CN105418742B - 1 function region variants JG135 of ray Angiostatin and its application - Google Patents

Abstract

The invention discloses a kind of 1 functional areas albumen of ray Angiostatin and its variants, and its application in the drug that preparation inhibits tumour growth.Make protein variant of the invention by eukaryotic expression, the inhibitory effect of cancer cell can be significantly improved relative to original protein.

Description

1 function region variants JG135 of ray Angiostatin and its application

Technical field

The invention belongs to field of biotechnology, specifically, the present invention relates to the changes of 1 functional areas of ray Angiostatin Body.

Background technique

1 functional areas of ray Angiostatin hereinafter referred to as ray functional areas, ray Angiostatin 1 are from the South Sea The protein (molecular weight 42KD) separated in a kind of ray tissue in sea area.Result of study is shown: ray Angiostatin 1 Significantly inhibit chick chorioallantoic membrane itself and KB cell's induction chick chorioallantoic membrane angiogenesis；Either Intraperitoneal injection or stomach-filling all significantly inhibit the growth and transfer of nude mouse Lewis lung cancer, reduce tumor tissues microvessel density, Lower the expression of angiogenic factors VEGF；Also there is high inhibition effect to the transfer of nude mouse Melanoma B16 cell；It lowers Promote the expression of transfer factor CD44v6 and ErBb2；It is more significant with effect when 5 FU 5 fluorouracil (5-FU) use in conjunction.Ray blood vessel is raw It is different from the anti-cancer drugs Avastin that gene technology research institute of the U.S. develops at the action target spot of inhibiting factor 1.Avastin It is gene engineering product, is the antibody of VEGF, action target spot is VEGF；Ray Angiostatin 1 is natural products, it VEGF is acted not only on, bFGF and PDGF are also acted on.

In the prior art, in order to improve the activity of albumen, for albumen specific site carry out specificity mutation and Replace, so that finding the higher variant of activity is conventional way.In order to further increase the bioactivity of the albumen, applicant Through a large number of experiments, a little dash forward for site specific in 1 function region amino acid sequence of ray Angiostatin Become, to obtain the variant that there is higher inhibitory activity than 1 functional areas of ray Angiostatin itself.

Summary of the invention

The present invention relates to the isolated variants of Parent Protease, in the functional areas that at least one corresponds to SEQ ID NO:1 Position 9R, 17F, 42H, 57S, 62R, 71K, 80N, 89P, 100G, 101R, 129A, 134E, 151Q, 163P, 189K or 217R Position include modification.Specifically, variant of the invention has improved inhibitory activity.

Preferably, the protein variant of the inhibitory activity improved applied to method of the invention, presentation is following any modification: 9R/G、17F/S、42H/D、57S/L、62R/V、71K/R、80N/W、89P/V、100G/E、101R/C、129A/G、134E/S、 151Q/N, 163P/K, 189K/R or 217R/ T.

The invention further relates to the method for generating protein variant of the invention, include (a) cultivating host cell；(b) it recycles The protein variant.

In production method of the invention, trained using method well known in the art in the nutrition for being suitable for generating the polypeptide It supports in base and cultivates cell.For example, can be by suitable culture medium and under conditions of allowing to express and/or separate the polypeptide In the shaking flask culture of progress and laboratory or industrial fermentation tank small-scale or large scale fermentation (including it is continuous, in batches, feed supplement In batches or solid state fermentation) cultivate cell.It is cultivated in suitable nutrient medium using methods known in the art, institute Stating nutrient medium includes carbon source and nitrogen source and inorganic salts.Suitable culture medium can obtain from commercial supplier or can basis Disclosed composition prepares (for example, in catalogue of American type culture collection).If polypeptide is secreted into nutrition culture In base, which can directly recycle from the culture medium.It, can be from cell lysate if polypeptide is not secreted (lysate) it recycles.

