CN106589100B - Anti-angiogenesis lamprey recombinant PR-1 protein and preparation method thereof - Google Patents

Abstract

The invention discloses a lamprey recombinant PR-1 protein capable of resisting angiogenesis, wherein the cDNA and the amino acid sequence of the lamprey recombinant PR-1 protein are respectively shown as SEQ ID No.1 and SEQ ID No. 2. Connecting PCR product (gene segment) of lamprey CRBGP to expression vector by molecular cloning method, and culturing in Escherichia coliRosetta blueThe recombinant protein of the lamprey PR-1 with a cDNA sequence containing 510 bp and totally encoding 170 amino acids is obtained after the induced expression and the denaturation and renaturation. The obtained lamprey recombinant PR-1 protein has the function of inhibiting angiogenesis, has high yield and small molecular weight compared with lamprey CRBGP recombinant protein, and is suitable for industrial large-scale production.

Description

Anti-angiogenesis lamprey recombinant PR-1 protein and preparation method thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a lamprey recombinant PR-1 protein with high yield and small molecular weight and capable of resisting angiogenesis and a preparation method thereof.

Background

Angiogenesis refers to the formation of new blood vessels from existing blood vessels. In vivo, angiogenesis is a complex process involving a variety of cells and molecules, including degradation of vascular basement membrane (activation phase), activation, proliferation, migration of endothelial cells (e.g., Human umbilical vein endothelial cells, HUVECs), and finally, the reconstruction and formation of new blood vessels and their network structures. Angiogenesis is important for embryonic development and tissue repair processes (e.g., wound healing and luteal formation) during normal physiological processes, but causes various diseases when angiogenesis-promoting factors and inhibitory factors are disturbed in vivo. In addition to the fact that abnormal angiogenesis has a significant influence on the occurrence, development and metastasis of tumors, there are successive research reports that the unexpected angiogenesis is closely related to various chronic inflammatory diseases, such as diabetes, age-related macular degeneration, choroidal neovascularization, psoriasis, myelodysplastic syndrome, ischemic diseases, rheumatoid arthritis and the like. The development of low-toxicity and high-efficiency anti-angiogenesis drugs is a hot spot for clinically treating diseases related to angiogenesis abnormality.

The lamprey CRBGP is prepared from Japanese lamprey (Lampetra japonica)Lampetra japonica) A novel secreted protein rich in Cysteine, which is separated and purified from oral glands, is named as secreted protein rich in Cysteine-rich oral gland (CRBGP) because of high homology with members of the CRISP family of secreted proteins rich in Cysteine (CRISPs) and 16 highly conserved Cysteine residues. The cDNA sequence (open reading frame) of the lamprey CRBGP contains 774bp, the protein of the lamprey CRBGP consists of 257 amino acids, and the molecular weight is 25.6 kDa through mass spectrometry. The lamprey protein rich in cysteine and secreted protein CRBGP is Na⁺Channel blocker capable of blocking Na not only in hippocampal neurons and dorsal root neurons⁺The electrical current can also inhibit the excitability of neurons by reducing the frequency and amplitude of the hippocampal and dorsal root neuron action units. The invention patent with the Chinese patent number of 201410528552.X discloses the application of lamprey CRBGP in the preparation of anti-angiogenesis drugs. However, the lamprey CRBGP gene sequence is long and has 8 pairs of disulfide bonds, the expression of recombinant protein is relatively difficult, the expression yield is low, and the application in industry is difficult. Although the gene segments are known to those skilled in the art to have the characteristics of small molecular weight and relatively easy protein expression, not all gene segments have the advantages of strong drug effect of expressed proteins, good targeting property, industrial value and the like. Therefore, no report about that the gene segment and the expression protein thereof in the lamprey CRBGP have the function of inhibiting angiogenesis exists so far.

Disclosure of Invention

The invention aims to solve the technical problems in the prior art and provides the lamprey recombinant PR-1 protein which has high yield and small molecular weight and can resist angiogenesis and a preparation method thereof.

The technical solution of the invention is as follows: an anti-angiogenic lamprey recombinant PR-1 protein is characterized in that the cDNA and amino acid sequence of the anti-angiogenic lamprey recombinant PR-1 protein are respectively shown as SEQ ID No.1 and SEQ ID No. 2.

