile?Gly?Ser?Gly?Val?Ser?Ile?Val?Lys?Val?Glu?Thr?Glu?Asp?Arg

195 200 205

ttc?gag?tgg?att?ggt?ggg?agc?tcc?att?gga?gga?ggc?acc?ttc?tgg?ggg 672

Phe?Glu?Trp?Ile?Gly?Gly?Ser?Ser?Ile?Gly?Gly?Gly?Thr?Phe?Trp?Gly

210 215 220

ctt?ggg?gct?ctg?ctc?acc?aaa?aca?aag?aag?ttt?gat?gag?ctg?ctg?cag 720

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

225 230 235 240

ctg?gct?tcc?aga?ggc?cgg?cac?gcc?aat?gtt?gac?atg?ctg?gta?caa?gac 768

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

245 250 255

atc?tat?gga?ggg?gcc?cac?cag?acc?ctg?ggg?ctg?agc?ggc?aat?ctc?atc 816

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

260 265 270

gca?agc?agt?ttt?ggg?aag?tca?gcc?act?gct?gac?aga?gag?ttc?tcc?aaa 864

Ala?Ser?Ser?Phe?Gly?Lys?Ser?Ala?Thr?Ala?Asp?Arg?Glu?Phe?Ser?Lys

275 280 285

gaa?gac?atg?gcc?aag?agc?ctg?ctg?cac?atg?atc?agc?aat?gac?att?gga 912

Glu?Asp?Met?Ala?Lys?Ser?Leu?Leu?His?Met?Ile?Ser?Asn?Asp?Ile?Gly

290 295 300

cag?ctc?gcc?tgt?ctc?tac?gcc?aag?ctt?cat?ggc?cta?gac?agg?gtc?tac 960

Gln?Leu?Ala?Cys?Leu?Tyr?Ala?Lys?Leu?His?Gly?Leu?Asp?Arg?Val?Tyr

305 310 315 320

ttt?ggg?ggt?ttc?ttc?atc?cgg?ggt?cac?cct?gtg?acc?atg?cgc?acc?atc 1008

Phe?Gly?Gly?Phe?Phe?Ile?Arg?Gly?His?Pro?Val?Thr?Met?Arg?Thr?Ile

325 330 335

acc?tac?agc?att?aac?ttc?ttc?tct?aag?ggt?gaa?gtc?cag?gca?ctc?ttc 1056

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

340 345 350

ctg?aga?cat?gaa?ggc?tac?ctg?gga?gcc?atc?ggg?gca?ttt?ttg?aaa?gga 1104

Leu?Arg?His?Glu?Gly?Tyr?Leu?Gly?Ala?Ile?Gly?Ala?Phe?Leu?Lys?Gly

355 360 365

gcc?gag?caa?gat?aat?cct?aac?cag?tac?agc?tgg?ggt?gag?aac?tac?gcg 1152

Ala?Glu?Gln?Asp?Asn?Pro?Asn?Gln?Tyr?Ser?Trp?Gly?Glu?Asn?Tyr?Ala

370 375 380

gcc?agc?tcc?ggg?ctg?atg?agc?acg?gcg?ccg?gag?ctg?tgc?ccg?aca?cag 1200

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

385 390 395 400

cgg?gca?agg?agc?ggc?aca?ttt?gac?ctg?ctg?gaa?atg?gac?cgc?ctg?gag 1248

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

405 410 415

aga?ccc?ctg?gtc?aac?ctg?ccc?ctc?ctc?ctg?gac?cca?tcc?tcc?tat?gtg 1296

Arg?Pro?Leu?Val?Asn?Leu?Pro?Leu?Leu?Leu?Asp?Pro?Ser?Ser?Tyr?Val

420 425 430

ccc?gac?acg?gta?gac?ctc?act?gac?gat?gct?ctg?gcc?cga?cag?tac?tgg 1344

Pro?Asp?Thr?Val?Asp?Leu?Thr?Asp?Asp?Ala?Leu?Ala?Arg?Gln?Tyr?Trp

435 440 445

ctc?aca?tgc?ttt?gag?gag?gct?ctg?gat?ggg?gtg?gtg?aag?cga?gct?gtg 1392

Leu?Thr?Cys?Phe?Glu?Glu?Ala?Leu?Asp?Gly?Val?Val?Lys?Arg?Ala?Val

450 455 460

gcc?agc?cag?cca?gaa?tcc?gtg?gat?gca?gct?gag?agg?gca?gag?aag?ttc 1440

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

465 470 475 480

