CN105481963A

CN105481963A - Ray angiogenesis inhibiting factor (1) functional domain variant JG50 and application thereof

Info

Publication number: CN105481963A
Application number: CN201610017406.XA
Authority: CN
Inventors: 张垒; 张勇
Original assignee: Individual
Current assignee: Chen Size; First Affiliated Hospital of Guangzhou Medical University
Priority date: 2014-06-06
Filing date: 2014-06-06
Publication date: 2016-04-13
Anticipated expiration: 2034-06-06
Also published as: CN105481967A; CN105481964A; CN105541985A; CN105461792A; CN105418753A; CN105481961A; CN105418753B; CN105481966B; CN105440117B; CN105481965B; CN105418742A; CN105541985B; CN105461792B; CN105440118B; CN105418742B; CN105481962B; CN105481964B; CN105418752A; CN105481962A; CN104031136B

Abstract

The invention discloses ray angiogenesis inhibiting factor (1) functional domain protein and a variant thereof and application of the angiogenesis inhibiting factor (1) functional domain protein and the variant thereof to preparation of medicine for inhibiting tumor growth. By means of the ray angiogenesis inhibiting factor (1) functional domain variant JG50 and application thereof, the protein variant is subjected to eukaryotic expression, and compared with original protein, the cancer cell inhibiting effect can be remarkably improved.

Description

Ray Angiostatin 1 functional zone variant JG50 and application thereof

Technical field

The invention belongs to biological technical field, specifically, the present invention relates to ray Angiostatin 1 functional zone variant.

Background technology

Ray Angiostatin 1 functional zone are hereinafter referred to as ray functional zone, and ray Angiostatin 1 is the protein (molecular weight 42KD) separated from a kind of ray tissue of South China Sea.Result of study shows: that ray Angiostatin 1 significantly suppresses chick chorioallantoic membrane itself and that KB cell is induced chick chorioallantoic membrane vasculogenesis; Be growth and the transfer that abdominal injection or gavage all significantly suppress nude mouse Lewis lung cancer, reduce tumor tissues microvessel density, lower the expression of angiogenic factors VEGF; Also high inhibition effect is had to the transfer of nude mouse Melanoma B16 cell; Lower the expression of short transfer factor CD44v6 and ErBb2; With more remarkable effect during 5 FU 5 fluorouracil (5-FU) combined utilization.The action target spot of ray Angiostatin 1 is different from the PTS Avastin that gene engineering institute of the U.S. develops.Avastin is gene engineering product, and be the antibody of VEGF, its action target spot is VEGF; Ray Angiostatin 1 is natural product, and it not only acts on VEGF, also acts on bFGF and PDGF.

In the prior art, in order to improve the activity of albumen, the specific site 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 is by a large amount of experiments, carry out point mutation for site specific in the aminoacid sequence of ray Angiostatin 1 functional zone, thus obtain the variant than ray Angiostatin 1 functional zone itself with higher inhibit activities.

Summary of the invention

The present invention relates to the variant of the separation of Parent Protease, its position corresponding to position 9R, 17F, 42H, 57S, 62R, 71K, 80N, 89P, 100G, 101R, 129A, 134E, 151Q, 163P, 189K or 217R of the functional zone of SEQIDNO:1 at least one comprises modification.Specifically, variant of the present invention has the inhibit activities of improvement.

Preferably, being applied to method of the present invention, presenting the protein variant of the inhibit activities of improvement is following arbitrary 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 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 for suppressing cancer.

Embodiment

Embodiment 1

1. the acquisition of ray Angiostatin 1 functional zone

Ray functional zone are made up of in the following order following amino-acid residue:

TLD1YKQLRDKETPSGFTLDDV1QTGVDNPGHPF1MTVGCVAGDEESYEVFKALFDPV1QDRHGGYKPTDKHKTDLNHENLKGGDDLDPNYVLSSRVRTGRS1KG1ALPPHCSRGERRLVEKLCLEGLATLTGEFQGKYYPLTTMSDAEQQQL1DDHFLFDKPVSPLLLASGMARDWPDARG1WHNNDKTFLVWVNEEDHLRV1SMQKGGNMKEVFRRFCVGLKK