Methods known in the art recycling can be used in gained polypeptide.For example, polypeptide can be by conventional method from nutrition It is recycled in culture medium, the conventional method includes but is not limited to centrifugation, filtering, extraction, spray drying, evaporation or precipitates.

Polypeptide of the invention can be purified by a variety of methods known in the art, the method includes but be not limited to chromatograph (for example, ion exchange, affine, hydrophobic, chromatofocusing and size exclusion), electrophoresis method are (for example, preparative (preparative) isoelectric focusing), differential solubility (for example, ammonium sulfate precipitation), SDS-PAGE or extract (see, e.g., Protein Purification, J.-C.Janson and Lars Ryden is compiled, VCH Publishers, New York, and 1989).

Polypeptide of the invention is used to prepare corresponding drug, goes for inhibiting cancer.

Specific embodiment

Embodiment 1

1. the acquisition of 1 functional areas of ray Angiostatin

Ray functional areas are made of in the following order following amino acid residue:

TLD1YKQLRDKETPSGFTLDDV1QTGVDNPGHPF1MTVGCVAGDEESYEVFKALFDPV1QDRHGGYKPTDKHKTDLN HENLKGGDDLDPNYVLSSRVRTGRS1KG1ALPPHCSRGERRLVEKLCLEGLATLTGEFQGKYYPLTTMSDAEQQQL1 DDHFLFDKPVSPLLLASGMARDWPDARG1WHNNDKTFLVWVNEEDHLRV1SMQKGGNMKEVFRRFCVGLKK

2, the building of 1 function region variants of ray Angiostatin

The introducing of catastrophe point

(1), corresponding mutagenic primer is designed according to corresponding mutational site, it is raw in ray blood vessel using PCR fixed-point mutation method The corresponding amino acid sites of wild type are mutated on gene corresponding to 1 functional areas of inhibiting factor；

(2), above-mentioned PCR product after purification by gel, carries out digestion with restriction enzyme, digestion identical as process After the connection of plasmid pmD-18T carrier segments, bacillus coli DH 5 alpha competent cell is converted；

(3), it identifies recombinant plasmid: recombinant plasmid being identified with digestion, PCR method, and inspection is sequenced, is thus obtained Nucleotide sequence after mutation.These nucleotide sequences say corresponding amino acid sequence respectively on the basis of SEQ ID NO:1 On, 9R/G, 17F/S, 42H/D, 57S/L, 62R/V, 71K/R, 80N/W, 89P/V, 100G/E, 101R/C, 129A/G, 134E/S, 151Q/N, 163P/K, 189K/R or 217R/ T location have carried out corresponding mutation.

(4), the expression of 1 function region variants of pPIC6 α-ray Angiostatin

The both ends for the 1 functional areas variant gene of ray Angiostatin that above-mentioned steps building obtains are introduced into digestion position Signal peptide sequence is added in upstream in point, and building obtains 1 functional areas variant vector of pPIC6 α-ray Angiostatin, identifies Afterwards, by plasmid transfection Pichia pastoris, and eukaryotic expression is carried out, using Blasticidin as selection markers.Induce it Secreting, expressing destination protein, discovery methanol concentration are that 0.5%, 4 days obtained expressing quantities of continuous induction are higher.Through SDS- The protein band that PAGE is separated by electrophoresis can react in Westernblot detection with the antiserum of rabbit-anti hCG.Experiment The result shows that recombinant plasmid pPIC6 α -1 function region variants of ray Angiostatin that we construct finish red ferment in Pasteur It is about 140 mg/ L that average expression quantity, which is expression quantity, in mother.