The preparation method of the lamprey recombinant PR-1 protein capable of resisting angiogenesis is characterized by comprising the following steps of:

step 1: carrying out PCR reaction by taking cDNA of the lamprey CRBGP as a template, wherein the sequence of a PCR reaction primer is as follows:

an upstream primer: 5' -CCGGAATTCACATCCGTCAACGACTGGAAGCTC-3

A downstream primer: 5'-ATTTGCGGCCGCGTAGGGTTTGTTGATCCTGGTGA-3'

Step 2: performing double enzyme digestion on the PCR product and the prokaryotic expression vector respectively, then connecting, and adopting CaCl as the connection product₂Method for transformation into clonal bacteriumRosetta blueScreening positive transformants;

step 3, carrying out induction expression on the positive transformant for 18 hours at 16 ℃ by using isopropyl- β -D-thiogalactoside with the final concentration of 0.3 mM;

and 4, step 4: centrifuging the bacteria liquid after induction expression at 4 ℃ at 7500 r/min for 15 min, and collecting the thallus precipitate;

and 5: washing the obtained thallus precipitate with 20 mM Tris-HCl with the pH value of 8.0, then resuspending thallus by adopting a thallus breaking solution, ultrasonically breaking the thallus on ice for 30 minutes, centrifuging the thallus precipitate for 20 minutes at 10000 r/min, and collecting the precipitate;

step 6: washing the precipitate with inclusion body washing liquid for 2 times, centrifuging at 10000 rpm for 10 minutes, and collecting the precipitate;

and 7: adding the precipitate into the denatured liquid A, stirring thoroughly with a magnetic stirrer at 4 deg.C to clarify, centrifuging at 10000 rpm for 30 min, collecting the supernatant, packaging the supernatant and the renaturation liquid A into a dialysis bag, and concentrating with polyethylene glycol;

and 8: putting the dialysis bag into 20 mM Tris-HCl buffer solution, dialyzing overnight at 4 ℃ to precipitate white protein liquid;

and step 9: centrifuging the separated white protein liquid at 10000 rpm for 30 minutes, collecting the precipitate, adding a denatured liquid B into the precipitate, fully stirring the mixture by using a magnetic stirrer at 4 ℃ until the mixture is transparent, centrifuging the mixture at 10000 rpm for 30 minutes, and collecting the supernatant;

step 10: adding the supernatant into the renaturation solution B, fully stirring for 24 hours at 4 ℃ by using a magnetic stirrer, transferring the mixture into a dialysis bag, placing the dialysis bag into 20 mM Tris-HCl with the pH value of 8.0, fully stirring for 5 hours at 4 ℃ by using the magnetic stirrer, centrifuging for 30 minutes at 10000 r/min, and collecting the supernatant;

the solutions were as follows:

bacteria liquid breaking: 50 mM Tris-HCl, 50 mM NaCl, 1 mM EDTA, pH 8.0;

the inclusion body washing solution comprises 50 mM Tris-HCl, 50 mM NaCl, 2M urea, 1 mM EDTA, 0.5% TritonX-100, 2 mM β -mercaptoethanol and pH 8.0;

denaturant A is 50 mM Tris-HCl, 50 mM NaCl, 100 mM β -mercaptoethanol, 7M guanidine hydrochloride, pH 8.0;

renaturation solution A, 50 mM Tris-HCl, 50 mM NaCl, 1 mM EDTA, 100 mM β -mercaptoethanol, pH 8.0;

denaturant B is 50 mM Tris-HCl, 50 mM NaCl, 100 mM β -mercaptoethanol, 8M urea, pH 8.0;

renaturation liquid B: 50 mM Tris-HCl, 0.75M L-Arg, 5 mM reduced glutathione, 0.5 mM oxidized glutathione, pH 8.0.

The invention designs a primer, connects a PCR product (gene segment) of the lamprey CRBGP to an expression vector by utilizing a molecular cloning means, and connects the PCR product (gene segment) to escherichia coliRosetta blueThe recombinant protein of the lamprey PR-1 with a cDNA sequence containing 510 bp and totally encoding 170 amino acids is obtained after the induced expression and the denaturation and renaturation. The obtained lamprey recombinant PR-1 protein has the function of inhibiting angiogenesis, has high yield and small molecular weight compared with lamprey CRBGP recombinant protein, and is suitable for industrial large-scale production.

