CN108823180A - Make the dephosphorylized albumen of protein kinase B specific site and its nucleic acid - Google Patents

Make the dephosphorylized albumen of protein kinase B specific site and its nucleic acid Download PDF

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CN108823180A
CN108823180A CN201810697347.4A CN201810697347A CN108823180A CN 108823180 A CN108823180 A CN 108823180A CN 201810697347 A CN201810697347 A CN 201810697347A CN 108823180 A CN108823180 A CN 108823180A
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acid sequence
amino acid
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akt
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罗开珺
张玮
朱启顺
蔡秋成
何浩娟
张力丹
张雪雯
崔继辉
陈昶旭
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Yunnan University YNU
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Abstract

This application provides make the dephosphorylized albumen of AKT albumen specific site and its nucleic acid.Amino acid sequence therein includes to claim amino acid sequence I, and the amino acid sequence I is the amino acid sequence such as at least one of (I), (II) and (III)：(I) amino acid sequence as shown in SEQ ID No.1；(II) in the polydnavirus of removing at least one of the Microplitis bicoloratus for parasitizing Microplitis as amino acid sequence shown in SEQ ID No.1 is with the same function shown in one kind in amino acid sequence；(III) with the consistency of the amino acid sequence as shown in SEQ ID No.1 95% or more, and the amino acid sequence with the amino acid sequence artificial mutation with the same function as shown in SEQ ID No.1.

Description

Make the dephosphorylized albumen of protein kinase B specific site and its nucleic acid
Technical field
This application involves a kind of amino acid sequence and its nucleic acid sequences, and especially it makes protein kinase B the 308th Soviet Union Application in the dephosphorylation of propylhomoserin site.
Background technique
PI3K is a kind of phosphatidyl inositol kinase intracellular, is made of adjusting subunit P85 and catalytic subunit P110, when by outer Come when stimulating, substrate phosphatidylinositol diphosphate (PIP2) by being converted into phosphatidyl-4 in conjunction with P85 subunit by P110 subunit Alcohol triphosphoric acid (PIP3), PIP3 can be combined with the N-terminal PH structural domain of protein kinase B (AKT), make AKT from Chromosome migration to On cell membrane, and in the auxiliary of phosphoinositide deopendent protein kinase 1 (PDKI) and phosphoinositide deopendent protein kinase 2 (PDK2) It helps down, makes two site phosphorylations of threonine (Thr308) and serine (Ser473) on AKT albumen respectively and activate.Pass through AKT albumen after phosphorylation activation can be activated with Direct Phosphorylation or inactivation tuberous sclerosis complex 2 (TSC2) enhancing indirectly Activate its substrate rapamycin target protein (mTOR), to promote cell Proliferation and inhibit Apoptosis, therefore, this be tumour, One of mechanism of generation of diseases such as cancer.In addition, tumor suppressor gene (PTEN) can turn and to PIP3 dephosphorylation Become PIP2, realize and the negativity of PI3K/AKT access is adjusted, inhibits cell Proliferation and promote Apoptosis.
Although PI3K/AKT signal path is existing, more in-depth study, the prior art can not be complete Illustrate the signal path.For example, in the prior art, although the dephosphorization to two phosphorylation sites in AKT albumen may be implemented Acidification, but these means are while by two site dephosphorylations that can not actually determine single locus in this way removes phosphoric acid Change the influence to AKT proteins downstream.
Summary of the invention
One of the application provides a kind of amino acid sequence, and it includes title amino acid sequence I, the amino acid sequence I to be Such as the amino acid sequence of at least one of (I), (II) and (III)：
(I) amino acid sequence as shown in SEQ ID No.1；
(II) nucleotide sequence is with the same function and as shown in SEQ ID No.1 parasitizes Microplitis (Microplitis) core in the polydnavirus of at least one of removing Microplitis bicoloratus (M.bicoloratus) One of nucleotide sequence；
(III) with the consistency of the amino acid sequence as shown in SEQ ID No.1 95% or more, and with such as SEQ ID The amino acid sequence of the artificial mutation with the same function of amino acid sequence shown in No.1；It is preferred that with such as SEQ ID No.1 institute The consistency of the amino acid sequence shown has identical function 98% or more, and with the amino acid sequence as shown in SEQ ID No.1 The amino acid sequence of the artificial mutation of energy；It is preferred that with the consistency of the amino acid sequence as shown in SEQ ID No.1 99.5% More than, and the amino acid sequence with the amino acid sequence artificial mutation with the same function as shown in SEQ ID No.1.
In a specific embodiment, the amino acid sequence I is and the amino acid sequence as shown in SEQ ID No.1 Consistency 98% or more, and with the amino acid sequence artificial mutation with the same function as shown in SEQ ID No.1 Amino acid sequence.
