CN108299546B

CN108299546B - Polypeptide, preparation method and application thereof, and pharmaceutical composition

Info

Publication number: CN108299546B
Application number: CN201710028133.3A
Authority: CN
Inventors: 肖承荣
Original assignee: Shenzhen Huazhong Medical Laboratory Co ltd
Current assignee: Shenzhen Huazhong Medical Laboratory Co ltd
Priority date: 2017-01-13
Filing date: 2017-01-13
Publication date: 2020-09-01
Anticipated expiration: 2037-01-13
Also published as: CN108299546A

Abstract

The invention belongs to the field of biomedicine, and particularly relates to a polypeptide, a preparation method and application thereof, and a pharmaceutical composition. The amino acid sequence of the polypeptide is SEQ ID NO: 1: ETIRKAYPDANLVNDRVLR are provided. The preparation method comprises the following steps: synthesizing a gene sequence for coding the polypeptide, and transferring the gene sequence into a pPIC9K plasmid to obtain a recombinant plasmid; amplifying the recombinant plasmid with E.coli DH5 a, and then extracting the recombinant plasmid; transferring the extracted recombinant plasmid into escherichia coli BL21 for gene expression, and extracting the polypeptide. The polypeptide can competitively inhibit the activity of Cyclin E protein, has good curative effect on treating human thyroid papilloma, can be used for preparing thyroid papilloma medicines, and provides a better choice for treating human thyroid papilloma by Cyclin E protein inhibitor polypeptide medicines.

Description

Polypeptide, preparation method and application thereof, and pharmaceutical composition

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to a polypeptide, a preparation method and application thereof, and a pharmaceutical composition.

Background

Thyroid cancer is a common malignant tumor in the field of endocrine systems, the incidence rate of the thyroid cancer is in the head and neck malignant tumor, the incidence rate of thyroid papillary carcinoma (PTC) accounts for about 80% of all types of thyroid cancer, and the thyroid cancer can be developed at any age and is often found in children or middle-aged and young women. The pathogenesis and the cause of the acute increase of the incidence of thyroid cancer are not clear, no obvious symptoms exist in the early stage, and the current treatment is mainly based on surgical clearance. The target therapy aims at blocking one or more signal paths in the process of generating tumors so as to achieve the purpose of treating the tumors, and the biological target therapy of the tumors is a promising treatment method.

Cyclin proteins control the cycle of rest, growth and division of cells. Wherein Cyclin E is a target for controlling the growth cycle of cells; cyclin E can participate in and control when cells begin to produce DNA and prepare for cell division to form new cells. Studies have shown that Cyclin E binds CDK2, rendering the latter kinase active. The cyclin E-CDK2 kinase catalyzes downstream p107 protein phosphorylation, the phosphorylated p107 protein loses the inhibition effect on E2F, E2F promotes cell replication related gene transcription, and cells are transformed from a G1 phase to an S phase, so that cell division is smoothly carried out. In many different types of tumors, Cyclin E is abnormally highly expressed, stimulating cells to divide too quickly to form tumors. Therefore, by blocking the binding of Cyclin E protein and CDK2, the transcription of genes related to DNA replication of tumor cells can be inhibited, thereby preventing the expansion of tumor cells. It is found that, during the development of fruit fly embryo, if Cyclin E is subjected to gene mutation, the cells of the embryo are stopped at the G1 stage; the specific antibody of anti-Cyclin E is injected by cell microinjection, and the injected cells stay in G1 phase.

In conclusion, Cyclin E can be used as a new target for treating human papillary thyroid carcinoma for research. However, no mature drug for treating human papillary thyroid cancer using Cyclin E protein inhibitor polypeptides has been developed.

Disclosure of Invention

The invention aims to overcome the defects in the prior art, provides a polypeptide, a preparation method and application thereof, and a pharmaceutical composition, and aims to overcome the defect that the existing clinical drug for treating human thyroid papillary carcinoma without Cyclin E protein inhibitor polypeptide is lacked.