3,1 function region variants of ray Angiostatin inhibit the growth and transfer of tumour

BALB/c nude mice is randomly divided into control group, the positive (cyclophosphamide) control group and experimental group, and every group 8, male and female are each Half.Every mouse dorsal sc is inoculated with 0.2ml Lewis lung carcinoma cell suspension (4 × 106 cell) Lewis lung carcinoma cell.Tumor inoculation The 7th day afterwards, various dose ray functional areas solution, 1 time a day, totally 14 times was injected intraperitoneally in experimental group；Equivalent is injected intraperitoneally in control group Solvent；Positive controls intraperitoneal injection of cyclophosphamide, 1 times a week, totally 2 times.21st day sacrifice mouse, removing tumour weighing (are used 10% neutral formalin is fixed for immunohistochemical experiment), it dissects, observe and record hepatic metastases nodule number.It is calculated as follows Inhibition rate of tumor growth and metastases inhibiting rate:

Concrete outcome is as follows:

4, the chick chorioallantoic membrane angiogenesis test of nasopharyngeal carcinoma cell (CNE-2Z) induction

Cleaning, uniform kind of eggshell homogeneous, gas chamber egg are selected, is hatched 1 week in 19200 type intelligence incubators, it is sterile Under the conditions of in embryo head end open a diameter 1cm aperture, form false gas chamber.It is inoculated with CNE-2Z cell (1.9 × 106/ embryo), uses transparent adhesive tape Band sealing.It sets after being cultivated 4 days in incubator, the filter paper of an aperture is played into center and is placed on the indoor chorioallantoic membrane of gas, respectively plus ray In on filter paper, control group then adds equivalent solvent by functional areas albumen and its 100 μ l of misfolded proteins medical fluid, 1 time a day, is administered 4 times altogether. Then remove adhesive tape, fix 12 minutes respectively with acetone and dehydrated alcohol.It cuts and is placed with the chorioallantoic membrane of filter paper and sets glass slide On, filter paper is abandoned or adopted, the branch that 5 visuals field counting visible vessels are randomly selected under microscope counts and takes a picture.Blood is calculated as follows Pipe generates inductivity and Agiogenesis inhibition rate:

Group	Chicken embryo number (only)	Dosage (μ g/ * days)	Vessel branch counts (X pulls out+S)	Inhibiting rate
					Control group	5	—	83.5	—
SEQ ID NO:1 albumen	5	100	42.2	49.5%
					JG1 (9R/G)	5	100	34.0	59.3%
JG2 (9R/W)	5	100	71.2	14.7%
					JG16 (17F/S)	5	100	28.3	66.1%
JG20 (42H/R)	5	100	83.5	0.0%
					JG23 (42H/D)	5	100	34.0	59.3%
JG30 (57S/P)	5	100	68.1	18.4%
					JG36 (57S/L)	5	100	28.2	66.2%
JG42 (62R/V)	5	100	39.4	52.8%
					JG50 (71K/R)	5	100	36.1	56.8%
JG55 (80N/W)	5	100	29.7	64.4%
					JG60 (89P/V)	5	100	34.3	58.9%
JG63 (100G/E)	5	100	35.3	57.7%
					JG70 (101R/C)	5	100	37.3	55.3%
JG80 (129A/S)	5	100	63.2	24.3%
					JG89 (129A/G)	5	100	38.1	54.4%
JG121 (134E/S)	5	100	26.7	68.0%
					JG135 (151Q/N)	5	100	36.0	56.9%
JG164 (163P/K)	5	100	25.0	70.1%
					JG175 (189K/R)	5	100	37.1	55.6%
JG196 (134E/S and 80N/W)	5	100	27.8	66.7%
					JG217 (217R/ T)	5	100	38.6	53.8%

From 3 and 4 experimental result can be seen that 9R/G, 17F/S, 42H/D, 57S/L, 62R/V, 71K/R, 80N/W, The variant of 89P/V, 100G/E, 101R/C, 129A/G, 134E/S, 151Q/N, 163P/K, 189K/R or 217R/ T both with respect to Original ray functional areas albumen is all significantly improved in cancer inhibition rate and vascular study rate, therefore achieves very well Effect.

