CN109628425A

CN109628425A - A kind of people's tropomyosin receptor kinase A mutant and application

Info

Publication number: CN109628425A
Application number: CN201910000843.4A
Authority: CN
Inventors: 周越
Original assignee: Individual
Current assignee: Individual
Priority date: 2019-01-02
Filing date: 2019-01-02
Publication date: 2019-04-16

Abstract

The present invention discloses a kind of people's tropomyosin receptor kinase A mutant, is related to genetically engineered drug field.It is characterized by: the mutant is the amino acid mutation in one or several sites of people's tropomyosin receptor kinase A prototype, the amino acid mutation site is located at tropomyosin receptor kinase A ligand binding region, including LEU486, GLY488, HIS489, ILE490, PHE521, LYS544, GLU560, LEU564, VAL573, PHE589, GLY667, ASP668, PHE669, GLY670；The beneficial effects of the present invention are in vitro and in vivo, which can induce, generates corresponding antibody, and corresponding antibody inhibits the tumour cell of a variety of tropomyosin receptor kinase A positives, safety, no cytotoxicity.

Description

A kind of people's tropomyosin receptor kinase A mutant and application

Technical field

The invention belongs to genetically engineered drug fields, and in particular to tropomyosin receptor kinase A mutant and its mutation Application of the body in oncotherapy.

Background technique

Tropomyosin receptor kinase A (Tropomyosin receptor kinase A, TrkA), and it is known as nerve 1 type of trophic factors tyrosine kinase receptor (neurotrophic tyrosine kinase receptor type 1, It NTRK1), is the 140kDa transmembrane glycoprotein encoded by NTRK1 gene.TrkA is that the specificity of nerve growth factor (NGF) is high Affine force receptor mediates the most of biological functions of NGF, is its functional receptor.NGF combination TrkA receptor can lead to receptor dimerization Change, activate inherent kinase activity, so as to cause the activation of many A signal pathways, including Ras/MAPK, PI3K/Akt and PLC γ is logical Road mediates a variety of pathologic, physiologic functions such as nerve, blood vessel and tumour.

Studies have shown that NGF-TrkA occurs to tumour, proliferation, angiogenesis and transfer are related.NGF and its TrkA expression In a variety of entity tumors including prostate cancer, cancer of pancreas, thyroid cancer, the cancer of the esophagus and lung cancer etc..TrkA is pernicious swollen It is activated in tumor by number of mechanisms, the mainly change of structural rearrangement and expression.Firstly, receptor encoding gene NTRK1 reset and The activation of TrkA is found in kinds of tumors.NTRK1 gene rearrangement generates chimeric oncogene, and tyrosine kinase composition is caused to swash It is living.Vaishnavi etc. has found new Gene Fusion in patients with lung cancer, wherein containing coding high-affinity receptor TrkA's NTRK1 gene.Gene Fusion results in the constitutively activated of TrkA kinase activity, to have carcinogenesis.Again, NGF with The overexpression of TrkA increases the migration of cancer cell, and with the lymphatic metastasis of tumour, at a distance send out, high TNM point Phase, low differentiation, low survival rate are related.Secondly, the exception of TrkA is not only shown in structure and expression, it is also manifested by tumour The exception of intracellular signaling pathway.The activation of TrkA receptor and PI3K/Akt and mitogen-activated protein kinase (mitogen Activated protein kinase, MAPK) access activation it is related, and this two accesses are strong survival-signals, Endogenous and exogenous apoptotic signal access can be blocked simultaneously.Therefore the activation of TrkA very likely passes through the accesses such as activation MAPK It stimulates cellular proliferation, to promote tumour growth.It can be seen that blocking it using TrkA inhibitor using TrkA receptor as target spot Signal path has become oncotherapy and provides new method.