In order to achieve the above purpose of the present invention, the adopted technical scheme is as follows:

in one aspect, the invention provides a polypeptide, wherein the polypeptide is Cyclin E protein inhibitory polypeptide, and the amino acid sequence of the Cyclin E protein inhibitory polypeptide is SEQ ID NO: 1: ETIRKAYPDANLVNDRVLR are provided.

The novel polypeptide provided by the invention can competitively inhibit the activity of Cyclin E protein, has a good curative effect on treating human thyroid papilloma, and provides a better choice for treating human thyroid papilloma by Cyclin E protein inhibitor polypeptide drugs.

In another aspect, the invention also provides a method for preparing the polypeptide. The method comprises the following steps:

synthesizing a gene sequence for coding the polypeptide, and transferring the gene sequence into a pPIC9K plasmid to obtain a recombinant plasmid;

amplifying the recombinant plasmid with E.coli DH5 a, and then extracting the recombinant plasmid;

transferring the extracted recombinant plasmid into escherichia coli BL21 for gene expression, and extracting the polypeptide.

The preparation method of the polypeptide provided by the invention is simple and easy to operate, has low cost, can obtain the polypeptide with high concentration, effectively keeps the activity of the polypeptide, and is beneficial to the subsequent development and research of the polypeptide medicament.

Correspondingly, the invention also provides application of the polypeptide in preparing a papillary thyroid tumor medicament.

The polypeptide can competitively inhibit the activity of Cyclin E protein, has good curative effect on treating human thyroid papilloma, and can be used for preparing thyroid papilloma medicines.

Finally, the invention also provides a pharmaceutical composition. The medicinal composition comprises the polypeptide and pharmaceutically acceptable auxiliary materials.

Because the polypeptide can competitively inhibit the activity of Cyclin E protein, the polypeptide can be combined with pharmaceutically acceptable auxiliary materials together to be used as a pharmaceutical composition for treating thyroid papilloma, and has good curative effect on treating human thyroid papilloma.

Detailed Description

In order to make the technical problems, technical solutions and advantageous effects to be solved by the present invention more clearly apparent, the present invention is further described in detail below with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

In one embodiment of the invention, a polypeptide is provided, the polypeptide is Cyclin E protein inhibitory polypeptide, and the specific amino acid sequence of the polypeptide is SEQ ID NO: 1: ETIRKAYPDANLVNDRVLR are provided.

The polypeptide provided by the embodiment can competitively inhibit the activity of Cyclin E protein, and has a good curative effect on treating human thyroid papilloma. The polypeptide can inhibit proliferation of thyroid papilloma Mino cells in vitro, successfully inhibit tumor growth in a nude mouse tumor-bearing model experiment, increase survival rate of mice and have potential new drug development value; provides a better choice for the Cyclin E protein inhibitor polypeptide drug to treat human thyroid papilloma.

In another embodiment of the invention, a method for preparing the polypeptide is also provided. The preparation method of the polypeptide comprises the following steps:

s01: synthesizing a gene sequence for coding the polypeptide, and transferring the gene sequence into a pPIC9K plasmid to obtain a recombinant plasmid;

s02: amplifying the recombinant plasmid of step S01 with E.coli DH5 α, and then extracting the recombinant plasmid;

s03: transferring the recombinant plasmid extracted in the step S03 into escherichia coli BL21 for gene expression, and extracting the polypeptide of the embodiment of the invention.

The method for preparing the polypeptide by using the genetic engineering technology provided by the embodiment is simple and easy to operate, has low cost, can obtain the polypeptide with high concentration, effectively maintains the activity of the polypeptide, and is beneficial to the subsequent development and research of the polypeptide medicament.

The embodiment of the invention also provides application of the polypeptide in preparing a papillary thyroid tumor medicament.

Finally, the embodiment of the invention also provides a pharmaceutical composition. The medicinal composition comprises the polypeptide and pharmaceutically acceptable auxiliary materials.

