CN115724994A - Endothelin receptor A fusion mutant protein and application thereof - Google Patents

Abstract

The invention discloses an endothelin receptor A fusion mutant protein and application thereof, relating to the technical field of protein expression, wherein the fusion mutant protein EDNRA is composed of the following components (1) - (3): (1) endothelin receptor a mutant sequences; (2) Replacing the signal peptide sequence of the N end with an HA signal peptide sequence, and then sequentially adding a TEV enzyme cutting site and a Flag label; (3) And adding a 3C enzyme cutting site at the C end, and then sequentially adding a GFP protein sequence and a 10His tag protein sequence. The invention utilizes the silkworm larva infected by the recombinant silkworm baculovirus to efficiently express the fusion protein, has higher activity than the activity directly expressed by prokaryotes and higher yield than the yield expressed by transgenic plants; the silkworm can be fed aseptically and massively, can be fed at low cost and massively, and does not cause public nuisance; the silkworm body contains multiple natural protein protective agents, which have protective effect on expression products and make the gene expression products more stable.

Description

Endothelin receptor A fusion mutant protein and application thereof

Technical Field

The invention relates to the technical field of protein expression, in particular to an endothelin receptor A fusion mutant protein and application thereof.

Background

Membrane proteins play an important role in maintaining the physiological functions and signaling of cells, more than 40% of FDA-approved drug targets are membrane proteins, and the family of G-protein-coupled receptors (GPCRs) is the largest membrane protein family in humans and is also an important target for many drugs. There are more than 800 members of this family. Statistically, a total of 475 FDA-approved drugs targeting GPCRs were obtained, accounting for 34% of all FDA-approved drugs. In recent 5 years, 69 new drugs targeting GPCRs have been approved by the FDA in the united states, and 321 drugs in the clinical phase are targeted to GPCRs, wherein 60 (19%) drugs target innovative GPCRs. The drug sales for targeting GPCRs account for 27% of the global market. GPCRs have been one of the important targets for drug development because they are able to regulate a wide variety of physiological processes and have druggable targets on the cell surface.

Endothelin (ET), a bioactive substance consisting of 21 amino acids, has significant vasoconstrictive activity, and can enhance smooth muscle and myocardial contraction, promote neuroendocrine function, induce angiogenesis, promote differentiation, and promote cell mitosis, and is also involved in the occurrence and development of various diseases, such as cancer, verification, hypertension, congestive heart failure, atherosclerosis, subarachnoid hemorrhage, and the like, and is involved in the physiological and pathological processes by binding to Endothelin receptors (EDNRs). Endothelin receptors belong to the GPCR family, having a size of 45-50KDa, and up to now a total of three homologous isomers have been found, namely Endothelin receptor A (EDNRA), endothelin receptor B (EDNRB), endothelin receptor C (EDNRC), wherein EDNRA and EDNRB are present in mammals and humans. Among them, EDNRA is found in various malignant diseased tissues such as: high expression is found in colorectal cancer, ovarian cancer and prostate cancer, which indicates that EDNRA is highly related to the pathology of these malignant tumors, so that the development of EDNRA-related small molecule inhibitors has unlimited prospects for the treatment of many malignant tumors.

The three-dimensional structure of the protein plays an important role in understanding the biological function and the design of the small molecule drug, the analysis of the crystal structure of the small molecule and protein compound can help people analyze how the small molecule and the protein are combined and interacted, then the small molecule drug is redesigned according to the obtained information, so that the small molecule drug with strong binding force and good specificity is obtained, a large amount of endothelin receptor A protein is needed for obtaining the crystal structure of the endothelin receptor A protein and the small molecule, however, the expression amount of the endothelin receptor A in insect cells and mammalian cells is very low, and the protein which is required to be purified to a sufficient amount is very difficult.