For TrkA receptor, the TrkA inhibitor started to develop has: entering the Trk inhibitor of clinical test earliest The CEP-751 and CEP-701 of Cephalon.These drugs are the derivatives of indole carbazole compound k252a, pass through competition The ATP-binding site of Trk receptor intracellular domain inhibits receptor tyrosine kinase activity, while to Flt3 and protein kinase C (PKC) also there is inhibiting effect.Such compound has anti-tumor activity.The efficient selective small molecule Trk of AstraZeneca A/B kinase inhibitor is about 2nM for the EC50 of TrkA in cell analysis.AZ-23 has good water-soluble, oral bio Utilize activity and PK profile.The kinase inhibiting properties and lestaurtinib and other indoles of AZ-23 Carbazole analog is different, such as does not detect to platelet derived growth factor (platelet-derived growth Factor, PDGF) and protein kinase C (protein kinase C, PKC) inhibiting effect, prompt its clinical activity and toxicity It is also likely to difference.Although AZ-23 selectivity with higher, nor absolute Trk receptor-specific inhibitor.

On November 26th, 2017, FDA ratify Larotrectinib (LOXO-101, trade name Vitrakvi) listing.In vitro Research: previous research in, evaluation concentration be 1000nM and ATP concentration be about Km LOXO-101 to non-TRK kinases group Miss the target kinase inhibitory activity.The result shows that LOXO-101 only has the inhibition greater than 50% to a kind of non-TRK kinases (TNK2), IC50 is 576nM.In vivo study: zooscopy discovery, LOXO-101 are able to suppress tumor growth in vivo.It is daily with LOXO-101 Oral medication injects nude mouse 2 weeks of KM12 cell, observes dose-dependent tumor suppression, shows that LOXO-101 has There is the ability for inhibiting tumor growth in vivo.Clinical test: the multicenter I phase dosage for having carried out patients with advanced solid tumors for 2014 is passed Increase research (ClinicalTrials.gov number: NCT02122913), to evaluate safety and the PK of LOXO-101.Patient is every Its dosage escalation successive administration 28 day once or twice.Preliminary PK and safety data show the free blood plasma water of LOXO-101 The flat biology related concentrations in inhibition TRK oncogene.Rapid clinical tumor regression is observed with LOXO-101 treatment.

Nevertheless, existing TrkA inhibitor is not absolute TrkA receptor-specific inhibitor, it may cause and miss the target Effect is also easy to produce drug resistance.And TrkA specific antibody, by the TrkA for inhibiting to be overexpressed, so that TrkA kinase activity is prevented, To inhibit the Proliferative Activated of tumour cell and migration, achievees the effect that antitumor, have the characteristics that high specificity.However due to TrkA specific antibody can only cause response to passive immunization, thus all there is dosages it is big, side effect is more the disadvantages of.With quilt It is dynamic immune to compare, body can be caused to generate the vaccine type medication of response to active immunization, dosage, safety and patient according to All there is greater advantage in terms of property.Since TrkA is autologous protein, the autoimmune tolerance phenomenon of body becomes TrkA vaccine The significant obstacle of preparation, so improving its immunogenicity becomes the key technology of associated treatment vaccine preparation.Mesh can be used The method of preceding common addition adjuvant improves immunogenicity, it can be difficult to the effectively generation of inducing specific antibody；And or Person can be transformed TrkA, merge T cell antigen epitope of certain external sources etc. to improve immunogenicity, though the method can produce Raw antibody, but height, and the transformation site limitation of TrkA are required to exogenous sequences, therefore application is limited.

Summary of the invention

Goal of the invention

The present invention in view of the drawbacks of the prior art, devises the tropomyosin receptor kinase A mutant of humanized a kind of, For inhibiting gastric cancer, prostate cancer, breast cancer, cancer of pancreas, the carcinoma of the rectum, lung cancer, the growth of the tumour cells such as thyroid cancer.

Technical solution

The technical solution of the present invention is to provide a kind of people's tropomyosin receptor kinase A mutant, the mutant is The amino acid mutation in one or several sites of tropomyosin receptor kinase A prototype, the amino acid mutation site are located at original Myosin receptor kinase A ligand binding region.The tropomyosin receptor kinase A ligand binding region includes LEU486, GLY488, HIS489, ILE490, PHE521, LYS544, GLU560, LEU564, VAL573, PHE589, GLY667, ASP668, PHE669, GLY670.

