CN106349366A - Human tumor suppression protein variant and application thereof - Google Patents
Human tumor suppression protein variant and application thereof Download PDFInfo
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- CN106349366A CN106349366A CN201610998421.7A CN201610998421A CN106349366A CN 106349366 A CN106349366 A CN 106349366A CN 201610998421 A CN201610998421 A CN 201610998421A CN 106349366 A CN106349366 A CN 106349366A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4702—Regulators; Modulating activity
- C07K14/4703—Inhibitors; Suppressors
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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Abstract
The invention provides a human tumor suppression protein variant. Compared with a wild type, the human tumor suppression protein variant has a better effect in inhibiting tumor cell growth. Variant proteins and derivatives of the variant proteins can be adopted to treat various tumors.
Description
Technical field
The invention belongs to biological technical field, specifically, the present invention relates to tumor suppressor protein variant.
Background technology
Kiaa0157 is positioned 10q26.13 (nm_032182.), mrna total length 3008bp, cdna1248bp, encodes 416
Individual aminoacid (sequence 2), molecular weight is the protein of 47kd.The kiaa0157 homology of mice, rat and people up to 90% with
On, illustrate that it is highly conserved during evolution, important biological function may be played.Shown using bioinformatic analysis
Show, there is a coiled coil domain at kiaa0157 active site of protein 215-266 amino acids, this domain is usual
It is present in monomeric protein, and highly conserved, the interaction between usual mediating proteins and albumen.To anti-apoptotic proteins
In the interaction protein screening process of bpe (brca complex subunit 4), kiaa0157 is screened to.This gene and albumen are
Full cell distribution, and substantially lower expression in kinds of tumors tissue.High expression kiaa0157 can suppress including hepatoma carcinoma cell lung
Cancerous cell neuroblastoma cell growth, and lead to cell occur the g0/g1 phase block.
In the prior art, in order to improve the activity of albumen, for albumen avtive spot carry out specific mutation and
Replace, thus finding the higher variant of activity is conventional way.In order to improve the biological activity of this albumen, applicant further
By substantial amounts of experiment, carry out point mutation for site specific in kiaa0157 aminoacid sequence, thus obtaining ratio
Kiaa0157 albumen has the variant of higher inhibitory activity in itself.
Detailed Description Of The Invention
The present invention relates to the detached variant of Parent Protease, it corresponds to the ripe many of seq id no:1 at least one
The position i40h of peptide, h57q, k85p, w94q, q110s, h113g, h141y, n151a, s159p, l160p, k190i, y213i,
v220f、a222y、r232p、v241q、r260p、s267t、d276e、s286l、d298s、d303l、y308k、p313k、
The position of q316s, t319h, h322s, s328a, m331q, r333r, g338a, p359i, g366f, s372i and p383l comprises
Modify.Specifically, the variant of the present invention has the inhibitory activity of improvement.
Preferably, the protein variant of heat stability and inhibitory activity be applied to the method for the present invention, assuming improvement comprises
At least 2 kinds arbitrary following modifications: i40h/a222y, h57q/v220f, k85p/h322s, w94q/s328a, q110s/h113g,
h141y/p383l、n151a/g366f、l160p/p383l、k190i/d298s、k85p/r232p、v241q/s286l、h57q/
r260p、s286l/p383l、w94q/d298s、y213i/d303l、q316s/s372i、q110s/g338a.
The invention still further relates to the method producing the protein variant of the present invention, cultivate host cell including (a);(b) reclaim
Described protein variant.
In the production method of the present invention, it is being suitable for producing the nutrition training of described polypeptide using method well known in the art
Cultured cells in foster base.For example, it is possible to by suitable culture medium and under conditions of permission expression and/or the described polypeptide of separation
Small-scale in the shake-flask culture carrying out, and laboratory or industrial fermentation tank or large scale fermentation (include continuous, in batches, feed supplement
In batches or solid fermentation) carry out cultured cells.Cultivated in suitable Nutrient medium using methods known in the art, institute
State Nutrient medium and comprise carbon source and nitrogen source and inorganic salt.Suitable culture medium can obtain from commercial supplier or can basis
Disclosed composition prepares (for example, in the catalogue of American type culture collection).If polypeptide is secreted into nutrition culture
In base, this polypeptide directly can reclaim from described culture medium.If polypeptide is not secreted, it can be from cell lysate
(lysate) reclaim.