Specifically, the dosage of the polypeptide in the medicinal composition is 6-24 mg/kg. Through research, when the dosage range of the polypeptide is 6-24mg/kg, the polypeptide has a very significant inhibiting effect on the growth of human thyroid papilloma cell Mino transplanted tumor. Compared with taxol, the polypeptide has no obvious influence on the body weight of experimental animals, has no obvious toxic or side effect, and obviously improves the survival rate.

Specifically, the auxiliary materials in the medicinal composition comprise at least one of a filling agent, a bonding agent, a lubricating agent, a disintegrating agent and a stabilizing agent. The auxiliary materials are selected conventionally and are determined according to the specific dosage form of the polypeptide.

Specifically, the pharmaceutical composition is in the form of injection. According to the practical application, the injection can be a solvent injection or a freeze-dried powder injection, the injection is a conventional choice of polypeptide drugs, and the activity of the polypeptide can be effectively maintained.

The invention is described in further detail with reference to a part of experimental results, which are described in detail with reference to specific examples.

Example 1

A polypeptide, the specific amino acid sequence of which is SEQ ID NO: 1: ETIRKAYPDANLVNDRVLR, the isoelectric point pI of the polypeptide is 8.69, the molecular weight Mw is 2243.55, and the preparation method is as follows.

S11: the gene sequence encoding the polypeptide is synthesized by the amino acid sequence of the polypeptide (SEQ ID NO:1), and the gene sequence is transferred into the pPIC9K plasmid to obtain a recombinant plasmid.

In the process, His-tag labels are respectively added in the front and back of a gene sequence for coding the polypeptide of the embodiment, a digestion enzyme cutting site is inserted between the gene sequence and the His-tag labels, and then the gene sequence is transferred into pPIC9K plasmid to obtain recombinant plasmid.

S12: the recombinant plasmid in the above step S11 was amplified with E.coli DH5 α, and then the E.coli DH5 α cells were lysed to extract a recombinant plasmid containing a gene sequence encoding the polypeptide.

In the above step S12, the process of DH5 α amplifying recombinant plasmid is:

after the recombinant plasmid transformed bacteria were activated overnight at 37 ℃, overnight bacterial liquid (250. mu.l) was aspirated at a ratio of 1:100 and added to 25ml LB liquid medium, followed by shaking culture at 37 ℃ and 200r/min for 2-3h to OD₆₀₀About 0.5. 25ml of the inoculum was transferred to a pre-cooled 50ml EP tube and allowed to stand on ice for 30min to cool the medium to 0 ℃. Centrifuging at 4 deg.C and 4000rpm for 10min, recovering cells, pouring out the culture medium, and placing the tube on absorbent paper for 1 min. 10ml of precooled 0.1mol/LCaCl per 50ml of bacterial liquid₂Resuspend each pellet in ice bath for 30 min. Centrifuging at 4000rpm for 10min at 4 deg.C, recovering cells, pouring out the culture solution, and placing the tube on absorbent paper for 1 min. Every 50ml of initial culture was 2ml of 0.1mol/L CaCl pre-cooled with ice₂Resuspend each cell pellet (containing 15% glycerol).

50 μ l of E.coli competent cells were taken, added to 4 μ l of plasmid, ice-washed for 30min, heat-shocked at 42 ℃ for 90s, immediately returned to ice, ice-washed for 2 min: adding 400 μ l LB culture medium, and culturing at 37 deg.C with shaking table for 45-60 min; 50-100. mu.l of the culture broth was applied to LB solid medium containing ampicillin, and cultured overnight at 37 ℃ in an inverted state. And (4) selecting a single colony growing on the LB solid culture medium, and being used for extracting the later-stage recombinant plasmid.

S13: transferring the extracted recombinant plasmid into escherichia coli BL21 for gene expression, finally cracking the escherichia coli BL21, and extracting the expressed polypeptide.