In a specific embodiment, the amino acid sequence I is and the amino acid sequence as shown in SEQ ID No.1 Consistency 99.5% or more, and with the amino acid sequence artificial mutation with the same function as shown in SEQ ID No.1 Amino acid sequence.
The two of the application provide a kind of nucleic acid sequence, and it includes capable of encoding such as the application for referred to as nucleic acid sequence I One of any one described in amino acid sequence nucleotide sequence.
In a specific embodiment, the nucleic acid sequence I is the nucleotide sequence as shown in SEQ ID No.2.
The three of the application provide the application of the amino acid sequence according to any one of one of the application.
In a specific embodiment, the application is the protein kinase B (AKT albumen) the 308th made in cell The dephosphorylized application in threonine site.
In a specific embodiment, the cell is insect cell.
The four of the application provide the application of two nucleic acid sequence according to the application.
In a specific embodiment, the application is two nucleic acid sequence by the application in cell Expression, so that protein kinase B the 308th dephosphorylized application in threonine site in the cell.
In a specific embodiment, the cell is insect cell.
In a specific embodiment, the nucleic acid sequence of the two of the application, which is connected to, can be used in the cell Expression vector on, and the expression vector can make the nucleic acid sequence be expressed as in the cell as the application it The albumen of amino acid sequence described in one any one.
The beneficial effect of the application：
More partitivirus (M.bicoloratuss of the inventor in the Microplitis bicoloratus for causing Apoptosis Bracovirus, MbBV) in obtain a kind of tyrosine-phosphatase PTP109 (SEQ ID No.1).When inventor will When expression of the PTP109 in cell, it is found surprisingly that PTP109 can be to the threonine phosphorylation sites (AKT on AKT albumen PThr308 dephosphorylation) is carried out, although there are one on AKT albumen Ser-phosphorylation site (AKT pSer473), PTP109 can not carry out effectively dephosphorylation to the Ser-phosphorylation site.And inventor be also found one it is important The phenomenon that, i.e. the dephosphorylation of the specificity of AKT pThr308 can lead to the cutting of Caspase3 albumen.And the skill of this field Art personnel are known, and the cutting that itself occurs after being activated for Caspase3 can cause Apoptosis.In fact, it is based on the application, Also observed in subsequent experimental makes the dephosphorylized cell of AKT pThr308 produce apoptosis phenomenon using PTP109.As it can be seen that The dephosphorylation of the specificity of AKT pThr308 can generate important influence to PI3K/AKT access.But how to influence Link in PI3K/AKT access between AKT albumen and Caspase3 albumen, can be by the method for application by AKT pThr308 Specific dephosphorylation carries out correlative study, with the details improved in PI3K/AKT access.
Detailed description of the invention
The total protein tag antibody that Fig. 1 is extracted after being pIZT/V5-His-ptp109 plasmid transfection Hi5 cell 72h The Western Blot result that Anti-V5 is carried out.The result shows that ptp109 can stablize expression about 44kDa in Hi5 cell The albumen of size.Wherein, M is albumen maker, and Ctrl is blank control without any processing, and EV is transfection pIZT/V5-His The negative control of empty plasmid, ptp109 are the experimental group for transfecting pIZT/V5-His-ptp109 plasmid, and Tubulin is in cell The tubulin of middle stable expression, as internal reference.
Fig. 2 is the dephosphorylation situation of two phosphorylation sites of the AKT albumen in the Hi5 cell that ptp109 is expressed. Wherein：
Fig. 2A is the Western Blot result of the AKT pThr308 of different disposal.As seen from the figure, AKT pThr308 water It puts down and is decreased obviously in the case where expressing ptp109, AKT Thr308 is in low phosphorylation level.Realize AKT pThr308 Dephosphorylation.
Fig. 2 B is the difference analysis of the phosphorylation level of the AKT pThr308 of different disposal, the knot examined according to t Fruit, * * indicate p<When 0.01, difference highly significant, NS expression is not significantly different.The result shows that the expression of ptp109 is to AKT PThr308 has significant dephosphorylation effect, and AKT Thr308 is made to be in low-down phosphorylation level.Realize AKT The dephosphorylation of pThr308.Wherein, the phosphorylation level of AKT pThr308 is the average value of independent repeated trials three times.
Fig. 2 C is the Western Blot result of the AKT pSer473 of different disposal.As seen from the figure, AKT pSer473 water It puts down and does not decline in the case where expressing ptp109, AKT pSer473 is still in high phosphorylation level.Do not reach and passes through The dephosphorylized purpose of AKT pSer473 is realized in the expression of ptp109.