2, the structure of ray Angiostatin 1 functional zone variant

The introducing of catastrophe point

(1), according to corresponding mutational site design corresponding mutagenic primer, adopt PCR fixed-point mutation method on the gene corresponding to ray Angiostatin 1 functional zone, 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 plasmid pmD-18T carrier segments of cutting through same enzyme, and transformation of E. coli DH5 α competent cell;

(3), qualification recombinant plasmid: with enzyme cut, PCR method identifies recombinant plasmid, and order-checking inspection, obtains the nucleotide sequence after suddenling change thus.These nucleotide sequences say that corresponding aminoacid sequence is respectively on the basis of SEQIDNO:1, has carried out corresponding sudden change in 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 position.

(4) expression of, pPIC6 α-ray Angiostatin 1 functional zone variant

Restriction enzyme site is introduced at two ends above-mentioned steps being built the ray Angiostatin 1 functional zone variant gene obtained, signal peptide sequence is added in upstream, structure obtains pPIC6 α-ray Angiostatin 1 functional zone variant vector, after qualification, by plasmid transfection Pichia pastoris, and carry out eukaryotic expression, adopt Blasticidin as selection markers.Induce its secreting, expressing target protein, find that the expressing quantity that methanol concentration is 0.5%, continuous induction obtains for 4 days is higher.The protein band obtained through SDS-PAGE electrophoretic separation can react with the antiserum(antisera) of the anti-hCG of rabbit in Westernblot detects.Experimental result shows that the expression amount average in pichia pastoris phaff of recombinant plasmid pPIC6 α-ray Angiostatin 1 functional zone variant that we build is that expression amount is about 140mg/L.

3, the growth of ray Angiostatin 1 functional zone variant Tumor suppression and transfer

BALB/c nude mice is divided into control group, the positive (endoxan) control group and experimental group at random, often organizes 8, male and female half and half.Every mouse dorsal sc inoculation 0.2mlLewis lung carcinoma cell suspension (4 × 106 cell) Lewis lung cancer cell.After tumor inoculation the 7th day, experimental group abdominal injection various dose ray functional zone solution, every day 1 time, totally 14 times; Control group abdominal injection equivalent solvent; Positive controls intraperitoneal injection of cyclophosphamide, 1 time weekly, totally 2 times.Within 21st day, sacrifice mouse, peel off tumour and weigh (being fixed for immunohistochemical experiment by 10% neutral formalin), dissect, observe and record hepatic metastases joint footing.Be calculated as follows inhibition rate of tumor growth and metastases inhibiting rate:

Concrete outcome is as follows:

4, the chick chorioallantoic membrane vasculogenesis that nasopharyngeal carcinoma cell (CNE-2Z) is induced is tested

Selection is clean, eggshell homogeneous, air chamber plant egg uniformly, and in 19200 types intelligence incubators, hatching 1 week, opens a diameter 1cm aperture at embryo head end under aseptic condition, form false air chamber.Inoculation CNE-2Z cell (1.9 × 106/ embryo), seals with scotch tape.Put in incubator and cultivate after 4 days, filter paper central authorities being made a call to an aperture is placed on the chorioallantoic membrane in air chamber, and add ray functional zone albumen and misfolded proteins liquid 100 μ l thereof respectively on filter paper, control group then adds equivalent solvent, every day 1 time, altogether administration 4 times.Then remove scotch tape, fix 12 minutes respectively with acetone and dehydrated alcohol.Cutting the chorioallantoic membrane being placed with filter paper puts on slide glass, abandons or adopts filter paper, and under microscope, the branch of random selecting 5 visual field counting visible vessels counts and takes a picture.Be calculated as follows vasculogenesis inductivity and Agiogenesis inhibition rate:

Group	Chicken embryo number (only)	Dosage (a μ g/ * days)	Vessel branch is counted (X pulls out+S)	Inhibiting rate
					Control group	5	—	83.5	—
SEQ ID NO:1 albumen	5	100	42.2	49.5% 3 -->
					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%

As can be seen from the experimental result of 3 and 4, the variant of 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 is all significantly improved in inhibition of cancer cell rate and vascular study rate relative to original ray functional zone albumen, therefore achieves good effect.