The tropomyosin receptor kinase A is humanized, the amino acid of tropomyosin receptor kinase A mutant Sequence are as follows:

SGLQGHIIENPQYFSDACVHHIKRRDIVLKWELGEGAFGKVFLAECHNKMLVVCAAALKEARQDFQREA ELLTMLQHQHIVRFFGVCTEGRPLLMVFEYMRHGDLNRFLRSHGPDAKLLAGGEDVAPGPLGLGQLLAVASQVAAGM VYLAGLHFVAGRDLATRNCLVGQGLVVKIGVAFGMSRDIYSTDYYRVGGRTMLPIRWMPPESILYRKFTTESDVWSF GVVLWEIFTYGKQPWYQLSN

Encode the nucleotide sequence of people's tropomyosin receptor kinase A mutant gene are as follows:

TCTGGTTTACAAGGCCATATTATCGAAAATCCTCAGTATTTTTCCGATGCTTGTGTTCACCATATAAAA CGTCGCGACATTGTCTTGAAGTGGGAGCTTGGAGAAGGGGCCTTCGGTAAAGTATTTCTCGCAGAGTGCCACAACAA GATGCTAGTGGCGGTTTGTGCTGCCGCACTGAAAGAAGCGCGACAAGATTTCCAGCGGGAGGCTGAATTATTGACTA TGCTTCAACATCAGCACATCGTCAGATTTTTCGGCGTATGCACCGAGGGAAGGCCCCTCCTAATGGTGTTTGAATAC ATGCGTCATGGGGACCTGAATCGCTTCTTACGATCACACGGTCCAGATGCCAAGTTGCTTGCAGGCGGAGAGGACGT TGCGCCGGGGCCTCTCGGTCTAGGCCAACTGTTAGCTGTCGCCTCGCAGGTAGCAGCGGGAATGGTGTATTTGGCTG GGCTTCATTTTGTTGCCGGTCGGGATCTCGCAACAAGAAACTGTCTAGTCGGCCAAGGACTGGTAGTGAAAATAGGG GTTGCGTTCGGTATGAGTAGGGACATTTACAGCACGGATTATTACCGTGTCGGCGGACGCACTATGTTACCCATCCG ATGGATGCCACCGGAATCTATATTGTATCGGAAGTTTACCACAGAGTCCGACGTATGGTCATTCGGGGTGGTTCTTT GGGAAATTTTTACGTACGGTAAACAGCCTTGGTATCAACTCTCGAATTAATAG

The application of people's tropomyosin receptor kinase A of the invention in anti-tumor drug, for treating gastric cancer, prostate Cancer, breast cancer, cancer of pancreas, the carcinoma of the rectum, lung cancer, thyroid cancer.

Beneficial effect

By pBR322-mNTRK1 plasmid construction, pBR322-mNTRK1 plasmid is transformed into E.coli DH5 α competence Cell, the thallus for filtering out high expression tropomyosin receptor kinase A mutant are obtained pure by the expression and purifying of albumen The tropomyosin receptor kinase A mutant that degree is 98%.The amino acid of tropomyosin receptor kinase A mutant of the invention Sequence is to have carried out targeted mutation in the sequence of the prototype albumen of tropomyosin receptor kinase A, the amino acid sequence It is classified as completely new sequence, meanwhile, encode the completely new sequence of the gene core of the mutant.

Tropomyosin receptor kinase A mutant of the invention for treating kinds of tumors, including lung cancer, thyroid cancer, Gastric cancer, the carcinoma of the rectum, breast cancer and uterine cancer.Test result shows that tropomyosin receptor kinase A mutant of the invention is in body Outer and internal can induce generates corresponding antibody, and the internal potency of antibody is 1:1000000.Tropomyosin receptor kinase A The corresponding antibody of mutant has the function of inhibition kinds of tumor cells, mainly acts on tropomyosin receptor kinase A sun Property tumour cell, according to the sequence of tumour inhibiting rate, (TrkA is positive, preceding by respectively MGC-803 (TrkA is positive, gastric cancer) > LNCaP Column gland cancer) (TrkA is positive, rectum by > MCF-7 (TrkA is positive, breast cancer) > MIAPaCa (TrkA is positive, cancer of pancreas) > HCT116 Cancer) > A549 (TrkA is positive, lung cancer) > FTC-133 (TrkA is positive, thyroid cancer).

