CN105461798A - Tumor arrestin variant DV92 and application thereof - Google Patents

Abstract

The invention provides a variant of tumor arrestin. The variant of tumor arrestin has the higher effect of restraining tumor cell growth relative to a wild arrestin variant. Variant protein and derivatives thereof can be used for treating multiple tumors.

Description

A kind of tumor suppressor protein modification D V92 and application thereof

Technical field

The invention belongs to biological technical field, specifically, the present invention relates to tumor suppressor protein variant.

Background technology

Tumor correlated albumen DENND2D-v2, be made up of 468 amino-acid residues, be uDENN structural domain from N-terminal the 47 to the 145 amino acids residue of sequence 4, being DENN structural domain from N-terminal the 146 to the 330 amino acids residue of sequence 4, is dDENN structural domain from N-terminal the 368 to the 435 amino acids residue of sequence 4.This tumor correlated albumen stablizes the vicious transformation that high expression level obviously can suppress this cell in l cell; Detect its expression of results in clone in mRNA level in-site to show, the expression level in lung cancer cell line is significantly lower than the normal bronchial epithelial cell of original cuiture; In lung cancer clinical tissue sample, detect its expression level result show, the expression level of cancerous lung tissue is starkly lower than the healthy tissues of its periphery.This tumor correlated albumen can be used for in-vitro diagnosis, the prognosis of tumour.Recombinant eukaryon expression vector containing above-mentioned tumor correlated albumen encoding gene in importing lung cancer cell line after, the growth of lung cancer cell line can be caused separately to slow down and tumorigenicity weakens.Therefore, this albumen has good tumour medicine application prospect.

In the prior art, in order to improve the activity of albumen, the avtive spot for albumen carries out specific sudden change and replacement, thus finds the way that active higher variant is routine.In order to improve the biological activity of this albumen further, applicant, by a large amount of experiments, has carried out point mutation for site specific in DENND2D-v2 aminoacid sequence, thus has obtained the variant than DENND2D-v2 albumen itself with higher inhibit activities.

Detailed Description Of The Invention

The present invention relates to the variant of the separation of Parent Protease, its position corresponding to position 53Y, 60K, 70P, 79R, 92R, 97I, 109A, 131K, 141A, 204S, 225E, 232L, 253K, 270A, 271A, 297C, 305G, 322V, 347L, 379F, 398Q, 433Q or 451L of the mature polypeptide of SEQIDNO:1 at least one comprises modification.Specifically, variant of the present invention has the inhibit activities of improvement.

Preferably, be applied to method of the present invention, present the protein variant of the inhibit activities of improvement and comprise at least a kind of arbitrary following modification: 53Y/F, 60K/C, 70P/V, 79R/K, 92R/K, 97I/F, 109A/S, 131K/R, 141A/P, 204S/T, 225E/A, 232L/S, 253K/V, 270A/R, 271A/S, 297C/R, 305G/S, 322V/L, 347L/R, 379F/S, 398Q/R, 433Q/S, 451L/V.

The invention still further relates to the method producing protein variant of the present invention, comprise (a) and cultivate host cell; (b) described protein variant is reclaimed.

In production method of the present invention, use method well known in the art culturing cell in the nutritional medium being suitable for producing described polypeptide.Such as; can by suitable culture medium and allow expression and/or the shake-flask culture that carries out under being separated the condition of described polypeptide, and small-scale in laboratory or industrial fermentation tank or large scale fermentation (comprise continuously, in batches, fed-batch or solid state fermentation) carry out culturing cell.Use methods known in the art to cultivate in suitable nutritional medium, described nutritional medium comprises Carbon and nitrogen sources and inorganic salt.Suitable substratum can obtain from commercial supplier or can according to disclosed composition preparation (such as, in the catalogue of American type culture collection).If polypeptide is secreted in nutritional medium, this polypeptide directly can reclaim from described substratum.If polypeptide is not secreted, it can reclaim from cell lysate (lysate).

Gained polypeptide can use methods known in the art to reclaim.Such as, polypeptide can be reclaimed from nutritional medium by ordinary method, and described ordinary method includes but not limited to centrifugal, filtration, extraction, spraying dry, evaporation or precipitation.

Polypeptide of the present invention can by multiple methods known in the art purifying, described method includes but not limited to that chromatography (such as, ion-exchange, affine, hydrophobic, chromatofocusing and size exclusion), electrophoresis method (such as, preparative (preparative) isoelectrofocusing), differential solubility (such as, ammonium sulfate precipitation), SDS-PAGE or extraction (see, such as, ProteinPurification, J.-C.Janson and LarsRyden compiles, VCHPublishers, NewYork, 1989).