The silkworm bioreactor is considered as the individual expression system with the most economic significance at present, is called as a gene engineering product 'micro fermentation tank', and has great superiority in expressing protein by using silkworm larvae: 1. under the regulation and control of a powerful polyhedrin promoter, the expression level of the exogenous gene is high and is 100-1000 times of that of insect cells and mammalian cell proteins, and the expression level can reach milligram-grade per silkworm; 2. the protein is effectively processed, transported, glycosylated, lipidated, phosphorylated and the like after being translated, and the biological activities of antigenicity, enzyme activity and the like of an expression product are comparable to those of a natural protein; 3. silkworm hemolymph has the capacity of storing protein, contains an inhibitor of protease in the hemolymph, plays a role in protecting target protein, and foreign protein is easily separated and purified from silkworm body fluid; 4. compared with insect and mammal cells, the silkworm has strong resistance to toxic protein; 5. the silkworm fat body cells contain rich lipid and have great effects on stabilizing membrane protein and easily degrading protein; 6. the silkworm is easy to be fed in large scale, and the cost is lower than that of the insect culture medium.

Based on the content, the method for efficiently expressing and producing the endothelin receptor A in the silkworm larvae by utilizing the silkworm-silkworm baculovirus expression system is provided.

Disclosure of Invention

The invention aims to provide a human endothelin receptor A fusion mutant protein and a method for efficiently expressing and producing an endothelin receptor A mutant in silkworm larvae by using a silkworm baculovirus expression system.

The invention realizes the purpose through the following technical scheme:

the invention provides an endothelin receptor A fusion mutant protein, which is EDNRA and consists of the following (1) to (3):

(1) An endothelin receptor A mutant sequence, wherein the mutation in the mutant sequence is one of (a) or (b):

(a) C379A, C a and C388A;

(b) R103Y, D A, Q252A, S A and I364A;

(2) Replacing the signal peptide sequence at the N end with an HA signal peptide sequence, and then sequentially adding a TEV enzyme cutting site and a Flag label;

(3) And adding a 3C enzyme cutting site at the C end, and then sequentially adding a GFP protein sequence and a 10His tag protein sequence.

The further improvement is that when the mutation in the mutant sequence in the fusion mutant protein EDNRA is (a) triple mutation, the amino acid sequence is shown as SEQ ID NO. 3; when the mutation in the mutant sequence in the fusion mutant protein EDNRA is (b) five mutations, the amino acid sequence of the fusion mutant protein EDNRA is shown in SEQ ID NO. 1.

The further improvement is that the signal peptide sequence at the N terminal is 20 amino acids at the N terminal of the wild type amino acid sequence of the endothelin receptor A.

The invention also provides a polynucleotide, which encodes the fusion mutant protein EDNRA.

In a further improvement, the sequence of the polynucleotide is shown as any one of SEQ ID NO.2 or SEQ ID NO. 4.

The invention also provides a recombinant plasmid which contains the polynucleotide and can translate and express the fusion mutant protein EDNRA.

In a further improvement, the plasmid is Bacmid.

The invention also provides an expression system of the fusion mutant protein EDNRA, which is a baculovirus cell transferred into the recombinant plasmid, namely recombinant baculovirus.

The further improvement is that the baculovirus cells are bombyx mori baculovirus BmNPV.

The invention also provides an expression method of the endothelin receptor A fusion mutant protein, which is characterized in that the expression method utilizes a silkworm larva expression system to express, and comprises the following steps:

(1) Injecting and inoculating the recombinant silkworm baculovirus into 5-instar silkworm larvae;

(2) The infected silkworm larva expresses the fusion mutant protein EDNRA efficiently.

The improvement is that the method comprises the following steps:

(1) Artificially synthesizing the gene for expressing the endothelin receptor A mutant protein, cloning the gene into a Bac-to-Bac silkworm baculovirus expression system carrying vector, and constructing to obtain a transfer expression vector, wherein the silkworm baculovirus carrying vector is preferably pFastBac1;

(2) Transforming the silkworm baculovirus carrier into escherichia coli, integrating the gene encoding the endothelin receptor A mutant protein on the transfer expression vector into baculovirus plasmids through site specific transposition to complete recombination of virus genomes, and obtaining recombinant virus plasmids through blue-white screening, wherein the baculovirus plasmids are preferably Bacmid;

(3) Transfecting the bombyx mori BmN cells with the baculovirus plasmid integrated with the gene of the endothelin receptor A mutant protein to obtain a recombinant baculovirus, amplifying the virus in the bombyx mori BmN cells, and inoculating bombyx mori larvae with the virus, wherein the bombyx mori larvae are preferably 5-instar bombyx mori larvae, and the inoculation amount of the virus is preferably 2-5 mu l;

(4) 4-5 days after virus inoculation, the silkworm larva expresses and generates endothelin receptor A mutant fusion protein.