Drawings

FIG. 1 is a schematic diagram showing that the lamprey recombinant PR-1 protein of the embodiment of the invention inhibits the cell proliferation of HUVECs.

FIG. 2 is a schematic diagram showing that the lamprey recombinant PR-1 protein inhibits the differentiation of HUVECs cells into tubules in the embodiment of the invention.

Detailed Description

The method comprises the following steps of:

step 1: carrying out PCR reaction by taking cDNA of the lamprey CRBGP as a template, wherein the sequence of a PCR reaction primer is as follows:

an upstream primer: 5' -CCGGAATTCACATCCGTCAACGACTGGAAGCTC-3

A downstream primer: 5'-ATTTGCGGCCGCGTAGGGTTTGTTGATCCTGGTGA-3'

Step 2: performing double enzyme digestion on the PCR product and a vector pET-42a respectively, performing overnight ligation, and adopting CaCl as a ligation product₂Method for transformation into clonal bacteriumRosetta blueScreening positive transformants;

step 3, carrying out induction expression on the positive transformant for 18 hours at 16 ℃ by using isopropyl- β -D-thiogalactoside (IPTG) with the final concentration of 0.3 mM;

and 4, step 4: centrifuging the bacteria liquid after induction expression at 4 ℃ at 7500 r/min for 15 min, and collecting the thallus precipitate;

and 5: washing the obtained thallus precipitate with 20 mM Tris-HCl with the pH value of 8.0, then resuspending thallus by adopting a thallus breaking solution, ultrasonically breaking the thallus on ice for 30 minutes, centrifuging the thallus precipitate for 20 minutes at 10000 r/min, and collecting the precipitate;

step 6: washing the precipitate with inclusion body washing liquid for 2 times, centrifuging at 10000 rpm for 10 minutes, and collecting the precipitate;

and 7: adding the precipitate into the denatured liquid A, stirring thoroughly with a magnetic stirrer at 4 deg.C to clarify, centrifuging at 10000 rpm for 30 min, collecting the supernatant, packaging the supernatant and the renaturation liquid A into a dialysis bag, and concentrating with polyethylene glycol to 17 ml;

and 8: putting the dialysis bag into 20 mM Tris-HCl buffer solution, dialyzing overnight at 4 ℃ to precipitate white protein liquid;

and step 9: centrifuging the separated white protein liquid at 10000 rpm for 30 minutes, collecting the precipitate, adding a denatured liquid B into the precipitate, fully stirring the mixture by using a magnetic stirrer at 4 ℃ until the mixture is transparent, centrifuging the mixture at 10000 rpm for 30 minutes, and collecting the supernatant;

step 10: adding the supernatant into the renaturation solution B, fully stirring the mixture for 24 hours at the temperature of 4 ℃ by using a magnetic stirrer, transferring the mixture into a dialysis bag, placing the dialysis bag into 20 mM Tris-HCl with the pH value of 8.0, fully stirring the mixture for 5 hours at the temperature of 4 ℃ by using the magnetic stirrer, centrifuging the mixture for 30 minutes at the speed of 10000 r/min, and collecting the supernatant.

The solutions were as follows:

bacteria liquid breaking: 50 mM Tris-HCl, 50 mM NaCl, 1 mM EDTA, pH 8.0;

the inclusion body washing solution comprises 50 mM Tris-HCl, 50 mM NaCl, 2M urea, 1 mM EDTA, 0.5% TritonX-100, 2 mM β -mercaptoethanol and pH 8.0;

denaturant A is 50 mM Tris-HCl, 50 mM NaCl, 100 mM β -mercaptoethanol, 7M guanidine hydrochloride, pH 8.0;

renaturation solution A, 50 mM Tris-HCl, 50 mM NaCl, 1 mM EDTA, 100 mM β -mercaptoethanol, pH 8.0;

denaturant B is 50 mM Tris-HCl, 50 mM NaCl, 100 mM β -mercaptoethanol, 8M urea, pH 8.0;

renaturation liquid B: 50 mM Tris-HCl, 0.75M L-Arg, 5 mM reduced glutathione, 0.5 mM oxidized glutathione, pH 8.0.

The collected supernatant is the target protein, and the cDNA and amino acid sequences of the target protein are respectively shown in SEQ ID NO.1 and SEQ ID NO.2 through sequencing, and the target protein is named as lamprey recombinant PR-1 protein (rL-PR-1).