cgt?cag?aag?tac?tgg?ggc?aaa?ctt?cag?act?ctg?agg?cac?cag?ccc?ttt 1488

Arg?Gln?Lys?Tyr?Trp?Gly?Lys?Leu?Gln?Thr?Leu?Arg?His?Gln?Pro?Phe

485 490 495

gct?tat?ggg?acg?ctg?act?gtg?cgc?agc?ctg?ttg?gac?aca?aga?gag?cac 1536

Ala?Tyr?Gly?Thr?Leu?Thr?Val?Arg?Ser?Leu?Leu?Asp?Thr?Arg?Glu?His

500 505 510

tgt?ttg?aat?gag?ttc?aac?ttc?cca?gac?ccc?tac?tcc?aag?gtg?aag?cag 1584

Cys?Leu?Asn?Glu?Phe?Asn?Phe?Pro?Asp?Pro?Tyr?Ser?Lys?Val?Lys?Gln

515 520 525

aaa?gaa?aac?ggc?ctc?gcg?cta?aag?tgc?ttt?cag?agc?gtg?act?cgc?tcg 1632

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

530 535 540

ctg?gac?tca?cta?ggc?tgg?gag?gag?cgg?cag?ctg?gcc?ctg?gtg?aag?ggg 1680

Leu?Asp?Ser?Leu?Gly?Trp?Glu?Glu?Arg?Gln?Leu?Ala?Leu?Val?Lys?Gly

545 550 555 560

ctg?tta?gct?ggg?aat?gtc?ttt?gac?tgg?gga?gcc?aag?gct?gtg?tct?gac 1728

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

565 570 575

gtc?ctg?gaa?tcg?gac?ccc?cag?ttt?ggg?ttt?gaa?gaa?gca?aag?aga?aaa 1776

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

580 585 590

ttg?caa?gaa?cgg?ccc?tgg?ctg?gtg?gat?tcc?tac?acc?aag?tgg?ctc?cag 1824

Leu?Gln?Glu?Arg?Pro?Trp?Leu?Val?Asp?Ser?Tyr?Thr?Lys?Trp?Leu?Gln

595 600 605

agg?tta?aag?ggg?ccc?cct?cat?aaa?tgt?gcc?tta?att?ttc?gca?gat?aac 1872

Arg?Leu?Lys?Gly?Pro?Pro?His?Lys?Cys?Ala?Leu?Ile?Phe?Ala?Asp?Asn

610 615 620

agt?gga?ata?gac?atc?att?ttg?gga?gtc?ttc?ccc?ttt?gtc?agg?gag?cta 1920

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

625 630 635 640

ctc?tgt?aga?ggg?ata?gag?gtc?atc?ttg?gca?tgc?aac?tca?ggc?cct?gcc 1968

Leu?Cys?Arg?Gly?Ile?Glu?Val?Ile?Leu?Ala?Cys?Asn?Ser?Gly?Pro?Ala

645 650 655

ctg?aac?gat?gtg?acc?tac?agt?gag?tct?ctc?att?gtg?gca?gaa?cgc?att 2016

Leu?Asn?Asp?Val?Thr?Tyr?Ser?Glu?Ser?Leu?Ile?Val?Ala?Glu?Arg?Ile

660 665 670

gca?gcc?atg?gac?ccc?atc?atc?tgc?act?gca?ctc?aga?gaa?gac?agg?cta 2064

Ala?Ala?Met?Asp?Pro?Ile?Ile?Cys?Thr?Ala?Leu?Arg?Glu?Asp?Arg?Leu

675 680 685

ctg?ctg?gtg?cag?acc?ggt?tcc?agt?ccc?cca?tgc?cta?gat?ctc?agc?cgc 2112

Leu?Leu?Val?Gln?Thr?Gly?Ser?Ser?Pro?Pro?Cys?Leu?Asp?Leu?Ser?Arg

690 695 700

cta?gat?aag?gga?ctg?gct?gtg?ctg?gtg?cgg?gag?cgt?ggt?gcc?gac?ctg 2160

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

705 710 715 720

gtg?gtc?atc?gag?gga?atg?ggc?cgt?gct?gtc?cac?acc?aac?tac?cat?gct 2208

Val?Val?Ile?Glu?Gly?Met?Gly?Arg?Ala?Val?His?Thr?Asn?Tyr?His?Ala

725 730 735

ttg?ctg?cga?tgt?gag?agt?ctc?aag?ctg?gcc?gtg?gtg?aag?aac?gcc?tgg 2256

Leu?Leu?Arg?Cys?Glu?Ser?Leu?Lys?Leu?Ala?Val?Val?Lys?Asn?Ala?Trp

740 745 750

ctg?gct?gag?cgt?ctg?ggt?ggc?cag?ctc?ttc?agt?gtc?atc?ttc?aag?tac 2304