Fig. 2 D be different disposal AKT p Ser473 phosphorylation level difference analysis, according to t examine as a result, NS expression is not significantly different.The result shows that the expression of ptp109 is to AKT pSer473 substantially without dephosphorylation effect, AKT PSer473 is still in high phosphorylation level.Do not reach and realizes that AKT pSer473 is dephosphorylized by the expression of ptp109 Purpose.Wherein, the phosphorylation level of AKT pSer47 is the average value of independent repeated trials three times.
Fig. 3 be in the Hi5 cell that ptp109 is expressed Cleaved-caspase3 (the Caspase3 albumen after cutting, Can be referred to as activated form band) detection case.Wherein signal egg of the Cleaved-caspase3 as cell late apoptic The white level for being used to detect Apoptosis on protein level.Wherein：
Fig. 3 A is the Western Blot of the Cleaved-caspase3 of different disposal as a result, as seen from the figure, after cutting Caspase3 protein level obviously rises in the case where expressing ptp109.
Fig. 3 B is the difference analysis of the Caspase3 protein level after the cutting of different disposal, the knot examined according to t Fruit, * indicate p<When 0.05, NS expression is not significantly different.The result shows that the expression of ptp109 can cause cutting for Caspase3 It cuts.
The total protein tag antibody that Fig. 4 is extracted after being pIZT/V5-His-ptp109 plasmid transfection Spli221 cell 72h The Western Blot result that Anti-V5 is carried out.Wherein, M is albumen maker, and Ctrl is blank control without any processing, EV is the negative control for transfecting pIZT/V5-His empty plasmid, and ptp109 is the reality for transfecting pIZT/V5-His-ptp109 plasmid Group is tested, Tubulin is the tubulin for stablizing expression in cell, as internal reference.
Fig. 5 is the dephosphorylation in two phosphorylation sites of the ptp109 AKT albumen in Spli221 cell expressed Situation.Wherein：
Fig. 5 A is the Western Blot of the AKT pThr308 of different disposal as a result, as seen from the figure, AKT pThr308 water It puts down and is decreased obviously in the case where expressing ptp109, AKT Thr308 is in low phosphorylation level.Realize AKT pThr308 Dephosphorylation.
Fig. 5 B is the difference analysis to the AKT pThr308 phosphorylation level of different disposal, according to t inspection as a result, * Indicate p<When 0.05, difference highly significant, NS expression is not significantly different, the results showed that the expression of ptp109 is to AKT PThr308 has significant dephosphorylation effect, and AKT Thr308 is made to be in low-down phosphorylation level.Realize AKT The dephosphorylation of pThr308.Wherein, the phosphorylation level of AKT pThr308 is the average value of independent repeated trials three times.
The Western Blot that Fig. 5 C is the AKT pSer473 of different disposal is horizontal, as seen from the figure, AKT pSer473 water It puts down and does not decline in the case where expressing ptp109, AKT pSer473 is still in high phosphorylation level.Do not reach and passes through The dephosphorylized purpose of AKT pSer473 is realized in the expression of ptp109.
Fig. 5 D is the difference analysis of the AKT p Ser473 phosphorylation level of different disposal, according to t inspection as a result, NS Expression is not significantly different, the results showed that the expression of ptp109 is to AKT p Ser473 substantially without dephosphorylation effect, AKT PSer473 is still in high phosphorylation level.Do not reach and realizes that AKT pSer473 is dephosphorylized by the expression of ptp109 Purpose.Wherein, the phosphorylation level of AKT pSer47 is the average value of independent repeated trials three times.
Fig. 6 is the detection case of Cleaved-caspase3 in the Spli221 cell of ptp109 expression.Cleaved- Signal protein level to detect on protein level Apoptosis of the caspase3 as cell late apoptic.Wherein：
Fig. 6 A is the Western Blot of the Cleaved-caspase3 of different disposal as a result, as seen from the figure, after cutting Caspase3 protein level obviously rises in the case where expressing ptp109.
Fig. 6 B be different disposal cutting after Caspase3 protein level difference analysis, according to t examine as a result, * Indicate p<When 0.05, NS expression is not significantly different, the results showed that the expression of ptp109 can cause the cutting of Caspase3.
Specific embodiment
The present invention is further explained in the light of specific embodiments, but following embodiment and not pairs of enough the application Limitation, all includes the technical solution within the scope of spirit herein, within the scope of protection of this application.
Embodiment 1
The design of 1.PTP109 special primer
Using primer-design software Primer Premier 5 design upstream primer ptp109-F (SEQ ID No.3) and under Trip primer ptp109-R (SEQ ID No.4) is used to clone the PTP109 gene sequence of Microplitis bicoloratus polydnavirus (PDV) It arranges (as shown in SEQ ID No.2), size 1032bp, encodes 344 amino acid altogether (as shown in SEQ ID No.1).