Safety evaluatio the results show that tropomyosin receptor kinase A mutant of the invention to tropomyosin polymeric immunoglobulin receptor The tumour cell no cytotoxicity of the kinases A positive, the tolerance test of mouse tail vein injection the result shows that, mutation of the invention Body is non-toxic, and the maximum tolerance consumption per day for extrapolating clinical adult intravenous injection is 4.17mg/Kg.

Detailed description of the invention

Fig. 1 TrkA wild-type amino acid sequence three-stage structure schematic diagram

Fig. 2 NTRK1 mutated genes electrophoretogram, 1-4 swimming lane are NTRK1 mutated genes.

Fig. 3 pBR322-mNTRK1 plasmid figure

Specific embodiment

Embodiment 1

PBR322-mNTRK1 plasmid construction

(1) it according to TrkA wild-type amino acid sequence (Fig. 1), is analyzed by software, filters out the active region in sequence. The one or more amino acid being mutated in the region, the amino acid sequence of the tropomyosin receptor kinase A mutant after mutation For SEQ ID NO:1.

(2) design of NTRK1 mutated genes (mNTRK1): according to the amino of tropomyosin receptor kinase A mutant Acid sequence will encode wild type gene (NTRK1) rite-directed mutagenesis of tropomyosin receptor kinase A, the nucleotides sequence of the gene It is classified as SEQ ID NO:2.

(3) the NTRK1 mutated genes designed by are synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd, set The gene of meter is on the plasmid HF392 that company provides.It is carried out according to gene order design primer, and with computer software OLIGO Reasonable evaluation.Primer sequence I: upstream primer P-F (5 '-TCTGGTTTACAAGGCCATATTATCGA-3 ') and downstream primer P-R(5′-CTATTAATTCGAGAGTTGATACCAAG-3′).Hind III and EcoRI restriction site are imported at 5 ' ends.With Plasmid HF392 is template, amplification system: 10 × PCR buffer, 0.2mmol/L dNTP, 100ng template DNA, 100 μ of total volume L, amplification condition be 93 DEG C 40 seconds, 55 DEG C 30 seconds, 72 DEG C 1 point 30 seconds, totally 35 circulation.Last 72 DEG C extend 10 minutes.It obtains PCR product after Hind III and EcoRI digestion, carry out gel electrophoresis, recycle glue, obtain NTRK1 mutated genes (Fig. 2).

(3) it pBR322-mNTRK1 plasmid construction: after the mNTRK1 of recycling, and is cut with EcoRI digestion and uses alkaline phosphatase Enzyme has made the rear pBR322 plasmid connection of dephosphorylation processing, obtains pBR322-mNTRK1 plasmid (Fig. 3).

Embodiment 2

The high expression bacterial strain screening of tropomyosin receptor kinase A mutant

(1) pBR322-mNTRK1 plasmid is transformed into E.coli DH5 α competent cell, be inoculated in containing ampicillin In LB culture medium, 37 DEG C of shaken overnights, next day will activate bacterium and be reinoculated in new culture solution through 1% ratio, in 37 DEG C Continue culture oscillation, when its absorbance A value (once claiming optical density OD value) (600nm) is up to 0.5~2, IPTG is added to final concentration For 1mmol/L, continue culture 5 hours, keep sample identification, screens high expression thallus.

(2) detection of tropomyosin receptor kinase A mutant: high expression thallus is collected by centrifugation, through ultrasonication, centrifugation Collect precipitating.It is washed with the 50mmol/L Tris-HCl (pH8.0) of the X-100 containing 0.5%triton, 1mmol/L EDTA solution 2~3 times, 10000r/min centrifuging and taking precipitating.With tropomyosin receptor kinase A mutation in indirect elisa method detection sediment The protein concentration of body, first antibody are the monoclonal antibody (McAb) of anti-TrkA, and secondary antibody is the anti-mouse IgG mcAb of enzyme mark, are obtained To the protein concentration of tropomyosin receptor kinase A mutant.Again with the concentration of Coomassie Brilliant Blue measurement total protein, calculate The purity of tropomyosin receptor kinase A mutant.To filter out the bacterium of high expression tropomyosin receptor kinase A mutant Body.