The invention has the following beneficial effects:

the silkworm larva infected by the recombinant silkworm baculovirus is used for efficiently expressing the fusion protein, the activity is higher than that directly expressed by a prokaryote, and the yield is higher than that expressed by a transgenic plant; the silkworm can be fed aseptically and massively, can be fed at low cost and massively, and does not cause public nuisance; the silkworm body contains multiple natural protein protective agents, which have protective effect on expression products and make the gene expression products more stable.

Drawings

FIG. 1 is a PCR validation scheme for the Bacmid plasmid;

FIG. 2 is a fluorescence chart of EDNRA after its expression in silkworm larvae;

FIG. 3 is an SDS-PAGE detection chart after the EDNRA protein is expressed in silkworm larvae;

FIG. 4a is SDS-PAGE of EDNRA protein expressed in silkworm in small test, and FIG. 4b is SDS-PAGE of EDNRA protein expressed in insect cell in small test;

FIG. 5 is a graph showing the Western Blot results for the EDNRA protein.

Detailed Description

The present application will now be described in further detail with reference to the drawings, it should be noted that the following detailed description is given for illustrative purposes only and is not to be construed as limiting the scope of the present application, as those skilled in the art will be able to make numerous insubstantial modifications and adaptations to the present application based on the above disclosure.

1. Material

The methods used in the present invention are conventional methods known to those skilled in the art unless otherwise specified, and are carried out under conventional conditions or conditions recommended by the manufacturer, and the reagents or equipment used are conventional products commercially available.

2. Method of producing a composite material

2.1 plasmid construction and preparation of silkworm baculovirus

All plasmids used in the invention are obtained by using a conventional molecular biology means to carry out primer design, PCR and vector fragment recombination construction, and all recombinant plasmids are completely consistent with a target sequence through sequencing verification.

The PCR and vector fragment recombination mode is that the gene of the modified human endothelin receptor A wild type (the wild type amino acid sequence of the endothelin receptor A is shown as SEQ ID NO.5, and the gene sequence is shown as SEQ ID NO. 6) and the mutant fusion protein is cloned into a pFastBac1 vector (wherein the gene of the mutant fusion protein comprises three mutations (the amino acid sequence is shown as SEQ ID NO.3, and the gene sequence is shown as SEQ ID NO. 4) and five mutations (the three mutations and the five mutations are only different, and the N end and the C end are identical), the amino acid sequence is shown as SEQ ID NO.1, and the gene sequence is shown as SEQ ID NO. 2), so that pFastBac 1-transfer plasmids are obtained, all the recombinant plasmids are completely consistent with the target sequence through sequencing verification, and the modified endothelin receptor A fusion protein is 75kDa.

2.1.1 transformation of E.coli competent cells with the endothelin receptor A fusion protein plasmid

Taking out the competent cells from a refrigerator at the temperature of-80 ℃, thawing the competent cells on ice, and transforming the recombinant plasmid into the BmDH10Bac escherichia coli competent cells in a super clean bench by using a conventional molecular biology method under the transformation conditions that:

(1) Taking 10 mul of baculovirus transfer wild type plasmid pFastBac1-Flag-TEV-EDNRA-3C-eGFP-10His, three mutant plasmid pFastBac1-Flag-TEV-EDNRA (C379A, C383A, C388A) -3C-eGFP-10His and five mutant plasmid pFastBac1-Flag-TEV-EDNRA (R103Y, D133A, Q252A, S325A, I364A) -3C-eGFP-10His to be added into BmDH10Bac competent cells, placing 30min on ice, water bath 90S at 42 ℃ and placing 2min on ice;

(2) Then adding 450 μ l SOC culture medium (SOC culture medium: 40g peptone, 10g yeast powder, 1.16g sodium chloride, 0.38g potassium chloride, 4.06g magnesium chloride hexahydrate, 4.92g magnesium sulfate heptahydrate, 7.2g glucose, water 2000 ml), shaking at 37 ℃ and 250rpm for 4h;