In the above step S13, the gene expression process in BL21 is: competent cells were first thawed on ice and mixed gently until homogeneous, and 100. mu.l was transferred to an EP tube. Adding the above extracted recombinant plasmid (below 10 ng), standing in ice for 30min, then at 42 deg.C for 45s, immediately transferring into ice and standing for 1-2 min. 900. mu.l of SOC medium pre-warmed at 37 ℃ was added and cultured with shaking at 37 ℃ for 1h (160-225 rpm). An appropriate amount of the culture broth was spread on an LB plate containing antibiotics and cultured overnight at 37 ℃. 50 μ l of Escherichia coli competent cells were taken, added with 4 μ l of recombinant plasmid, ice-washed for 30min, heat-shocked at 42 ℃ for 90s, immediately returned to ice, and ice-washed for 2 min. Adding 400 μ l LB culture medium, and culturing at 37 deg.C with shaking table for 45-60 min; 50-100. mu.l of the culture broth was applied to LB solid medium containing ampicillin, and cultured overnight at 37 ℃ in an inverted state.

In the above step S13, the BL21 cracking process is: single colonies grown on LB solid medium were picked and inoculated into 20ml of LB liquid medium (containing Amp100ug/ml) and cultured overnight (about 12-14hr) at 37 ℃ under 250rmp shaking. The culture solution was collected, centrifuged at 4 ℃ for 10min, and the precipitated cells were collected. 1-2g of the cells were mixed with 10ml of disruption buffer on ice for 45 min. The mixed thallus is crushed for 20 minutes in ice water by an ultrasonic probe, the crushed thallus is crushed for four times, the interval between the crushed thallus and the ice water is 2min, and the pH value is controlled to be 7-8. The disruption solution was then centrifuged at 12000g at 4 ℃ for 10min, and 0.12ml of a 2M imidazole solution was added to the disrupted and centrifuged supernatant to give a final concentration of 20mM, and the total volume of the sample was 10 ml.

In the step S13, the polypeptide extraction process includes: an equilibration buffer (50 mM phosphate buffer, pH7.4, containing 0.5M NaCl and 20mM imidazole) and an elution buffer (50 mM phosphate buffer, pH7.4, containing 0.5M NaCl and 500mM imidazole) were prepared. 1ml of nickel sepharose FF prepacked column was equilibrated with 10ml of equilibration buffer, and then 10ml of the disrupted supernatant was sampled at 0.5ml/min and collected in 2ml tubes. Unadsorbed samples were washed off with 15ml of equilibration buffer, flow 1-2ml/min, 2ml tube collection. Washing off unadsorbed sample with 5ml of elution buffer at a flow rate of 1-2ml/min, collecting with a 2ml tube, equilibrating the column with 5ml of equilibration buffer, filling with 20% ethanol, and sealing for next use.

Example 2

Experiment for verifying that polypeptide (SEQ ID NO:1) competitively inhibits the binding of Cyclin E protein and CDK2 protein. In the experiment, competitive enzyme-linked immunosorbent assay is adopted to detect the competitive inhibition effect of the polypeptide on the combination of Cyclin E protein and CDK2 protein.

First, 35. mu.g/ml CDK2 protein was injected into 24-well plates at 100. mu.l/well. The polypeptide and the Cyclin E protein labeled with horseradish peroxidase were diluted to 35ug/ml and mixed at 1: 1. After incubation at 37 ℃ was complete, dilutions were added to 24-well plates (200. mu.l/well) to allow competitive binding to CDK2 protein. An enzyme-labeled Cyclin E protein solution (100. mu.l/well) was added to the control group, and an equal amount of solvent was mixed to directly bind CDK2 protein. Respectively washing to remove unbound components, adding excessive substrate o-biphenylmethylamine, fully developing and washing, and measuring absorbance value (measuring wavelength is 425nm) with an enzyme-labeling instrument. The influence of the polypeptide on competitive inhibition of the binding of Cyclin E protein and CDK2 protein is reflected by calculating the absorbance ratio.

The inhibition ratio (PI) is calculated by the following formula: PI (%) ═ 1-experimental/control. Finally, the results of the polypeptide competitively inhibiting the binding of Cyclin E protein and CDK2 protein are shown in Table 1; from the data in table 1, it can be seen that: when 35 mug/ml of the polypeptide is added, the polypeptide has obvious inhibition effect on the binding activity of Cyclin E protein.