Embodiment 3

The expression and purifying of tropomyosin receptor kinase A mutant

By the tropomyosin receptor kinase A mutant culture solution of acquisition, centrifuging and taking bacterial sediment, ultrasonication, centrifugation Supernatant is taken, supernatant is purified.Supernatant is passed through into Q Sepharose Fast Flow in AKTA protein purification instrument first Monitoring under,

Main albumen is eluted using the Tris-HCl buffer containing pH8.0,20mM that concentration is 0.2M NaCl.Secondly will Eluent containing main albumen is purified by Ni-NTA, and pH8.0,20mM of 50mM imidazoles, 500mM imidazoles is respectively adopted Tris-HCl buffer elutes foreign protein and main albumen.Gained protein purification liquid is subjected to indirect elisa method and Mas bright blue Method measurement, detection purity reach 98%.

Embodiment 4

The detection of tropomyosin receptor kinase A mutant external activity

Fresh human peripheral blood 5mL is taken, is centrifugated to specifications using lymph separating liquid, finally obtains 10⁶A/mL people Lympho-mononuclear cells.The cell liquid average mark that separation is obtained after incubator culture 4h, is separately added into sheep in six orifice plates The 20 μ g/mL of high-purity tropomyosin receptor kinase A mutant that anti-human 2 μ g/mL of IgM costimulation agent and embodiment 3 obtain, and The tropomyosin receptor kinase A of prototype.Incubator culture 96h takes 200 μ L cell liquid respectively, and centrifuging and taking supernatant carries out ELISA Experiment.(table 1) as the result is shown, prototype tropomyosin receptor kinase A does not generate antibody, and tropomyosin receptor kinase A is prominent Variant stimulates cell to generate antibody, and albumen prototype group has statistical difference (P < 0.01) compared with mutant group.

1 cell of table generates antibody OD value

Embodiment 5

High-purity tropomyosin receptor kinase A mutation induction mouse that embodiment 3 obtains generate tropomyosin by The titration of body kinases A antibody.

Using ICR mouse as animal subject, 20 mouse, half male and half female, weight 18-22g, in test the 1st, 14 are taken (the 1st day is complete Freund assistant with Freund's adjuvant to the Freund's adjuvant of the mutant prepared with 28 days subcutaneous multi-point injections containing embodiment 3 Agent and mutant are with the mixing of 1:1 ratio；14th and 28 day is incomplete Freund's adjuvant and mutant with 1:1 ratio with Freund's adjuvant Mixing).42nd day, blood is taken using eyeground vein clump, carries out ELISA test, mutant prepared by the measurement embodiment of the present invention 1 lures Lead the potency that mouse generates tropomyosin receptor kinase A IgG antibody.

Titration step: (1) antigen coat: TrkA is diluted as antigen with coating buffer l:8000, and 100 holes μ L/ are added In 96 hole reaction plate of polystyrene.4 DEG C stand overnight, and cleaning solution is washed 3 times；(2) it closes: adding the hole l00 μ L/ confining liquid (containing 1% N Seralbumin, the PBS of 0.1%Tween20), it is placed at room temperature for 1h, is washed 3 times with cleaning solution；(3) by immune serum according to The mice serum of extension rate and blank negative controls are added separately to instead by doubling dilution at different extension rates, (4) It answers in plate, 100 holes μ L/, 37 DEG C, reacts 2h, washed 3 times with cleaning solution；(5) enzyme mark antiantibody: rabbit anti-mouse igg-HRP, with envelope Liquid l:8000 dilution is closed, 100 holes μ L/ cover 37 DEG C of insulating boxs and incubate 1h, washed 3 times with cleaning solution；(6) develop the color: tmb substrate is molten 10min is placed in 100 hole μ L/ of liquid, room temperature dark place；(7) it terminates reaction: adding 50 hole μ L/ terminate liquids；(8) 450nm is measured with microplate reader Locate light absorption value, is positive reaction, to detect positive reaction most when test sample absorbance/negative sample absorbance > 2.5 Big dilution is the potency of mice serum antibody.