Gained polypeptide can be reclaimed using methods known in the art.For example, polypeptide can be by conventional method from nutrition
Reclaim in culture medium, described conventional method is including but not limited to centrifuged, filters, extracting, being spray-dried, evaporating or precipitation.
The polypeptide of the present invention can be by multiple methods known in the art purification, and methods described includes but is not limited to chromatograph
(for example, ion exchange, affine, hydrophobic, chromatofocusing and size exclusion), electrophoresis method (for example, preparative
(preparative) isoelectrofocusing), differential solubility (for example, ammonium sulfate precipitation), sds-page or extract (see, e.g.,
Protein purification, j.-c.janson and lars ryden compiles, vch publishers, new york, and 1989).
The polypeptide of the present invention is used for preparing corresponding medicine, for treating cancer it is preferred that treating hepatocarcinoma.
Specific embodiment
The acquisition of embodiment 1:kiaa0157 gene
Propose the total rna of human bone marrow cell, carry out reverse transcription by reverse transcription reagent box (promega company) description.Take 1 μ g
Rna after quantitation, the benefit that adds water to 10.5 μ l, 70 DEG C, 10min, add the 10 × buffer of 2 μ l, the mgcl2 of 2 μ l, 2 μ l
Dntp, the oligo dt of 1 μ l, the rrnasin of 1 μ l, the amv (15u) of 1.5 μ l, reaction system totally 20 μ l;42℃、1h;94℃、
5min;4℃、5min.Rna is reverse transcription is cdna.With 5 '-cggaattcaatgtcctacag agagcaggtt-3 ';5’-
Ggggatccttaaatctgggagg tctgagtg-3 ' expands purpose fragment for primer pcr.Pcr condition is:
cycle 1(1x)
94℃ 5min
cycle 2(30x)
94℃ 1min
55℃ 30s
72℃ 1min
cycle 3(1x)
72℃ 10min
Pcr product carries out 1% agarose gel electrophoresiies, and result produces special dna band, by this dna fragment and pmd-
18-t carrier (takara biotech firm) connects, and connection product converts e.coli jm109, and bacterium colony pcr is accredited as positive colony
Afterwards, then it is sequenced after enzyme action identification.Sequence after sequencing is shown in sequence 1.
Embodiment 2: the preparation method of the kiaa0157 after corresponding mutation
1. the introducing of catastrophe point
(1) corresponding mutagenic primer is designed according to corresponding mutational site, using pcr fixed-point mutation method in kiaa0157 base
Because the corresponding amino acid sites of upper wild type are mutated;
(2) above-mentioned pcr product through glue reclaim after purification, carries out enzyme action with restricted enzyme, and through identical enzyme action
After plasmid ppic9k fragment connects, convert escherichia coli dh5 α competent cell;
2. identify recombiant plasmid: with enzyme action, pcr method, recombiant plasmid is identified, and the inspection that is sequenced, thus dashed forward
Nucleotide sequence after change.
Embodiment 3, kiaa0157 variant overexpression suppression cell colony are formed
Have an impact to study kiaa0157 variant cell proliferation, we construct kiaa0157 variant-myc/hisb+
Carrier for expression of eukaryon, and its propagation after kiaa0157 variant overexpression in hepg2 cell be have detected using Colony forming experiment
Situation of change.Result is as follows:
Table 1 kiaa0157 variant overexpression suppresses cell colony formational situation
The expression of embodiment 4 kiaa0157 variant causes the hepg2 cell g0/g1 phase to block
For understanding the impact to hepg2 cell cycle for the kiaa0157 variant, our band gfp marks in hepg2 cell transfecting
The carrier for expression of eukaryon of the kiaa0157 signing, the distribution of each phase of flow cytomery cell cycle.It is found through experiments, with
Transfection kiaa0157 original protein is compared, and after the carrier for expression of eukaryon of transfection kiaa0157 variant, the g0/g1 phase in hepg2 cell
The change of retardance, result is as follows:
The impact level that table 2 kiaa0157 variant blocked for the hepg2 cell g0/g1 phase
Sequence table
<110>Zhu Yupan
A kind of < 120 > human tumor inhibiting protein variant and its application
〈160〉1
〈210〉1
〈211〉416
〈212〉prt
< 213 > ethnic group (homo sapiens.)