Toxicological experiment

Acute toxicity test

Using healthy Kunming mouse, 19~22 grams of weight, half male and half female.Fasting 4h before administration (po), use are interior for 24 hours more Secondary dose regimen takes the circumstances into consideration supply feed therebetween, can't help water.Dosage is the effective agent of ray functional areas original series and variant sequence thereof 100 times of (1p, 20mg/kg of amount；Po, 40mg/kg), it is observed 7 days after administration.Day by day the toxic reaction of animal is recorded during observation The distribution of situation and dead animal.Because not finding dead mouse, LD50 is not measured, changes then and does " mtd test ", one 300 times of (1p, 60mg/kg of secondary property ray functional areas and its effective dose of variant；Po, 120mg/kg) 0.5ml and 0.8ml points Not Gei the injection of 20 mouse peritoneals or stomach-filling, observe 14 days, as a result discovery mouse toxicity reaction or dead yet.

Stability

Ray functional areas and its variant sample are made using Freeze Drying Technique, are saved 12 months in -20 DEG C of refrigerators, are given birth to Object activity (inhibits angiogenesis, inhibits in vitro culture oncocyte and mice transplanted tumor growth) nothing to be substantially reduced.

Ray functional areas and its variant can be mixed or be dissolved with pharmaceutical carrier, and the drug of the various tumours for the treatment of, packet is made Include various dosage forms, which can be used for leukaemia, also can be used for various entity tumors, as cancer of the esophagus, liver cancer, gastric cancer, cancer of pancreas, Colon and rectum carcinoma, cervical carcinoma, oophoroma, breast cancer, nasopharyngeal carcinoma, carcinoma of mouth, lip cancer, cutaneum carcinoma, bladder cancer, on chorion The treatment of skin cancer, carcinoma of parotid gland, lymthoma, myomata, melanoma and various intracranial tumors etc., and its in the people for needing this treatment In application.In made various combination of oral medication, the content of ray functional areas and its variant is that daily every Kg weight is big About 0.001-100mg, preferably from about 0.01-50mg, more excellent about 0.05-30mg, most preferably about 0.1-10mg.Make for every bu 2-4 times With, 10-30 days as a treatment course, 3-6 course for the treatment of of general medication, each course for the treatment of interval 10-30 days.Made injectable drug group Close object, for intramuscular injection or intravenous drip, wherein the content of ray functional areas and its variant be daily every Kg weight about 0.001-50mg, preferably from about 0.01-25mg, more excellent about 0.05-15mg, most preferably about 0.1-3mg.Every bu 1-3 times use, 10-30 days as a treatment course, 3-6 course for the treatment of of general medication, each course for the treatment of interval 10-30 days.However, accurate dosage should be by doctor It determines, depends primarily on age, weight, the state of an illness, reaction of patient etc..

Sequence table

110 > of < brave

120 > ray Anti-angiogenesis factors of <, 1 function region variants JG135 and its application

〈160〉1

〈210〉1

〈211〉225

〈212〉PRT

213 > ray of <

〈400〉1

TLDIYKQLRD KETPSGFTLD DVIQTGVDNP GHPFIMTVGC VAGDEESYEV FKALFDPVIQ 60

DRHGGYKPTD KHKTDLNHEN LKGGDDLDPN YVLSSRVRTG RSIKGIALPP HCSRGERRLV 120

EKLCLEGLAT LTGEFQGKYY PLTTMSDAEQ QQLIDDHFLF DKPVSPLLLA SGMARDWPDA 180

RGIWHNNDKT FLVWVNEEDH LRVISMQKGG NMKEVFRRFC VGLKK 225

Claims (3)

1. a kind of 1 functional areas protein variant of ray Angiostatin, which is characterized in that the ammonia shown in SEQ ID NO:1 On the basis of base acid, replaced accordingly in 151Q/N.

2. protein variant as described in claim 1 inhibits the purposes in tumour medicine in preparation, the tumour is lung cancer or nose Pharynx cancer.

3. a kind of anti-tumor medicinal preparation, it contains protein variant described in claim 1 and pharmaceutically suitable carrier.