Polypeptide of the present invention, for the preparation of corresponding medicine, goes to be used for the treatment of cancer.

Embodiment

The acquisition of embodiment 1:DENND2D-v2 gene

DENND2D-v2：

Upstream primer 5 ' CACTCCAGGGGCCATGGATG3 '

Downstream primer 5 ' GTCATTCTTATTCACCACAGCTC3 '

Use above-mentioned primer, with people normal lung tissue cDNA library (Clontech:K1420-1) for template carries out pcr amplification reaction, reaction conditions is as follows:

Reaction volume 50 μ l, wherein contains:

50 μ l volumes are complemented to distilled water.

Temperature of reaction, time: 94 DEG C, sex change 5 minutes; Then 94 DEG C of sex change 30 seconds, 57 DEG C of annealing 30 seconds, 72 DEG C extend 1 minute, 30 circulations of increasing; Finally 72 DEG C of downward-extensions 10 minutes.

Amplified production is 3 ' with 3 ' the outstanding cohesive end fragment of base A, test kit (Qiagen is reclaimed with QIAquick glue, 28706) purifying is carried out by product description, then the linear pGEM-TEASY carrier (Promega of base T is had with 3 ', A1360) at 16 DEG C, 8 hours are connected, use 2mm pole cup, 2500V transformation of E. coli DH5 α, conversion product is growing containing on the LB plate culture medium of penbritin, selected clone, extract plasmid, use AbIPRISM3700DNA analyser (Perkin-Elmer/AppliedBiosystem) order-checking, obtain the full-length cDNA of DENND2D-v2 gene, length is 1905bp, its encoding sequence is 5 ' end the 57 to 1463 deoxyribonucleotide from sequence, to encode 468 amino acid.

Embodiment 2: the preparation method of the DENND2D-v2 gene after corresponding sudden change

1. the introducing of catastrophe point

(1) design corresponding mutagenic primer according to corresponding mutational site, adopt PCR fixed-point mutation method on DENND2D-v2 gene, amino acid sites corresponding for wild-type to be suddenlyd change;

(2) above-mentioned PCR primer reclaims after purifying through glue, carries out enzyme cut with restriction enzyme, after being connected with the pPIC9k fragment of cutting through same enzyme, and transformation of E. coli DH5 α competent cell;

2. identify recombinant plasmid: with enzyme cut, PCR method identifies recombinant plasmid, and order-checking inspection, obtains the nucleotide sequence after suddenling change thus.

Embodiment 3, DENND2D-v2 variant are to the effect of lung cancer cell line H1299 growth-inhibiting

After nucleotide sequence after the sudden change obtain embodiment 2 reclaims, directly forward is connected into pcDNA3.1/V5-HisTOPOTA (Invitrogen, K4800) carrier for expression of eukaryon, through two-way order-checking and enzyme cut qualification errorless, obtain the cDNA gene plasmid of the nucleotide sequence after forward insertion mutation, called after pcDNA3.1/V5-HisTOPOTA-DENND2D-v2, is called for short pcDB3.1-DENND2D-v2.

1, apoptotic observation and detection:

(1) structure of positive control plasmid pcDB3.1-BAX plasmid

PcDB3.1-BAX is the positive control of apoptosis experiment, build by the following method: with people normal lung tissue cDNA library (Clontech:K1420-1) for template, utilize Taq enzyme to carry out the full-length cDNA gene (GenbankNo.NM_138761) of PCR reaction amplification BAX.Agarose electrophoresis reclaims PCR primer, be connected in pGEM-T-easy carrier and check order, the correct fragment restriction enzyme EcoRI (Promega) of order-checking is cut from pGEM-T-Bax carrier, cut eukaryotic expression vector pcDNA3.1/mycHis (-) B (Invitrogen with EcoRI enzyme simultaneously, V85520), the cDNA gene fragment of the Bax after being cut by enzyme is connected 8 hours with carrier at 16 DEG C, transformation of E. coli DH5 α, conversion product is growing containing on the LB plate culture medium of penbritin, select the bacterium colony of growth, extract plasmid, cut with EcoRI enzyme, digestion products agarose gel electrophoresis is identified, select the positive colony of Insert Fragment, by order-checking (using ABIPRISM3700DNA analyser), select the cDNA gene plasmid that correct forward inserts Bax, called after pcDB3.1-BAX.PcDB3.1-BAX is without c-myc and his label.