Experiment:

firstly, the MTT method is used for measuring the inhibition effect of rL-PR-1 on the cell proliferation of HUVECs.

(1) HUVECs cells were first placed in RPMI 1640 medium containing 10% Fetal Bovine Serum (FBS) and seeded into 96-well cell culture plates for 24 hours;

(2) adding PBS and rL-PR-1 with different concentrations into HUVECs cells to continue culturing for 24 hours;

(3) MTT solution was added to a final concentration of 0.5 mg/ml, CO at 37 deg.C₂Continuously culturing for 4 hours in the incubator;

(4) gently sucking out the culture solution, adding dimethyl sulfoxide into a 96-well plate, and performing shake culture in a shaker at 37 ℃ for 10 minutes;

(5) detecting the absorbance value of the HUVECs at 492 nm by using a microplate reader (the average value of each group of experiments is obtained by 3 times of experiments);

(6) HUVECs in the PBS-treated group were used as a control group, and the absorbance (OD value) at 492 nm was defined as 100%, and the proliferation rate (%) of HUVECs in the rL-PR-1-treated group was (%) = OD value in the rL-PR-1-treated group/OD value in the PBS-treated group × 100%.

The results are shown in FIG. 1, which shows that rL-PR-1 of the invention can inhibit the proliferation of Human Umbilical Vein Endothelial Cells (HUVECs) in a dose-dependent manner, and the IC thereof is₅₀At 2. mu.M.

Secondly, the in-vitro anti-angiogenesis function of rL-PR-1 is determined by using HUVECs cell tubule formation experiment

(1) Firstly, adding artificial basement membrane Matrigel to the surface of a 24-well plate, and incubating for 30 minutes at 37 ℃;

(2) HUVECs cells were suspended in RPMI 1640 medium containing 10% FBS;

(3) mixing cultured HUVECs with PBS and rL-PR-1 (1.5 μ M and 3.0 μ M) with different concentrations, adding into the 24-well culture plate, and culturing for 16 hr;

(4) photographing in three visual field areas randomly by adopting an inverted microscope;

(5) the surface area of the tubules formed by the differentiation of HUVECs cells was analyzed by NIS-Elements D software, and the results are shown in FIG. 2.

Endothelial cell differentiation to form tubular structures is reported to be one of the key steps in the angiogenic process. In an in vitro artificial basement membrane (Matrigel) tubule formation experiment, HUVECs cells in the PBS-treated group were able to differentiate into tubules, and these tubules were connected to each other to form a cross-linked network structure. At this time, the number of tubules formed by differentiation of the HUVECs is relatively large, and the surface area is relatively large. It can be seen from FIG. 2 that in the same experiment, the number and surface area of tubules formed by differentiation of HUVECs after the HUVECs are treated with 1.5. mu.M of rL-PR-1 are significantly reduced, and the tubulesThe network structure formed by the mutual connection is destroyed. When HUVECs cells were treated with 3. mu.M rL-PR-1, essentially no intact tubule structures and network structures formed therefrom were observed in the microscope. rL-PR-1 at 1.5. mu.M or 3. mu.M was able to reduce the surface area of the tubules to 30.07% + -4.79%, respectively, (of the control group)P<0.01) and 89.47% + -6.67% ((R)P<0.001). Indicating that rL-PR-1 can inhibit HUVECs from differentiating into tubules in vitro in a dose-dependent manner.

Sequence listing

<110> university of Liaoning teacher

<120> anti-angiogenesis lamprey recombinant PR-1 protein and preparation method thereof

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<170>PatentIn version 3.2

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gtg gtg ccc acc gcc agc aac atg ctc aag atg gcg tac aac gaa

cag gca gca gag acc agc cgc ttg tgg gcc gcc gcc tgc agc ttc

tcg cac agc ccc agc aac acg cgc acc tgg aag acg ccg caa gca

gag tgg gac tgc gga gag aac ctc ttc atg tcc agc aac cca cgg

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ccc ggc ttc cag tac ggc acg ggg gct gtg ggg ccc ggg gcc gtg

gga cac tac act cag gtg gtg tgg tac aag tcc cac cag gtg ggc

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ctc tac gtg tgc cac tac tgc ccc gca ggg aac ctg gtc acc agg

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T S V N D W K L L D T K L S A

N R K V I V D V H N E L R R G

V V P T A S N M L K M A Y N E

Q A A E T S R L W A A A C S F

S H S P S N T R T W K T P Q A

E W D C G E N L F M S S N P R

S W D E A V R S W Y D E V T S

P G F Q Y G T G A V G P G A V

G H Y T Q V V W Y K S H Q V G

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atttgcggccgcgtagggtttgttgatcctggtga 35

Claims (2)

1. An anti-angiogenic lamprey recombinant PR-1 protein is characterized in that the cDNA and amino acid sequence of the anti-angiogenic lamprey recombinant PR-1 protein are respectively shown as SEQ ID No.1 and SEQ ID No. 2.