〈400〉1
met ala ala ser ile ser gly tyr thr phe ser ala val cys phe his 16
ser ala asn ser asn ala asp his glu gly phe leu leu gly glu val 32
arg gln glu glu thr phe ser ile ser asp ser gln ile ser asn thr 48
glu phe leu gln val ile glu ile his asn his gln pro cys ser lys 64
leu phe ser phe tyr asp tyr ala ser lys val asn glu glu ser leu 80
asp arg ile leu lys asp arg arg lys lys val ile gly trp tyr arg 96
phe arg arg asn thr gln gln gln met ser tyr arg glu gln val leu 112
his lys gln leu thr arg ile leu gly val pro asp leu val phe leu 128
leu phe ser phe ile ser thr ala asn asn ser thr his ala leu glu 144
tyr val leu phe arg pro asn arg arg tyr asn gln arg ile ser leu 160
ala ile pro asn leu gly asn thr ser gln gln glu tyr lys val ser 176
ser val pro asn thr ser gln ser tyr ala lys val ile lys glu his 192
gly thr asp phe phe asp lys asp gly val met lys asp ile arg ala 208
ile tyr gln val tyr asn ala leu gln glu lys val gln ala val cys 224
ala asp val glu lys ser glu arg val val glu ser cys gln ala glu 240
val asn lys leu arg arg gln ile thr gln arg lys asn glu lys glu 256
gln glu arg arg leu gln gln ala val leu ser arg gln met pro ser 272
glu ser leu asp pro ala phe ser pro arg met pro ser ser gly phe 288
ala ala glu gly arg ser thr leu gly asp ala glu ala ser asp pro 304
pro pro pro tyr ser asp phe his pro asn asn gln glu ser thr leu 320
ser his ser arg met glu arg ser val phe met pro arg pro gln ala 336
val gly ser ser asn tyr ala ser thr ser ala gly leu lys tyr pro 352
gly ser gly ala asp leu pro pro pro gln arg ala ala gly asp ser 368
gly glu asp ser asp asp ser asp tyr glu asn leu ile asp pro thr 384
glu pro ser asn ser glu tyr ser his ser lys asp ser arg pro met 400
ala his pro asp glu asp pro arg asn thr gln thr ser gln ile 415
Claims (5)
1. a kind of human tumor inhibiting protein, its aminoacid sequence is shown in seq id no:1.
2. a kind of human tumor inhibiting protein variant is it is characterised in that on the basis of the aminoacid shown in seq id no:1,
Replaced accordingly on y213i/d303l.
3. purposes in preparation suppression tumour medicine for the protein variant as claimed in claim 2.
4. purposes as claimed in claim 3 it is characterised in that: tumor be hepatocarcinoma.
5. a kind of anti-tumor medicinal preparation, it contains protein described in claim 2 and pharmaceutically suitable carrier.
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Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101712958A (en) * | 2008-10-08 | 2010-05-26 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Novel human tumor inhibiting gene KIAA0157 and application thereof |
CN103951733A (en) * | 2014-04-02 | 2014-07-30 | 华国光 | Protein variant capable of inhibiting human tumor and application thereof |
CN103966224A (en) * | 2014-05-12 | 2014-08-06 | 华国光 | Aptamer as well as screening method and application thereof |
-
2016
- 2016-11-14 CN CN201610998421.7A patent/CN106349366A/en active Pending
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101712958A (en) * | 2008-10-08 | 2010-05-26 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Novel human tumor inhibiting gene KIAA0157 and application thereof |
CN103951733A (en) * | 2014-04-02 | 2014-07-30 | 华国光 | Protein variant capable of inhibiting human tumor and application thereof |
CN103966224A (en) * | 2014-05-12 | 2014-08-06 | 华国光 | Aptamer as well as screening method and application thereof |
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