(3) Taking 4 mu l of the mixture, coating an LB plate (the LB plate contains 2g of peptone, 1g of yeast powder, 2g of sodium chloride, 3g of agar powder and 200ml of water, sterilizing the mixture for 20min at 121 ℃, taking the mixture out, adding 600ul of three antibodies, 1ml of X-gal and 33ul of IPTG (isopropyl-beta-D-thiogalactoside) when the temperature is reduced to 50-60 ℃ (the mixture can bear about 5s by hand holding), and carrying out inverted culture for 2d after the final concentration is 50 mu g/ml of kanamycin, 7 mu g/ml of gentamicin, 10 mu g/ml of tetracycline, 100 mu g/ml of X-gal and 40 mu g/ml of IPTG (isopropyl-thiogalactoside) in a 37 ℃ incubator for 30min in the forward direction;

2.1.2 extraction of recombinant baculovirus plasmid Bacmid and preparation of silkworm baculovirus

(1) Performing blue-white spot screening on the bacterial colonies cultured in the step (3) in the step 2.1.1, selecting a single white bacterial colony to be cultured in an LB liquid culture medium containing a three-antibody overnight, then extracting Bacmid to obtain a recombinant virus plasmid Bacmid, performing PCR verification through an M13 primer to obtain a target band (4361 bp) with the same molecular weight as the theoretical molecular weight, and indicating that the recombinant Ba cmid contains a target gene, wherein the target band is shown in figure 1;

(2) 1.5ml of EP tube is taken in a biological safety cabinet, 100 mul of transfection medium, 15 mul of Bacmid (evenly and lightly flicked by a gun head) and 7 mul l X-treme transfection reagent (evenly and lightly flicked by a gun head) are sequentially added into each tube, and the mixture is incubated for 20min at room temperature. And taking the BmN cells passaged one day before from a shaking table, sucking 200 mu l of the cells in a safety cabinet, taking 20 mu l of trypan blue by using a 20 mu l pipette to cover a centrifuge tube, uniformly blowing the cells, taking 20 mu l of the cells to cover, uniformly mixing with the trypan blue, taking 20 mu l of sample, pouring the sample into a hole of a cell counting plate, wherein air bubbles are not available, pouring the sample into the plate, and counting by using a counter for 3-8 minutes. Diluting the cell density to 1.0X 10 ⁶ . 3ml of the diluted cells were pipetted into a 25ml shake flask in a safety cabinet. At the end of the incubation period, the X-treme-DNA complex was added dropwise to SF9 cells and mixed slowly. The shake flask was placed in a shaker for culture at 27 ℃ and 100rpm for 4 days to obtain P0 virus.

(3) 1ml P0/50ml (1.5X 10) ⁶ cell/ml) cells, and P0 virus was added. Culturing in constant temperature shaking table at 27 deg.C, 100rpm,72h, collecting P1 virus, counting cells, measuring cell concentration, cell activity and diameter, and recording.

(4) Per 4ml P1/200ml (1.5X 10) ⁶ cell/ml) cells, and P1 virus was added. Culturing in constant temperature shaking table at 27 deg.C, 100rpm,72h, collecting P2 virus, counting cells, measuring cell concentration, cell activity and diameter, and recording.

2.2 expression of the EDNRA protein in silkworm larvae

(1) Taking 500ul or 1ml micro-injector, taking prepared P2 virus, injecting diluted virus (10-20 ul) from the back internode of silkworm to the blood cavity, placing the injected silkworm into a corresponding prepared cage box, and replacing 10 silkworms with new injectors every time. 3-5 days after virus injection, the silkworm larvae are observed under ultraviolet light or blue light to see whether green fluorescence exists, silkworms with green fluorescence indicate that the EDNRA protein fused with GFP expresses, as shown in figure 2, the silkworms infected with silkworm baculovirus emit green fluorescence, and the experimental result shows that the silkworm injected with wild type EDNRA protein virus basically has no color, and the silkworm injected with three-mutation EDNRA protein virus and the silkworm injected with five-mutation EDNRA protein virus all have green fluorescence. Furthermore, it is found from the fluorescence intensity that the five-mutation EDNRA protein has higher expression in silkworm larvae than the three-mutation.