TABLE 1

Example 3

Test for verifying the Mino migration effect of polypeptide (SEQ ID NO:1) on human thyroid papilloma cells. This experiment used a scratch test to detect the mobility of the polypeptide inhibiting the Mino cells.

Firstly, a marker pen is used at the back of a 12-hole plate, the 12-hole plate is touched by a ruler, transverse lines are uniformly drawn, the transverse lines cross through holes every 0.5-1 cm, each hole penetrates 3 lines, and the Mino cells in logarithmic phase are drawn to be 1.0 × 10⁵Adding into 12-hole culture plate, and culturing overnight for 12 hr. The next day, the ruler is compared with a 10-microliter gun head, the scratch is vertical to the transverse line at the back, and the intersection point of the scratch and the transverse line at the back is used as a fixed observation point; adding experimental drug (polypeptide) and positive control drug (vincristine) with different concentrations into the experimental hole and the positive control hole respectively; adding solvent with the same volume into the blank group, and arranging five multiple holes in each hole; adding 5% CO at 37 deg.C₂And (5) an incubator for culture. Sampling at 0, 6, 12, 24 and 48 hours, and taking a picture; scratch widths were measured at 0, 6, 12, 24, 48 hours. At different time points, the change in scratch width at three fixed positions per well was recorded as the cell migration distance.

The Mobility (MR) calculation formula is: mobility (MR) ═ scratch width at experimental hour 0-scratch width at experimental hour n)/scratch width at blank group hour 0 × 100%. Finally, the polypeptide competitively inhibits the Mino migration inhibition of human thyroid papilloma cells as shown in Table 2; when the dosage of the polypeptide is 35 mu g/ml, no significant difference exists, the effect is equivalent to that of a positive group, and the fact that the polypeptide can inhibit migration of human thyroid papilloma cells Mino is shown.

TABLE 2

Example 4

Test for verifying the proliferation effect of polypeptide (SEQ ID NO:1) on human thyroid papillary carcinoma cells Mino. The activity of the polypeptide for inhibiting the growth of Mino cells is detected by adopting an MTT method in the experiment.

Mino cells at 37 ℃ with 5% CO₂When the cells were cultured in the incubator of (1) to a confluency of 90% or more, they were digested with trypsin and collected, and the cells were resuspended in a culture medium and counted under a microscope to adjust the cell concentration to 2 × 10⁴One/ml, the cell suspension was seeded into 96-well plates at 100. mu.l/well and at 37 ℃ in 5% CO₂The culture was carried out overnight in an incubator. The polypeptides were diluted with culture medium to various predetermined concentrations. The concentration was diluted to the final concentration with the culture broth. After the cells were fully adherent, each dilution was added to a 96-well plate (100. mu.l/well). The polypeptide diluent is added as an administration group, paclitaxel and endostatin are added as a positive control group, and the culture solution without any medicine is added as a negative control group. At 5% CO₂Incubate at 37 ℃ for 48 h. To a 96-well plate, 20. mu.l of MTT was added at 5mg/ml per well, and the culture was continued for 4 hours. Absorbing the culture medium, adding 150 mu l DMSO into each hole for dissolving, and shaking for 10 minutes to mix gently; the absorbance was measured at a measurement wavelength of 570nm with a microplate reader.

The formula of the growth inhibition rate (PI) is as follows: PI (%) ═ 1-dose group/negative group; the results obtained from the experiments are expressed as mean ± SD and subjected to a statistical T-test, with 0.01 + P <0.05 for significant differences and 0.01 for very significant differences. The result of the inhibition effect of the polypeptide on human thyroid papillary carcinoma Mino cell proliferation is shown in Table 3; as can be seen from the data in Table 3, compared with the negative control, the polypeptide can significantly inhibit the proliferation of Mino cells in vitro and presents obvious dose dependence.