2. the extraction of cocoon bee viral DNA
From its ovary is taken out after 50-100 emergence in the queen bee of 3-4d, the PBS (formula of 100 μ l is added：NaCl：8g； KCl：0.2g；Na2HPO4：1.42g；KH2PO4：0.27g；It is settled to 1L, pH7.4), with the micro syringe pressure-vaccum ovum of 100 μ l Nest is collected into the EP pipe of 2ml, is centrifuged under conditions of 3000rpm, 3min, 4 DEG C, collect supernatant to fragmentation.
Following steps need to be completed in superclean bench：With the ovum and other cell fragments in 0.45 μm of membrane filtration supernatant Deng being centrifuged under conditions of 15,000rpm, 15min, 4 DEG C, abandon supernatant, sequentially add 200 μ l's in precipitating after centrifugation PDV-DNA lysate (formula：NaCl：0.5844g；Tris/HCl：0.1214g；EDTA：0.9306g；SDS：0.5g；Distilled water Be settled to 100ml, pH be adjusted to 8.0), the Proteinase K of 25 μ l it is (final concentration of：2.5mg/ml is purchased from the limited public affairs of Tiangeng biochemical technology Department), 8 μ l sarcosyls, in 55 DEG C of 3h；Then the RNaseA that 40 μ l are added is (final concentration of：1mg/ml, purchased from green The skies), in 37 DEG C of 1h；It is primary with equivalent phenol/chloroform (+300 μ l chloroform of 300 μ l phenol) extracting, at 1,0000rpm from Heart 10min collects supernatant；It is primary with the chloroform of equivalent, it is centrifuged 10min at 1,0000rpm, collects supernatant；With equal bodies (100% ethyl alcohol and 1/10 volume ratio of sodium acetate are 9 for long-pending 100% ethyl alcohol and 1/10 mixture of sodium acetate:1) it, mixes In -70 DEG C of precipitating 10min after even, it is then centrifuged 10min at 12,000g, abandons supernatant；5min is cleaned with ethyl alcohol, is dried in vacuo 10min；It is finally dissolved in the pure water of 20 μ l, obtains PDV DNA solution.1 μ l PDV DNA solution NanoDrop is taken to detect DNA Concentration and purity.1 μ l PDV DNA solution is taken to be detected on 1% Ago-Gel.
The gene fragment clone of 3.PTP109
Using above-mentioned PDV DNA solution as DNA profiling, with ptp109-F (SEQ ID No.3) and ptp109-R (SEQ ID No.4) it is primer pair, carries out PCR amplification.The genetic fragment for obtaining ptp109 is 1032bp, and PCR product is recycled, and is connected to On cloning vector pMD19-T carrier, after sequencing determines correctly, the pMD19- containing PTP109 full length gene target fragments is obtained Ptp109 plasmid.
The genetic fragment of 4.PTP109 is subcloned
Band restriction enzyme site primer is designed using primer-design software Primer Premier 5：Upstream primer E-ptp109-F (SEQ ID No.6 contains Not I enzyme by (SEQ ID No.5 contains EcoR I restriction enzyme site) and downstream primer E-ptp109-R Enzyme site).Use above-mentioned purified PMD19-ptp109 plasmid as DNA profiling, with E-ptp109-F (SEQ ID No.5) and E-ptp109-R (SEQ ID No.6) is primer pair, carries out PCR amplification, obtains the band of 1045bp size, and PCR product is returned It receives, and is connected on cloning vector pMD19-T carrier, after sequencing determines correctly, obtain containing PTP109 full length gene target The pMD19-ptp109 plasmid of segment.
The building of 4.PTP109 carrier for expression of eukaryon
EcoR I and Not I double digestion are carried out to pMD19-ptp109 plasmid, obtained with restriction enzyme site cohesive end Ptp109 genetic fragment.The resulting ptp109 genetic fragment with restriction enzyme site cohesive end is connected to equally via EcoR On the pIZT/V5-His expression vector (commercially available acquisition) of I and Not I double digestion.By to this recombinant plasmid product into Row double digestion, PCR detection and sequencing analysis it is correct after, obtain positive plasmid pIZT/V5-His-ptp109.
5. expressing PTP109
1, pIZT/V5-His-ptp109 plasmid is transfected
(1) prepare the good good Hi5 cell of Spli221 and three group of growth conditions of three groups of growth conditions, be layered on 6 respectively In orifice plate, every hole about 2 × 105A cell.Wherein it is not processed as blank control group for one group；In other two groups, wherein one Group transfection pIZT/V5-His plasmid is as unloaded control group, and another group of transfection pIZT/V5-His-ptp109 plasmid is as processing Group.
