CN104592355A - Anti-tumor peptide variant NC3 and application thereof - Google Patents

Abstract

The invention provides an anti-tumor peptide variant. The protein variant has a stronger effect of inhibiting tumor cell growth in comparison with a wild type protein. The variant proteins and derivatives thereof can be used for treating liver cancer, lung cancer, breast cancer and other tumors.

Description

A kind of natineoplaston variant NC3 and application thereof

related application

the application is number of patent application is 201410312285.2, name is called the divisional application of " a kind of natineoplaston variant and application thereof ".

Technical field

The invention belongs to biological technical field, specifically, the present invention relates to natineoplaston variant.

Background technology

The octapeptide that ND100 is made up of 1Ala, 1Glu, 1Gly, 1Leu, 2Pro, 1Thr, 1Tyr.External and in vivo test all shows that ND100 has the effect of obvious inhibition tumor cell growth, can be used to prepare antitumor drug, is used for the treatment of tumour.

In the prior art, in order to improve the activity of polypeptide, carry out specific change for peptide sequence, thus find the way that active higher variant is routine.In order to improve the biological activity of this polypeptide further, applicant, by a large amount of experiments, has carried out amino acid whose change for peptide sequence, thus has obtained the variant than original polypeptide itself with higher inhibit activities.

Summary of the invention

The polypeptide that the present invention relates to, sequence is as follows:

NC： AlaGluGlyLeuProProThrTyr；

（NC1）：AlaGluGlyLeuProProThrIle；

（NC2）：AlaSerGlyLeuProProThrTyr；

（NC3）：AlaGluGlyLeuProSerThrTyr；

（NC4）：AlaGluGlyGlyLeuProProThrTyr；

（NC5）：AlaGluGlyGlyLeuProProProThrTyr；

（NC 6）：GlyAlaGluGlyLeuProProProThrTyr；

（NC7）：AlaGlyGlyLeuProProThrTyr；

（NC8）：GlyAlaGlyGlyLeuProProThrTyr；

（NC 9）：AlaGlyGlyLeuProSerThrTyr；

（NC 10）：GlyAlaGlyGlyLeuProSerThrTyr；

（NC 11）：GlyAlaGlyGlyLeuProProProThrTyr；

（NC 12）：AlaGlyGlyLeuProProProThrTyr；

（NC 13）：AlaGlyGlyLeuProProProThrIle；

Contrast 1:AlaGluGlyLeuProGlyThrTyr;

Contrast 2:AlaLeuGlyLeuProProThrTyr;

After Peptide systhesis of the present invention, free state form can be made, also can make cationic salts, as: tfa salt (trifluoroacetate), HCl salt (hydrochloride), acetate form.

Polypeptide of the present invention, can select to combine with one or more pharmaceutical carriers, make multi-medicament formulation, be used for the treatment of.These pharmaceutical dosage forms are contained: formulation local or the Formulations for systemic administration such as injection solution, tablet, creme, capsule, ointment, lotion, tongue lozenge.

Embodiment

Embodiment 1: the artificial chemistry synthesis of polypeptide of the present invention

The preparation of peptide of the present invention adopts liquid phase synthesizing method and Acibenzolar growth method one by one; second that the C of peptide of the present invention is held amino acid whose amino Boc base (tertiary butyloxycarbonyl acyl group) protection; carboxyl HOSu (N-hydroxy-succinamide) activation, then at KHCO ₃hold with C first amino acid 30 DEG C of water-baths in solution, react 2 days.Pump organic solvent, in ice bath, adjust pH3 ~ 4 (when precipitating maximum) with 2mol/LHCl, use ethyl acetate extracting, saturated NaCl washes 3 times, anhydrous Na ₂sO ₄drying, drains, and anhydrous diethyl ether grinds, must with the dipeptides of protecting group.Process with 50%TFA (trifluoracetic acid); slough Boc base; again with N end band Boc protecting group; C end the 3rd amino acid condensation of the C end of HOSu activation, uses the same method and connects amino acid one by one, finally slough the protecting group on Glu side chain with catalytic hydrogenation; the Boc base of peptide N of the present invention end is sloughed with 50%TFA; obtain thick peptide, thick peptide HPLC carries out separation and purification, can obtain the polypeptide of the present invention of 98% purity.Through electrospray ionization mass spectrum measure, its sequence and target sequence completely the same.

The anti-tumor activity of embodiment 2:ND100

(1) active determination in vitro:

Adopt cell cultures and Cytometric method, human liver cancer cell 7721, stomach cancer cell MKN, lung cell A549 are with substratum conventional separately, the calf serum of about 10% is added depending on different situations, with T-25 culturing bottle at 37 DEG C, monolayer culture in 5%CO2 incubator.

Digest after Growth of Cells becomes individual layer, make cell suspension with the nutrient solution containing 1% calf serum, be inoculated on 24 well culture plates by every hole 1ml, 37 DEG C, 5%CO ₂after cultivating 4h in incubator, add the polypeptide of the application of various dose, control group and experimental group all establish three repeating holes, and control group adds the PBS of 10 μ l, test group adds the polypeptide of the application of various dose, continue cultivation after 72 hours, sucking-off nutrient solution, washes with PBS, then every hole adds 200 μ l Digestive system digestion, after cell rounding, add the PBS of 800 μ l, count with blood counting chamber under inverted microscope.