(2) transient transfection H1299 cell: H1299 cell is imported in six orifice plates with 1 × 105,24 hours after cell attachment growth, according to the working method that Lipofectamine2000 specification sheets provides, by pcDNA3.1/mycHis (-) B, pcDB3.1-DENND2D-v2 variant, pcDB3.1-BAX plasmid is each separately transfected into each porocyte, and the amount of plasmid DNA used and transfection reagent is 1/2 of recommended amounts to reduce cytotoxicity.4 hours replaced medium after transfection, collect cell in latter 36 hours to be transfected.Illustrate according to AnnexinVFITCApoptosisDetectionKit (CALBIOCHEM, PF032) and carry out the two dye of AnnexinVFITC and PI, flow cytomery.Result is as shown in the table.

2, the observation of cell cycle and detection:

PI flow cytometry carries out cell cycle detection: by empty carrier H1299-vector cell, the H1299-DENND2D-v2 variant monoclonal cell stablizing high expression level DENND2D-v2 variant imports in 60mm Tissue Culture Dish with 2 × 105, and 37 DEG C of 5%CO2 are cultured to fusion rate and reach about 80%.Cell is collected in digestion, and 80% ethanol (preparing with PBS)-20 DEG C fixedly spends the night (being greater than 8 hours).After PI dyeing, upper machine testing.As shown in the table, H1299 can be significantly arrested in the G1/S phase by the exogenous high expression level of DENND2D-v2 variant.

3, soft-fractrue rock mass experiment: adopt empty carrier H1299-vector cell, H1299-DENND2D-v2 wild-type cell, the H1299-DENND2D-v2 variant monoclonal cell strain screening the stably express DENND2D-v2 variant obtained is tested.Experiment is carried out with six orifice plates, every hole first adds 1.5 milliliters of DMEM substratum containing 10% foetal calf serum, 0.5% agar, after room temperature cooling, by the stably transfected cell line built up, each 5000 cells are added in the DMEM substratum containing 10% foetal calf serum, 0.35% agarose, be added on above-mentioned bottom substratum after mixing, cultivate 2-3 week, every ware adds 0.5ml0.2%P-iodonitrontetraziliumchiloride and dyes more than 1 hour; Microscopic observation is taken a picture, the clone that counting is greater than 200 microns.Often organize repetition three holes.Bore hole and light Microscopic observation, three groups of parallel laboratory test Colony forming result numbers are compared as follows shown in table.

Above embodiment illustrates, DENND2D-v2 genetic mutation can be apoptosis-induced, has clear and definite cell-cycle arrest ability in the cell cycle G1/S phase, and this is the major cause of its cell growth inhibiting multiplication capacity.Proceed to the more non-mutain of the ability of independent growths in soft agar of the cell after DENND2D-v2 variant obviously to weaken simultaneously.

Sequence table

<110> Ma Hengbiao

< 120 > tumor suppressor protein modification D V92 and application thereof

〈160〉1

〈210〉1

〈211〉468

〈212〉PRT

< 213 > ethnic group (HomoSapiens.)

〈400〉1

1MDGLGRRLRASLRLKRGHGGPPQDNSGEAL30KEPERAQEHSLPNFAGGQHFFEYLLVVSLK

61KKRSEDDYEPIITYQFPKRENLLRGQQEEE90ERLLKAIPLFCFPDGNEWASLTEYPRETFS

121FVLTNVDGSRKIGYCRRLLPAGPGPRLPKV150YCIISCIGCFGLFSKILDEVEKRHQISMAV

181IHPFMQGLREAAFPAPGKTVTLKSFIPDSG210TEFISLTRPLDSHLEHVDFSSLLHCLSFEQ

241ILQIFASAVLERKIIFLAEGLSTLSQCIHA270AAALLYPFSWAHTYIPVVPESLLATVCCPT

301PFMVGVQMRFQQEVMDSPMEEVLLVNLCEG330TFLMSVGDEKDILPPKLQDDILDSLGQGIN

361ELKTAEQINEHVSGPFVQFFVKIVGHYASY390IKREANGQGHFQERSFCKALTSKTNRRFVK

421KFVKTQLFSLFIQEAEKSKNPPAGYFQQKI450LEYEEQKKQKKPREKTVK

Claims (4)

1. a tumor suppressor protein, its aminoacid sequence is for shown in SEQIDNO:1.

2. a human tumor inhibiting protein variant, is characterized in that, on the amino acid whose basis shown in SEQIDNO:1, replaces accordingly at 92R/K.

3. protein variant as claimed in claim 2 suppresses the purposes in lung-cancer medicament in preparation.

4. an anti-tumor medicinal preparation, it contains protein variant according to claim 2 and pharmaceutically suitable carrier.