2. The method for preparing the anti-angiogenic lamprey recombinant PR-1 protein according to claim 1, which comprises the following steps:

step 1: carrying out PCR reaction by taking cDNA of the lamprey CRBGP as a template, wherein the sequence of a PCR reaction primer is as follows:

an upstream primer: 5'-CCGGAATTCACATCCGTCAACGACTGGAAGCTC-3'

A downstream primer: 5'-ATTTGCGGCCGCGTAGGGTTTGTTGATCCTGGTGA-3'

Step 2: performing double enzyme digestion on the PCR product and a prokaryotic expression vector pET-42a respectively, then connecting, and adopting CaCl as the connecting product₂Method for transformation into clonal bacteriumRosetta blueScreening positive transformants;

step 3, carrying out induction expression on the positive transformant for 18 hours at 16 ℃ by using isopropyl- β -D-thiogalactoside with the final concentration of 0.3 mM;

and 4, step 4: centrifuging the bacteria liquid after induction expression at 4 ℃ at 7500 r/min for 15 min, and collecting the thallus precipitate;

and 5: washing the obtained thallus precipitate with 20 mM Tris-HCl with the pH value of 8.0, then resuspending thallus by adopting a thallus breaking solution, ultrasonically breaking the thallus on ice for 30 minutes, centrifuging the thallus precipitate for 20 minutes at 10000 r/min, and collecting the precipitate;

step 6: washing the precipitate with inclusion body washing liquid for 2 times, centrifuging at 10000 rpm for 10 minutes, and collecting the precipitate;

and 7: adding the precipitate into the denaturation solution A, and stirring thoroughly at 4 deg.C with a magnetic stirrer

Clarifying, centrifuging at 10000 rpm for 30 minutes, collecting supernatant, filling the supernatant and renaturation liquid A into a dialysis bag, and concentrating with polyethylene glycol;

and 8: putting the dialysis bag into 20 mM Tris-HCl buffer solution, dialyzing overnight at 4 ℃ to precipitate white protein liquid;

and step 9: centrifuging the separated white protein liquid at 10000 rpm for 30 minutes, collecting the precipitate, adding a denatured liquid B into the precipitate, fully stirring the mixture by using a magnetic stirrer at 4 ℃ until the mixture is transparent, centrifuging the mixture at 10000 rpm for 30 minutes, and collecting the supernatant;

step 10: adding the supernatant into the renaturation solution B, fully stirring for 24 hours at 4 ℃ by using a magnetic stirrer, transferring the mixture into a dialysis bag, placing the dialysis bag into 20 mM Tris-HCl with the pH value of 8.0, fully stirring for 5 hours at 4 ℃ by using the magnetic stirrer, centrifuging for 30 minutes at 10000 r/min, and collecting the supernatant;

the solutions were as follows:

bacteria liquid breaking: 50 mM Tris-HCl, 50 mM NaCl, 1 mM EDTA, pH 8.0;

inclusion body washing solution 50 mM Tris-HCl, 50 mM NaCl, 2M urea, 1 mM EDTA, 0.5% Triton X-100, 2 mM β -mercaptoethanol, pH 8.0;

denaturant A is 50 mM Tris-HCl, 50 mM NaCl, 100 mM β -mercaptoethanol, 7M guanidine hydrochloride, pH 8.0;

renaturation solution A, 50 mM Tris-HCl, 50 mM NaCl, 1 mM EDTA, 100 mM β -mercaptoethanol, pH 8.0;

denaturant B is 50 mM Tris-HCl, 50 mM NaCl, 100 mM β -mercaptoethanol, 8M urea, pH 8.0;

renaturation liquid B: 50 mM Tris-HCl, 0.75M L-Arg, 5 mM reduced glutathione, 0.5 mM oxidized glutathione, pH 8.0.

Images