Inhibiting rate result is as follows:

Polypeptide (dosage 1mg/ml)	Human liver cancer cell 7721	Stomach cancer cell MKN	Lung cell A549
				NC	85.2%	74%	70%
NC1	97%	92%	90%
				NC2	98%	94%	91%
NC3	98%	93%	92%
				NC4	99%	92%	91%
NC5	98%	95%	93%
				NC6	98%	94%	92%
NC7	98%	93%	91%
				NC8	97%	93%	93%
NC9	98%	94%	94%
				NC10	98%	92%	93%
NC11	97%	96%	92%
				NC12	99%	95%	92%
NC13	98%	91%	93%
				Contrast 1	35%	19%	11%
Contrast 2	29%	20%	19%

(2) activity in vivo measures:

30 healthy Kun Ming mice, body weight 20 ± 2 grams, average mark three groups, often organizes 10, male and female half and half.To inoculate the tumor-bearing mice execution of human liver cancer cell 7721, stomach cancer cell MKN, lung cell A549, its ascites is extracted in aseptic technique, by 1: 3 dilution proportion, is mixed with cell suspension, in the right fore armpit subcutaneous injection 0.2ml tumor cell suspension of every laboratory mice, oncocyte is no less than 10 ⁵, after planting knurl 24hr, administration group carries out intravenous injection with various dose respectively, and using physiological saline as negative control group, CTX is positive controls, and continuously injection is after 7 days, puts to death mouse, take subcutaneous tumors block, claim knurl weight, calculate tumour inhibiting rate when drug withdrawal 3 days.For lung cancer, after planting knurl 24hr, after administration group, physiological saline and CTX group sample inject 10 days continuously, when drug withdrawal 4 days, put to death mouse, take subcutaneous tumors block, claim knurl weight, calculate tumour inhibiting rate.

Detected result shows, and the tumour inhibiting rate of the NC of 15mg/kg dosage is respectively: liver cancer 77%, lung cancer 78%, cancer of the stomach 75%.And the tumour inhibiting rate of three of NC1-13 kinds of cancers is close, be approximately respectively liver cancer 99%, lung cancer 98%, cancer of the stomach 96%.And contrast 1 and contrast 2 almost there is no tumour inhibiting rate.

As can be seen from the above results, NC1-13 can as medicine for the preparation of antitumour drug.There is good application prospect.

Sequence table

<110> horse sea otter

<120> natineoplaston variant NC3 and application thereof

<160> 16

<210> 1

<211> 8

<212> PRT

<213> artificial sequence

<400> 1

AlaGluGlyLeuProProThrTyr

<210> 2

<211> 8

<212> PRT

<213> artificial sequence

<400> 2

AlaGluGlyLeuProProThr Ile

<210> 3

<211> 8

<212> PRT

<213> artificial sequence

<400> 3

Ala SerGlyLeuProProThrTy

<210> 4

<211> 8

<212> PRT

<213> artificial sequence

<400> 4

AlaGluGlyLeuPro SerThrTyr

 

<210> 5

<211> 9

<212> PRT

<213> artificial sequence

<400> 5

AlaGluGly GlyLeuProProThrTyr

 

<210> 6

<211> 10

<212> PRT

<213> artificial sequence

<400> 6

AlaGluGly GlyLeuProPro ProThrTyr

 

<210> 7

<211> 10

<212> PRT

<213> artificial sequence

<400> 7

GlyAlaGluGlyLeuProPro ProThrTyr

<210> 8

<211> 27

<212> PRT

<213> artificial sequence

<400> 8

Ala GlyGlyLeuProProThrTyr

 

<210> 9

<211> 9

<212> PRT

<213> artificial sequence

<400> 9

GlyAla GlyGlyLeuProProThrTyr；

<210> 10

<211> 8

<212> PRT

<213> artificial sequence

<400> 10

Ala GlyGlyLeuPro SerThrTyr

 

<210> 11

<211> 9

<212> PRT

<213> artificial sequence

<400> 11

GlyAla GlyGlyLeuPro SerThrTyr；

<210> 12

<211> 10

<212> PRT

<213> artificial sequence

<400> 12

GlyAla GlyGlyLeuProPro ProThrTyr；

<210> 13

<211> 9

<212> PRT

<213> artificial sequence

<400> 13

Ala GlyGlyLeuProPro ProThrTyr；

<210> 14

<211> 9

<212> PRT

<213> artificial sequence

<400> 14

Ala GlyGlyLeuProPro ProThr Ile；

<210> 15

<211> 8

<212> PRT

<213> artificial sequence

<400> 15

AlaGluGlyLeuPro GlyThrTyr

<210> 16

<211> 8

<212> PRT

<213> artificial sequence

<400> 16

Ala LeuGlyLeuProProThrTyr；

 

 

 

Claims (4)

1. a natineoplaston, its aminoacid sequence is SEQ ID NO:1, shown in 4.

2. tumour peptide variant as claimed in claim 1 is preparing the purposes in Tumor suppression medicine.

3. purposes as claimed in claim 2, is characterized in that: described tumour is liver cancer, cancer of the stomach or lung cancer.

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