Toxicological experiment

Acute toxicity test

Adopt healthy Kunming mouse, body weight 19 ~ 22 grams, male and female half and half.The front fasting 4h of administration (po), adopts multiple dosing method in 24h, takes the circumstances into consideration therebetween to supply feed, can't help water.Dosage is 100 times of (1p, 20mg/kg of the effective dose of ray functional zone original series and variant sequence thereof; Po, 40mg/kg), to observation post administration 7 days.Viewing duration records the toxic reaction situation of animal and the distribution of dead animal day by day.Because do not find dead mouse, do not measure LD50, then change and do " mtd test ", disposable 300 times of (1p, the 60mg/kg using the effective dose of ray functional zone and variant thereof; Po, 120mg/kg) 0.5ml and 0.8ml give the injection of 20 mouse peritoneals or gavage respectively, observe 14 days, result does not find mouse toxicity reaction or death yet.

Stability

Adopt Freeze Drying Technique to make ray functional zone and variant sample thereof, preserve 12 months in-20 DEG C of refrigerators, its biological activity (inhibiting angiogenesis, suppression vitro culture oncocyte and mice transplanted tumor growth) is without obviously reducing.

Ray functional zone and variant thereof can mix with pharmaceutically useful carrier or dissolve, the medicine of the various tumour of obtained treatment, comprise various formulation, this medicine can be used for leukemia, also can be used for various noumenal tumour, as the treatment of esophagus cancer, liver cancer, cancer of the stomach, carcinoma of the pancreas, colon and rectum carcinoma, cervical cancer, ovarian cancer, mammary cancer, nasopharyngeal carcinoma, oral carcinoma, lip cancer, skin carcinoma, bladder cancer, chorioepithelioma, carcinoma of parotid gland, lymphoma, myomata, melanoma and various intracranial tumorss etc., and the application in the people needing this treatment.In made various combination of oral medication, the content of ray functional zone and variant thereof is the about 0.001-100mg of every Kg body weight every day, preferably about 0.01-50mg, is more excellently about 0.05-30mg, and the best is 0.1-10mg about.Every bu 2-4 time uses, and within 10-30 days, is a course for the treatment of, general medication 3-6 course for the treatment of, each course for the treatment of interval 10-30 days.Made medicinal composition for injections, can supply intramuscular injection or intravenous drip, wherein the content of ray functional zone and variant thereof is the about 0.001-50mg of every Kg body weight every day, preferably about 0.01-25mg, is more excellently about 0.05-15mg, and the best is 0.1-3mg about.Every bu 1-3 time uses, and within 10-30 days, is a course for the treatment of, general medication 3-6 course for the treatment of, each course for the treatment of interval 10-30 days.But accurate dosage should be determined by doctor, depend primarily on the age of patient, body weight, the state of an illness, reaction etc.

Sequence table

< 110 > opens bravely

< 120 > ray Anti-angiogenesis factors 1 functional zone variant JG50 and application thereof

〈160〉1

〈210〉1

〈211〉225

〈212〉PRT

< 213 > ray

〈400〉1

TLDIYKQLRDKETPSGFTLDDVIQTGVDNPGHPFIMTVGCVAGDEESYEVFKALFDPVIQ60

DRHGGYKPTDKHKTDLNHENLKGGDDLDPNYVLSSRVRTGRSIKGIALPPHCSRGERRLV120

EKLCLEGLATLTGEFQGKYYPLTTMSDAEQQQLIDDHFLFDKPVSPLLLASGMARDWPDA180

RGIWHNNDKTFLVWVNEEDHLRVISMQKGGNMKEVFRRFCVGLKK225

Claims

1. a ray Angiostatin 1 functional zone albumen, its aminoacid sequence is for shown in SEQIDNO:1.

2. a ray Angiostatin 1 functional zone protein variant, is characterized in that, on the amino acid whose basis shown in SEQIDNO:1, replaces accordingly at 71K/R.

3. protein variant as claimed in claim 2 is preparing the purposes in Tumor suppression medicine.

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