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

755 760 765

gag?gtc?cca?gcc?gag?tga 2322

Glu?Val?Pro?Ala?Glu

770

<210>2

<211>773

<212>PRT

＜213〉rat

<400>2

Met?Ala?Glu?Cys?Arg?Ala?Ser?Gly?Gly?Gly?Ser?Gly?Gly?Asp?Ser?Leu

1 5 10 15

Asp?Lys?Ser?Ile?Thr?Leu?Pro?Pro?Asp?Glu?Ile?Phe?Arg?Asn?Leu?Glu

20 25 30

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

35 40 45

Ala?Tyr?Tyr?Ser?Thr?Val?Gln?His?Lys?Val?Ala?Lys?Val?Arg?Ser?Phe

50 55 60

Asp?His?Pro?Gly?Lys?Asp?Ala?Glu?Gln?Asp?His?Glu?Pro?Pro?Tyr?Glu

65 70 75 80

Ile?Ser?Val?Gln?Glu?Glu?Ile?Thr?Ala?Arg?Leu?His?Phe?Ile?Lys?Phe

85 90 95

Glu?Asn?Thr?Tyr?Met?Glu?Ala?Cys?Leu?Asp?Phe?Ile?Arg?Asp?His?Leu

100 105 110

Val?Asn?Thr?Glu?Thr?Lys?Val?Ile?Gln?Ala?Thr?Gly?Gly?Gly?Ala?Tyr

115 120 125

Lys?Phe?Lys?Asp?Leu?Ile?Glu?Glu?Lys?Leu?Arg?Leu?Lys?Val?Asp?Lys

130 135 140

Glu?Asp?Val?Met?Thr?Cys?Leu?Ile?Lys?Gly?Cys?Asn?Phe?Val?Leu?Lys

145 150 155 160

Asn?Ile?Pro?His?Glu?Ala?Phe?Met?Tyr?Gln?Lys?Asp?Ser?Asp?Pro?Glu

165 170 175

Phe?Arg?Phe?Gln?Thr?Asn?His?Pro?Asn?Ile?Phe?Pro?Tyr?Leu?Leu?Val

180 185 190

Asn?Ile?Gly?Ser?Gly?Val?Ser?Ile?Val?Lys?Val?Glu?Thr?Glu?Asp?Arg

195 200 205

Phe?Glu?Trp?Ile?Gly?Gly?Ser?Ser?Ile?Gly?Gly?Gly?Thr?Phe?Trp?Gly

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

Ala?Ser?Ser?Phe?Gly?Lys?Ser?Ala?Thr?Ala?Asp?Arg?Glu?Phe?Ser?Lys

275 280 285

Glu?Asp?Met?Ala?Lys?Ser?Leu?Leu?His?Met?Ile?Ser?Asn?Asp?Ile?Gly

290 295 300

Gln?Leu?Ala?Cys?Leu?Tyr?Ala?Lys?Leu?His?Gly?Leu?Asp?Arg?Val?Tyr

305 310 315 320

Phe?Gly?Gly?Phe?Phe?Ile?Arg?Gly?His?Pro?Val?Thr?Met?Arg?Thr?Ile

325 330 335

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

340 345 350

Leu?Arg?His?Glu?Gly?Tyr?Leu?Gly?Ala?Ile?Gly?Ala?Phe?Leu?Lys?Gly

355 360 365

Ala?Glu?Gln?Asp?Asn?Pro?Asn?Gln?Tyr?Ser?Trp?Gly?Glu?Asn?Tyr?Ala

370 375 380

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

385 390 395 400

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

405 410 415

Arg?Pro?Leu?Val?Asn?Leu?Pro?Leu?Leu?Leu?Asp?Pro?Ser?Ser?Tyr?Val

420 425 430

Pro?Asp?Thr?Val?Asp?Leu?Thr?Asp?Asp?Ala?Leu?Ala?Arg?Gln?Tyr?Trp

435 440 445

Leu?Thr?Cys?Phe?Glu?Glu?Ala?Leu?Asp?Gly?Val?Val?Lys?Arg?Ala?Val

450 455 460

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

465 470 475 480

Arg?Gln?Lys?Tyr?Trp?Gly?Lys?Leu?Gln?Thr?Leu?Arg?His?Gln?Pro?Phe

485 490 495

Ala?Tyr?Gly?Thr?Leu?Thr?Val?Arg?Ser?Leu?Leu?Asp?Thr?Arg?Glu?His

500 505 510

Cys?Leu?Asn?Glu?Phe?Asn?Phe?Pro?Asp?Pro?Tyr?Ser?Lys?Val?Lys?Gln

515 520 525