(2) when the cell density in 6 orifice plates reaches 80%-90%, old culture medium is removed, new culture is added Base gently blows and beats suspension cell with pipette tips, and trypan blue counts；
(3) by 27 DEG C of adhere-wall culture 2h or more of cell after the counting in 6 orifice plates until cell is adherent, in good condition；? It is added before the transfection reagent mixed liquor in below step, is incubated for 30min with double no culture mediums, and transfection reagent mixing is being added Before liquid, it is sucked out double without culture medium.
(4) transfection reagent mixed liquor is prepared, that is, draws the double of 100 μ L and is placed in 1.5mL without (serum-free, no antibiosis) culture medium In EP pipe, the plasmid of 1 μ g is then added；In addition by the double without culture medium and 5 μ l transfection reagent Cell fection II of 100 μ L (purchased from grace of speeding) is added in the EP pipe of another 1.5mL, and concussion mixes；Two EP pipes are merged, are stored at room temperature after mixing 45min, and within the standing phase of the 45min, concussion in every 15 minutes several times, the double without culture medium of 800 μ L is added later, is formed and is turned Transfection reagent mixed liquor is incubated for 30min, then transfection reagent mixed liquor is added dropwise to the above-mentioned steps (3) with cell It is sucked out in double 6 orifice plates without culture medium, is then put on shaking table and shakes gently (about 21rpm/min), make transfection reagent mixed liquor It is uniformly mixed in 6 orifice plates, then continues to cultivate in 27 DEG C of standings.It carries out changing liquid after 4h-5h, i.e., liquid is sucked out, addition contains There is the culture medium of 10% serum.It is shaked gently on shaking table uniform to culture medium, is then placed in 27 DEG C of incubators, is started with this Timing cultivates 72h with the extraction of the total protein of cell for subsequent step.
2. the extraction of total protein of cell
(1) after transfecting 72h, culture medium is discarded, is cleaned 1 time with 1 × PBS, 1mL1 × PBS is then added, it is light with liquid-transfering gun Lightly adherent cell is blown afloat, is moved in 1.5ml EP pipe, is centrifuged 5min at 3000g and 4 DEG C, discards supernatant, sunk It forms sediment；
(2) PMSF is added in RIPA tissue/cell lysate (being purchased from Suo Laibao), obtains efficient RIPA tissue/cell (PMSF is protease inhibitors to lysate, the final concentration of 10 μ L/ml in efficient RIPA tissue/cell lysate, current existing Add PMSF)；Then according to the amount of precipitating, the efficient RIPA tissue/cell cracking of 50-70 μ L is added into the precipitating of step (1) Liquid controls the concentration of total protein within 10mg/ml.
(3) gently pressure-vaccum precipitates, and shakes 30s, makes cell even suspension, and placing 5-10min on ice cracks cell sufficiently； 13000g, 4 DEG C of centrifugation 10min, taking supernatant is total protein.
Determination of protein concentration and denaturation
Egg concentration mensuration is carried out using BCA determination of protein concentration kit：
(I) total protein obtained above is added in 96 orifice plates, 1 μ L sample is loaded in each hole, and it is double to add 9 μ L sterilizing Steam water；It carries out in group three times in parallel to reduce error；
(II) standard curve is drawn with matched standard protein solution B SA in BCA kit (being purchased from ancient cooking vessel state), by albumen mark Quasi- liquid is added in 96 orifice plates according to 0,2,4,6,8,10 microlitre, supplies 10 μ L with sterilizing distilled water；
(III) after proportionally mixing A/B solution in kit (50 volume A, 1 volume B), A/B is added in every hole Mixed liquor 200 μ L, 37 DEG C of reaction 30min；
(IV) its light absorption value is measured under microplate reader 562nm wavelength, the concentration of total protein is calculated by standard curve；
(V) after the total protein that above-mentioned steps (3) obtain being mixed in proportion with 5 × SDS-PAGE sample-loading buffer, 90 DEG C It is heat-treated 15min；
(VI) cool down immediately, then carry out PAGE gel electrophoretic analysis or deposit in -80 DEG C it is spare.
3.SDS-PAGE proteins gel electrophoresis
Prepare SDS-PAGE glue and electrophoresis：
The polyacrylamide gel condensed is removed from gel maker, is put into electrophoresis tank, and is added into electrophoresis tank Comb, albumen loading electrophoresis are extracted after entering 1 × electrophoretic buffer.Race glue first is carried out with 80V, crosses after the glue of upper layer and uses to band 100V voltage carries out about 90min.