The potency that the mutation induction mouse that as the result is shown prepared by embodiment 1 generates TrkA antibody is 1:1000000.

Embodiment 6

The effect for the high-purity tropomyosin receptor kinase A mutant antibodies cell proliferation that embodiment 3 obtains

(1) the high-purity tropomyosin receptor kinase A mutant that the specialized company of antibody obtains according to embodiment 3 is prepared The monoclonal antibody of the tropomyosin receptor kinase A mutant of Antibody preparation specific recognition people.

(2) MTT cell Proliferation is carried out using the monoclonal antibody for the tropomyosin receptor kinase A mutant being prepared Test, evaluates the antibody to the cel l proliferation of the TrkA positive.

Test cell: MCF-7 (TrkA is positive, breast cancer), MIAPaCa (TrkA is positive, cancer of pancreas), LNCaP (TrkA sun Property, prostate cancer), A549 (TrkA is positive, lung cancer), FTC-133 (TrkA is positive, thyroid cancer), MGC-803 (TrkA is positive, Gastric cancer), HCT116 (TrkA is positive, the carcinoma of the rectum) and U937 (TrkA is negative, histocytic lymphoma's cell)；

Grouping: 1: antibody group (potency is respectively as follows: 1:1000,1:10000,1:100000)；2: blank group.

Step:

(1) logarithmic phase tumor cell is collected respectively, adjusts concentration of cell suspension, and 100ul is added in every hole, 5000 thin Born of the same parents/hole.

(2) 5%CO2,37 DEG C of incubations, until cell monolayer is paved with bottom hole (96 hole flat underside), is added the former flesh of different potency The monoclonal antibody of Ig receptor kinases A mutant, if 5 multiple holes, while setting the control wells (drug of cell, same concentrations Dissolving medium, culture solution, MTT, dimethyl sulfoxide).

(3) 5%CO2,37 DEG C are incubated for 48 hours, and 20ulMTT solution (5mg/ml, i.e. 0.5%MTT) is added in every hole, continue Cultivate 4h.

(4) culture is terminated, culture solution in hole is carefully sucked.150ulDMSO is added in every hole, sets low-speed oscillation on shaking table 10min dissolves crystal sufficiently.The light absorption value in each hole is measured at enzyme-linked immunosorbent assay instrument OD570nm.Calculate tumour cell Proliferation inhibition rate (%)=(one experimental group absorbance value of control group absorbance value)/control group absorbance value × 100.

As a result: table 2 shows the inhibiting rate of the antibodies on tumor cell of TrkA mutant preparation of the present invention, and the antibody is to TrkA Positive tumour cell is effective.In addition, according to the sequence of inhibiting rate: MGC-803 (TrkA is positive, gastric cancer) > LNCaP (TrkA The positive, prostate cancer) > MCF-7 (TrkA is positive, breast cancer) > MIAPaCa (TrkA is positive, cancer of pancreas) > HCT116 (TrkA sun Property, the carcinoma of the rectum) > A549 (TrkA is positive, lung cancer) > FTC-133 (TrkA is positive, thyroid cancer), it is seen that the antibody is swollen to difference The inhibiting rate of tumor is different.

The inhibiting rate (%) of the antibodies on tumor cell of the TrkA mutant of the present invention of table 2 preparation

Embodiment 7

The Cytotoxic evaluation for the high-purity tropomyosin receptor kinase A mutant that embodiment 3 obtains.

Using CCK8 method, the high-purity tropomyosin receptor kinase A mutant point that the embodiment of the present invention 3 obtains is measured It is other to MCF-7 (TrkA positive, breast cancer), MIAPaCa (TrkA is positive, cancer of pancreas) and LNCaP (the TrkA positive, prostate cancer) Cytotoxicity.