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

530 535 540

Leu?Asp?Ser?Leu?Gly?Trp?Glu?Glu?Arg?Gln?Leu?Ala?Leu?Val?Lys?Gly

545 550 555 560

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

565 570 575

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

580 585 590

Leu?Gln?Glu?Arg?Pro?Trp?Leu?Val?Asp?Ser?Tyr?Thr?Lys?Trp?Leu?Gln

595 600 605

Arg?Leu?Lys?Gly?Pro?Pro?His?Lys?Cys?Ala?Leu?Ile?Phe?Ala?Asp?Asn

610 615 620

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

625 630 635 640

Leu?Cys?Arg?Gly?Ile?Glu?Val?Ile?Leu?Ala?Cys?Asn?Ser?Gly?Pro?Ala

645 650 655

Leu?Asn?Asp?Val?Thr?Tyr?Ser?Glu?Ser?Leu?Ile?Val?Ala?Glu?Arg?Ile

660 665 670

Ala?Ala?Met?Asp?Pro?Ile?Ile?Cys?Thr?Ala?Leu?Arg?Glu?Asp?Arg?Leu

675 680 685

Leu?Leu?Val?Gln?Thr?Gly?Ser?Ser?Pro?Pro?Cys?Leu?Asp?Leu?Ser?Arg

690 695 700

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

705 710 715 720

Val?Val?Ile?Glu?Gly?Met?Gly?Arg?Ala?Val?His?Thr?Asn?Tyr?His?Ala

725 730 735

Leu?Leu?Arg?Cys?Glu?Ser?Leu?Lys?Leu?Ala?Val?Val?Lys?Asn?Ala?Trp

740 745 750

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

755 760 765

Glu?Val?Pro?Ala?Glu

770

Claims (9)

1, a kind of isolated nucleic acid molecule, it comprises and has the polynucleotide that are selected from as a kind of nucleotide sequence of next group, and described group is:

(a) coding has the full-length polypeptide of the aminoacid sequence shown in SEQ ID NO:2

Nucleotide sequence;

(b) be complementary to the nucleotide sequence of the nucleotide sequence of above (a);

(c) under stringent condition with the nucleotide sequence of (a) nucleotide sequence hybridization.

2, nucleic acid molecule as claimed in claim 1, wherein said polynucleotide comprise the nucleotide sequence shown in the SEQ IDNO:1.

3, a kind of expression vector that comprises the nucleic acid molecule of claim 1 or 2.

4, the expression vector of claim 3, it is carrier for expression of eukaryon pcDNA3.1-pank4.

5, by the expression vector conversion of claim 3 or the host cell of transfection.

6, a peptide species, it is total length pank4 albumen or its fragment with biologic activity with the complete amino acid sequence shown in the SEQ ID NO:2.

7, the antibody of the polypeptide of anti-claim 6.

8, claim 1 or 2 nucleic acid molecule are as the purposes of the target spot for the treatment of diabetes.

9, the purposes of the polypeptide of claim 1 or 2 nucleic acid molecule or claim 6 in the medicine of preparation treatment diabetes.

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