4. transferring film and subsequent operation
(1) first pvdf membrane is soaked in methyl alcohol and soaks 1min in methyl alcohol.And pvdf membrane is covered on glue in order. 100min is carried out with 100V voltage in ice-water bath；(2) pvdf membrane is taken out after the completion of transferring film, closes liquid chamber with 5% skimmed milk power Warm close membrane 1h；(3) with 1 × PBST clean closed pvdf membrane twice, each 5min；(4) dilute with the antibody containing primary antibody Releasing liquid, (wherein, the volume ratio of primary antibody and antibody diluent is 1:2000, antibody diluent is purchased from Suo Laibao) it is incubated overnight (4 ℃)；(5) dilution containing an antiantibody is recycled, next cleans film three times with 1 × PBST, each 5min；(6) with containing (wherein, the volume ratio of secondary antibody and confining liquid is 1 to the confining liquid of secondary antibody:5000, confining liquid be containing 3% skimmed milk power 1 × PBST) it is incubated at room temperature film 1h；(7) film is cleaned three times with 1 × PBST, each 10min；(8) by Pierce ECL Western After Blotting Substrate kit A, B liquid mixes in equal volume, it is added dropwise on pvdf membrane；In FluorChem EFE0511 Middle exposure image；Gray analysis is carried out using Image J software, to determine that expressing quantity becomes with the presence or absence of significant otherness Change.
According to above-mentioned Western Blot operating procedure, result as shown in Figure 1 and Figure 4 is obtained, wherein M is albumen Marker, Anti-V5 (Thermo, R96025) are the antiantibody used, and Tubulin is internal reference albumen.As seen from the figure, The albumen of 44kDa or so size is obtained, illustrates pIZT/V5-His-ptp109 successful expression in insect cell line.
According to above-mentioned Western Blot method, it is incubated for AKT pThr308 (abcam, ab66134), AKT respectively The specific primary antibody of pSer473 (Servicebio, GB13012-3), Cleaved-caspase3 (ImmunoWay, YC0006) Antibody detects the PTP109 albumen of expression to AKT pThr308, AKT pSer473, Cleaved-caspase protein level It influences, carries out gray analysis using Image J software, see Fig. 2, Fig. 3, Fig. 5, Fig. 6.The result shows that PTP109 expression may be implemented To the dephosphorylation of AKT pThr308 specificity, the ATK albumen of Thr308 low phosphorylation level is obtained, while can be caused The cutting of Caspase3 albumen, but it can not achieve the dephosphorylation to AKT pSer473.Thus conclusion can be from which further followed that, The dephosphorylation of AKT pThr308 specificity can cause the cutting of Caspase3 albumen.
According to the subsequent morphology of the cell of the culture of processing group and blank control group and zero load control group it is found that The expression of PTP109 can cause Apoptosis.And the cutting of Caspase3 albumen can cause Apoptosis, in conjunction with above-mentioned PTP109 Expression can cause Caspase3 albumen cutting conclusion, the expression for further demonstrating PTP109 caused by cascading Final Apoptosis.
Although the application is described referring to specific embodiment, it should be appreciated by those skilled in the art In the case where no real spirit and scope for being detached from the application, the various changes that can carry out.Furthermore, it is possible to this Shen Main body, spirit and scope please are variously changed to adapt to specific situation, material, material compositions and method.All These changes are included in the range of claims hereof.
Sequence table
<110>Yunnan University
<120>Make the dephosphorylized albumen of protein kinase B specific site and its nucleic acid
<130> LHA1860403
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 343
<212> PRT
<213>Microplitis bicoloratus virus (Microplitis bicoloratus bracovirus)
<400> 1
Met Gly Asn His Ser Phe Lys Thr Leu Ser Ile Val Asp Phe Leu Lys
1 5 10 15
Leu Thr Ser Gln Pro Asp Phe Ser Lys Leu Ile Glu Lys Glu His Ala
20 25 30
Gln Ile Leu Ala Val Gln Leu Pro Gly Thr Phe Ala Ser Phe Ser Arg
35 40 45
His Glu Asn Ser Ser Lys Asn Arg His Tyr Asp Val Pro Cys Trp Asp