Step:

(1) cell suspension is respectively prepared in different types of cell, it is outstanding in the cell that 100 μ L are added in 96 orifice plates Liquid, cell concentration 5*10⁵A/hole.By culture plate at incubator preculture 24 hours (37 DEG C, 5%CO2).

(2) dosing: being added culture medium or tropomyosin receptor kinase A mutant to culture plate respectively, if 5 multiple holes, And culture plate is incubated for 48 hours in incubator.

(3) CCK8 is detected:

Culture medium is discarded, and washs cell twice with culture medium, new 100ul culture medium is then added, is added to every hole 10 μ LCCK8 solution.Wherein CCK8 is added as blank group in the reserved hole PBS (+the CCK8 containing serum is free of cell).By culture plate 1-4h is incubated in incubator.The absorbance at 450nm is measured with microplate reader.

(4) cell activity calculates: cell viability (%)=[OD (dosing)-OD (blank)]/[OD (0 dosing)-OD is (empty It is white)] × 100 (OD (dosing): the absorbance in the hole with cell, CCK8 solution and drug solution；OD (blank): there is culture Base and CCK8 solution are without the absorbance in the hole of cell；OD (0 dosing): have cell, CCK8 solution without the hole of drug Absorbance cell viability).

The results show that the obtained high-purity tropomyosin receptor kinase A mutant of embodiment 3 is to three kinds of TrkA positive The cell viability of cell MCF-7, MIAPaCa and LNCaP are respectively 106.8,106.8 and 95.5% (tables 3), with culture medium feminine gender It compares, without statistical difference (P > 0.05), it can be seen that, the high-purity tropomyosin receptor kinase A that embodiment 3 obtains Mutant not cell effect with the TrkA positive itself, no cytotoxicity.

3 Cytotoxic evaluation of table

Embodiment 8

The acute toxicity test for the high-purity tropomyosin receptor kinase A mutant that embodiment 3 obtains.

Tropomyosin receptor kinase A mutant of the present invention is evaluated using the acute toxicity test of mouse tail vein injection In body toxicity.

Method: by ICR mouse 40, half male and half female, weight 18-22g is randomly divided into control group and experimental group, and every group 20, experimental group is using maximum concentration (10mg/ml), the tropomyosin receptor kinase A mutant one of maximum volume (0.1ml) Secondary property mouse tail vein injection, control group give same amount of normal saline, behavior, appearance after observation mouse administration in 2 weeks, into Food, excreta, main organs substantially with pathological change and death condition, and calculate mouse to nano silver maximum be resistant to Amount.

Test result: after tropomyosin receptor kinase A is mutated body disposable mouse tail vein injection, behavior is without exception, No significant difference (P > 0.05), each main organs Non Apparent Abnormality change compared with the control group for food ration, weight etc., and 2 Without death in all.Thus it calculates, the maximum tolerance consumption per day of clinical adult intravenous injection is 4.17mg/Kg.