TABLE 3

Example 5

Test for verifying the inhibiting effect of polypeptide (SEQ ID NO:1) on the growth of human thyroid papillary carcinoma cell Mino nude mouse xenograft tumor.

Preparing human papillary thyroid tumor cell Mino cell strain of logarithmic growth phase into 5 × 10 under aseptic condition⁷The cell suspension is inoculated in 0.1ml under the right axilla of the nude mouse. Measuring the diameter of the transplanted tumor of the nude mouse by using a vernier caliper until the tumor grows to 100-³Animals were then randomized into groups. The antitumor effect of the tested polypeptide is dynamically observed by using a method for measuring the tumor size. Tumor diameter was measured 1 time every 2 days. The administration mode adopts tail vein injection. Injecting equal amount of normal saline into the negative control group for 1 time per day; paclitaxel group 12mg/kg, administered 1 time per week; the dose of the Endoku group is 3mg/kg, and the administration is carried out for 1 time per day; the administration of the polypeptide group at 24mg/kg, 12mg/kg, and 6mg/kg is performed 1 time per day. After the experiment was completed, the mice were sacrificed and the tumor mass was surgically removed and weighed.

The result of the inhibition effect of the polypeptide on the growth of the human thyroid papilloma cell Mino nude mouse xenograft tumor is shown in Table 4.

TABLE 4

The above results show that: the tumor inhibition rate of the taxol 12mg/kg group to human thyroid papilloma cell Mino nude mouse transplanted tumor is 73.16%; the tumor inhibition rate of the Endu 3mg/kg group to human thyroid papilloma cell Mino nude mouse transplanted tumor is 32.56%; the tumor inhibition rates of the high, medium and low dosage polypeptide groups on human thyroid papilloma cell Mino nude mouse transplanted tumor reach 75.21%, 73.68% and 67.73% respectively. However, the toxicity of the taxol is high, the weight of the animal is reduced, the animal dies in the experimental process, and the polypeptide has no significant influence on the weight of a nude mouse. Therefore, compared with a negative control group, the polypeptides of 24mg/kg, 12mg/kg and 6mg/kg have extremely significant inhibition effect on the growth of the human thyroid papilloma cell Mino transplantation tumor. Compared with the positive control group paclitaxel, the polypeptide has no obvious influence on the body weight of the experimental animal, has no obvious toxic or side effect, and improves the survival rate.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

SEQUENCE LISTING

<110> Shenzhen Huazhong medical inspection institute Limited

<120> polypeptide, preparation method and application thereof, and pharmaceutical composition

<160>1

<170>PatentIn version 3.3

<210>1

<211>19

<212>PRT

<213> Artificial sequence

<400>1

Glu Thr Ile Arg Lys Ala Tyr Pro Asp Ala Asn Leu Val Asn Asp Arg

1 5 10 15

Val Leu Arg

Claims

1. A Cyclin E protein inhibitory polypeptide is characterized in that the amino acid sequence is shown as SEQ ID NO. 1.

2. A method for preparing a polypeptide, comprising the steps of:

synthesizing a gene sequence for coding the Cyclin E protein inhibitory polypeptide according to claim 1, and transferring the gene sequence into a pPIC9K plasmid to obtain a recombinant plasmid;

transferring the extracted recombinant plasmid into escherichia coli BL21 for gene expression, and extracting the Cyclin E protein inhibitory polypeptide.

3. The use of the Cyclin E protein-inhibiting polypeptide of claim 1 in the preparation of a medicament for the treatment of thyroid papilloma.

4. A pharmaceutical composition comprising the Cyclin E protein inhibiting polypeptide of claim 1 and a pharmaceutically acceptable excipient.

5. The pharmaceutical composition of claim 4, wherein said Cyclin E protein-inhibiting polypeptide is administered in an amount of 6-24 mg/kg.

6. The pharmaceutical composition of claim 4, wherein the excipient comprises at least one of a filler, a binder, a lubricant, a disintegrant, and a stabilizer.

7. The pharmaceutical composition of claim 4 or 5, wherein the pharmaceutical composition is in the form of an injection.