50 55 60
Leu Ser Arg Val Ile Leu Thr Pro Arg Ser Ala Ile Tyr Asp Asp Glu
65 70 75 80
Asp Trp Asn Leu Ser His Ile Thr Thr Thr Ser Ala Glu Ile Ala Ser
85 90 95
Thr Tyr Ile His Ala Asn Phe Val Asp Gly Phe Glu Glu Ile Asn Lys
100 105 110
Phe Ile Cys Ser Gln Gly Pro Lys Lys Asn Ser Cys Glu Asp Phe Trp
115 120 125
Arg Met Val Leu Glu Gln Glu Ser Cys Ile Ile Val Ser Leu Thr Glu
130 135 140
Thr Asp Asp Glu Asp Gln Val Cys Tyr Glu Tyr Trp Val Lys Glu Glu
145 150 155 160
Asp Tyr Glu Leu Val Phe Gly Arg Tyr Ile Val Lys Thr Leu Glu Ile
165 170 175
Ile Glu Glu Ser Ser Phe Thr Arg Thr Arg Leu Arg Leu Thr Asp Val
180 185 190
Asn Ser Gly Thr Ser Arg Glu Ile His His Phe Trp Tyr Pro His Trp
195 200 205
Pro Asp Tyr Gly Asn Pro Thr Asn Pro Ala Glu Ile Leu Asn Leu Ile
210 215 220
Ser Gln Val Asn Gln Lys Arg Lys Glu Met Lys Lys Thr Ala Asp Ser
225 230 235 240
Gln Pro Gly Pro Ile Val Val His Cys Ser Ala Gly Ile Gly Arg Thr
245 250 255
Gly Thr Phe Cys Thr Ile Asp Asn Ala Leu Cys Gln Leu Arg Lys Gln
260 265 270
Lys Ser Val Ser Leu Pro Gln Thr Val Leu Lys Ile Arg Arg Gln Arg
275 280 285
His Ser Ser Val Phe Leu Pro Glu Gln Tyr Ala Phe Cys Tyr Lys Thr
290 295 300
Leu Lys His Ala Leu Met Thr Glu Val Lys Glu Ile Ala Ala Asn Gln
305 310 315 320
Leu Asn Leu Asp Ala Ile Thr Leu Glu Pro Glu Thr His Asn Ser Gln
325 330 335
Arg Lys Arg Leu Arg Val Leu
340
<210> 2
<211> 1032
<212> DNA
<213>Microplitis bicoloratus virus (Microplitis bicoloratus bracovirus)
<400> 2
atggggaatc atagcttcaa gacactcagc atcgtggact tcctgaagct gacaagtcag 60
ccggacttct caaaactcat tgaaaaagag cacgcccaga ttttggctgt acaattacca 120
ggtacttttg ctagtttttc aagacacgaa aattcatcaa agaatcggca ttacgatgtt 180
ccgtgttggg atctttctag agtaatttta accccacgca gtgcaatata cgatgacgaa 240
gattggaatt tatcacatat aactacgaca tctgcagaaa tagcatcaac ttacatacat 300
gcgaactttg ttgatggatt tgaggaaata aataaattta tttgcagcca gggtccgaag 360
aaaaattcgt gtgaagactt ttggaggatg gttttagagc aagaatcttg tattatcgtg 420
tcactgacgg aaacagatga tgaagatcaa gtgtgttatg aatattgggt taaagaagag 480
gattatgaac tagtatttgg aagatatatt gtaaaaaccc ttgaaattat agaagagtct 540
agttttacca gaacgcgatt gcgactcact gatgtgaaca gtggtacttc acgggagatc 600
caccactttt ggtatcctca ctggcccgac tatggtaatc ctaccaatcc ggcggaaata 660
ttaaatctaa tatcgcaagt gaaccaaaaa cgaaaagaga tgaaaaagac agcagactct 720
caaccgggac cgatagtagt ccactgttct gcaggaattg gtcggacagg aacattttgt 780
actatagaca atgcactatg ccagttacgg aaacaaaagt cagtgtcgct tccacagacg 840
gtgctaaaaa ttcgaagaca aagacattca agtgtgtttc ttcctgaaca gtatgcattc 900
tgttacaaaa cattaaagca tgctttgatg acagaagtca aagaaatagc tgccaatcaa 960
ctgaacctgg acgctataac tttggagcca gaaacccata attcacagag aaagaggttg 1020
agagtgctat aa 1032
<210> 3
<211> 17
<212> DNA
<213>Artificial sequence (non)
<400> 3
atggggaatc atagctt 17
<210> 4
<211> 20
<212> DNA
<213>Artificial sequence (non)
<400> 4
ttatagcact ctcaacctct 20
<210> 5
<211> 23
<212> DNA
<213>Artificial sequence (non)
<400> 5
gaattcatgg ggaatcatag ctt 23
<210> 6
<211> 16
<212> DNA
<213>Artificial sequence (non)
<400> 6
gcggccgcat agcact 16

Claims (10)

1. a kind of amino acid sequence, it includes claim amino acid sequence I, the amino acid sequence I to be as in (I), (II) and (III) At least one amino acid sequence：
(I) amino acid sequence as shown in SEQ ID No.1；
(II) nucleotide sequence is with the same function and as shown in SEQ ID No.1 parasitizes Microplitis (Microplitis) polydnavirus of at least one of the insect of removing Microplitis bicoloratus (M.bicoloratus) In one of nucleotide sequence；
(III) with the consistency of the amino acid sequence as shown in SEQ ID No.1 95% or more, and with such as SEQ ID No.1 Shown in amino acid sequence artificial mutation with the same function amino acid sequence；It is preferred that with as shown in SEQ ID No.1 The consistency of amino acid sequence 98% or more, and with as amino acid sequence shown in SEQ ID No.1 it is with the same function The amino acid sequence of artificial mutation；It is preferred that with the consistency of the amino acid sequence as shown in SEQ ID No.1 99.5% or more, And the amino acid sequence with the amino acid sequence artificial mutation with the same function as shown in SEQ ID No.1.