Sequence table

<110>Zhou Yue

<120>a kind of people's tropomyosin receptor kinase A mutant and application

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 243

<212> PRT

<213>artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 1

Ser Gly Leu Gln Gly His Ile Ile Glu Asn Pro Gln Tyr Phe Ser Asp

1 5 10 15

Ala Cys Val His His Ile Lys Arg Arg Asp Ile Val Leu Lys Trp Glu

20 25 30

Leu Gly Glu Gly Ala Phe Gly Lys Val Phe Leu Ala Glu Cys His Asn

35 40 45

Lys Met Leu Val Val Cys Ala Ala Ala Leu Lys Glu Ala Arg Gln Asp

50 55 60

Phe Gln Arg Glu Ala Glu Leu Leu Thr Met Leu Gln His Gln His Ile

65 70 75 80

Val Arg Phe Phe Gly Val Cys Thr Glu Gly Arg Pro Leu Leu Met Val

85 90 95

Phe Glu Tyr Met Arg His Gly Asp Leu Asn Arg Phe Leu Arg Ser His

100 105 110

Gly Pro Asp Ala Lys Leu Leu Ala Gly Gly Glu Asp Val Ala Pro Gly

115 120 125

Pro Leu Gly Leu Gly Gln Leu Leu Ala Val Ala Ser Gln Val Ala Ala

130 135 140

Gly Met Val Tyr Leu Ala Gly Leu His Phe Val Ala Gly Arg Asp Leu

145 150 155 160

Ala Thr Arg Asn Cys Leu Val Gly Gln Gly Leu Val Val Lys Ile Gly

165 170 175

Val Ala Phe Gly Met Ser Arg Asp Ile Tyr Ser Thr Asp Tyr Tyr Arg

180 185 190

Val Gly Gly Arg Thr Met Leu Pro Ile Arg Trp Met Pro Pro Glu Ser

195 200 205

Ile Leu Tyr Arg Lys Phe Thr Thr Glu Ser Asp Val Trp Ser Phe Gly

210 215 220

Val Val Leu Trp Glu Ile Phe Thr Tyr Gly Lys Gln Pro Trp Tyr Gln

225 230 235 240

Leu Ser Asn

<210> 2

<211> 738

<212> DNA

<213>artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 2

tctggtttac aaggccatat tatcgaaaat cctcagtatt tttccgatgc ttgtgttcac 60

catataaaac gtcgcgacat tgtcttgaag tgggagcttg gagaaggggc cttcggtaaa 120

gtatttctcg cagagtgcca caacaagatg ctagtggcgg tttgtgctgc cgcactgaaa 180

gaagcgcgac aagatttcca gcgggaggct gaattattga ctatgcttca acatcagcac 240

atcgtcagat ttttcggcgt atgcaccgag ggaaggcccc tcctaatggt gtttgaatac 300

atgcgtcatg gggacctgaa tcgcttctta cgatcacacg gtccagatgc caagttgctt 360

gcaggcggag aggacgttgc gccggggcct ctcggtctag gccaactgtt agctgtcgcc 420

tcgcaggtag cagcgggaat ggtgtatttg gctgggcttc attttgttgc cggtcgggat 480

ctcgcaacaa gaaactgtct agtcggccaa ggactggtag tgaaaatagg ggttgcgttc 540

ggtatgagta gggacattta cagcacggat tattaccgtg tcggcggacg cactatgtta 600

cccatccgat ggatgccacc ggaatctata ttgtatcgga agtttaccac agagtccgac 660

gtatggtcat tcggggtggt tctttgggaa atttttacgt acggtaaaca gccttggtat 720

caactctcga attaatag 738

Claims

1. a kind of people's tropomyosin receptor kinase A mutant, it is characterised in that: be tropomyosin in the mutant by The amino acid mutation in one or several sites of body kinases A prototype, the amino acid mutation site is located at tropomyosin polymeric immunoglobulin receptor Kinases A ligand binding region.

2. people's tropomyosin receptor kinase A mutant according to claim 1, which is characterized in that the former flesh ball Protein receptor kinases A is humanized's.

3. people's tropomyosin receptor kinase A mutant according to claim 1, it is characterised in that: the former flesh ball Protein receptor kinases A ligand binding region includes LEU486, GLY488, HIS489, ILE490, PHE521, LYS544, GLU560, LEU564, VAL573, PHE589, GLY667, ASP668, PHE669, GLY670.

4. people's tropomyosin receptor kinase A mutant according to claim 1, it is characterised in that: the former flesh ball The amino acid sequence of protein receptor kinases A mutant is SEQ ID NO:1.

5. people's tropomyosin receptor kinase A mutant according to claim 4, which is characterized in that it is former to encode the people The nucleotides sequence of myosin receptor kinase A mutant gene is classified as SEQ ID NO:2.

6. application of people's tropomyosin receptor kinase A mutant in anti-tumor drug described in claim 1-5.

7. application as claimed in claim 6, which is characterized in that the tumor disease includes gastric cancer, prostate cancer, mammary gland Cancer, cancer of pancreas, the carcinoma of the rectum, lung cancer, thyroid cancer.