2. amino acid sequence according to claim 1, which is characterized in that the amino acid sequence I for such as SEQ ID No.1 institute The consistency of the amino acid sequence shown has identical function 98% or more, and with the amino acid sequence as shown in SEQ ID No.1 The amino acid sequence of the artificial mutation of energy.
3. amino acid sequence according to claim 2, which is characterized in that the amino acid sequence I for such as SEQ ID No.1 institute The consistency of the amino acid sequence shown 99.5% or more, and with the amino acid sequence as shown in SEQ ID No.1 have it is identical The amino acid sequence of the artificial mutation of function.
4. a kind of nucleic acid sequence, it includes capable of encoding such as any one of claims 1 to 3 institute for referred to as nucleic acid sequence I The nucleotide sequence for the amino acid sequence stated.
5. nucleic acid sequence according to claim 4, which is characterized in that the nucleic acid sequence I is as shown in SEQ ID No.2 Nucleotide sequence.
6. the application of amino acid sequence as claimed in any of claims 1 to 3.
7. application according to claim 6, which is characterized in that the application is protein kinase B the 308th made in cell The dephosphorylized application in threonine site.
8. application according to claim 7, which is characterized in that the cell is insect cell.
9. the application of nucleic acid sequence according to claim 4 or 5.
10. application according to claim 9, which is characterized in that the application is by as described in claim 4 or 5 Nucleic acid sequence is expressed in cell, is answered so that the protein kinase B in the cell the 308th threonine site is dephosphorylized With；
It is preferred that the cell is insect cell；
It is preferred that nucleic acid sequence as described in claim 4 or 5 is connected on the expression vector that can be used in the cell, and institute Stating expression vector can be such that the nucleic acid sequence is expressed as described in any one of claims 1 to 3 in the cell Amino acid sequence albumen.
CN201810697347.4A 2018-06-29 2018-06-29 Make the dephosphorylized albumen of protein kinase B specific site and its nucleic acid Pending CN108823180A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114703154A (en) * 2022-03-30 2022-07-05 云南大学 Polypeptide, protein containing polypeptide and application of polypeptide

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1082110A (en) * 1992-07-31 1994-02-16 法米塔利亚·卡洛·埃巴有限责任公司 Separation and clone have the protein of tyrosine-phosphatase activity
WO2009069980A2 (en) * 2007-11-29 2009-06-04 Proteogen, Inc Protein chip for determining kinase or phosphatase activity
CN101570557A (en) * 2009-06-09 2009-11-04 中国人民解放军第二军医大学 Compound in cyclocarya paliurus and application thereof in medicament field
CN103525781A (en) * 2013-10-09 2014-01-22 中国科学院微生物研究所 Mycobacterium tuberculosis secretory protein PtpA capable of dephosphorylating p-JNK and p-P38

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1082110A (en) * 1992-07-31 1994-02-16 法米塔利亚·卡洛·埃巴有限责任公司 Separation and clone have the protein of tyrosine-phosphatase activity
WO2009069980A2 (en) * 2007-11-29 2009-06-04 Proteogen, Inc Protein chip for determining kinase or phosphatase activity
CN101570557A (en) * 2009-06-09 2009-11-04 中国人民解放军第二军医大学 Compound in cyclocarya paliurus and application thereof in medicament field
CN103525781A (en) * 2013-10-09 2014-01-22 中国科学院微生物研究所 Mycobacterium tuberculosis secretory protein PtpA capable of dephosphorylating p-JNK and p-P38

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
刘樾彤: "茧蜂病毒蛋白PTP109、Vank86和Vank92对寄主免疫抑制的作用", 《中国优秀硕士学位论文全文数据库 农业科技辑》 *
鲁云霞等: "蛋白酪氨酸磷酸酶1B的研究进展", 《实用糖尿病杂志》 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114703154A (en) * 2022-03-30 2022-07-05 云南大学 Polypeptide, protein containing polypeptide and application of polypeptide
CN114703154B (en) * 2022-03-30 2024-01-09 云南大学 Polypeptide, protein containing same and application

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