To further verify whether the green-fluorescent protein was the target protein, 1 silkworm larva was each taken, the fat body cells were collected, washed with PBS and placed in an EP tube prepared in advance, the collected fat body was homogenized with a tissue homogenizer and 5ml of a buffer (100 mM Tris-HCl (pH 7.5), 150mM NaCl,5% glycerol,1% DDM,0.2% CHS, SMNE,1mM MgCl, 1% DDM, 1% CHS, SMNE,1mM MgCl, and the like ₂ 1mM PTU,1mM PMSF) were sonicated. The centrifuged supernatant was transferred to a tube to which 200. Mu.l of pretreated Flag filler was added, incubated at 4 ℃ for 2 hours, added with 100. Mu.l of an eluent (100 mM Tris-HCl (pH 7.5), 150mM NaCl,5% glycerol,0.05% DDM,0.005% CHS,200ug/ml Flag peptide) and incubated for 30 minutes, centrifuged at 4500rpm, and the supernatant was subjected to SDS-PAGE. The results of the experiment are shown in FIG. 3, no significant protein expression was observed in silkworm fat injected with wild-type EDNRA protein, and the injection of both triple-and penta-mutant EDNRA proteins had bands with a molecular weight of about 78kDa, indicating that both proteins were significantly expressed. And the expression level of the protein of the five mutations is higher than that of the protein of the three mutations.

2.3 comparison of the yields of the EDNRA protein in silkworm larvae and insect cells

From the results in 2.2, it was found that the EDNRA five mutant protein was expressed in a high amount, and in order to further compare the EDNRA expression in different expression cells, the EDNRA five mutant protein was used to simultaneously compare the EDNRA expression in silkworm cells and insect cells.

2.3.1EDNRA five mutations in silkworm system

Extracting EDNRA protein: taking 5 diseased, green-fluorescing, uniformly sized silkworm larvae, cutting off the head and tail, cutting open along the ventral midline, removing the intestinal tract, mariothis tube, silk gland, collecting adipose body cells, washing with PBS, placing into an EP tube prepared in advance, homogenizing the collected adipose bodies with a tissue homogenizer, adding 5ml of a buffer (100 mM Tris-HCl (pH 7.5), 150mM NaCl,5% glycerol,1% DDM,0.2% CHS, SMNE,1mM MgCl, 1% DDM,0.2% CHS, SMNE, and ₂ 1mM PTU,1mM PMSF), using a single channel sonicator 200-300w, sonicated for 3min,16000rpm, centrifuged for 15min at 4 ℃. The centrifuged supernatant was transferred to a tube to which 200. Mu.l of pretreated Flag filler was added, incubated at 4 ℃ for 2 hours, added with 100. Mu.l of an eluent (100 mM Tris-HCl (pH 7.5), 150mM NaCl,5% glycerol,0.05% DDM,0.005% CHS,200ug/ml Flag Peptide) and incubated for 30 minutes, and centrifuged at 4500rpm to obtain a supernatant.

10ul of the above supernatant was sampled and subjected to SDS-PAGE to verify the expression, as shown in FIG. 4a, a band was evident at 75kDa, which was consistent with the theoretical molecular weight, and the concentration was measured by Nanodrop to be 0.52mg/ml, and the expression amount per silkworm larva was calculated to be about 100. Mu.g. In order to further verify whether the EDNRA protein is correctly expressed in the silkworm larvae, western blot verification is carried out, and as shown in figure 5, specific bands of anti-Flag antibodies appear at the position of about 78KDa of the EDNRA protein, so that the EDNRA protein is correctly expressed in the silkworm larvae.

2.3.2EDNRA five mutations in silkworm system

SF21 cells were counted and cell concentration, cell viability and diameter were determined. The cell concentration is required to be 1.8-2.2X 10 ⁶ cell/ml, dividing the cells into 250ml sterile culture flasks, 30ml per flask, inoculating the P2 virus in 2.1 to SF21 cells at a ratio of 3ul/0.5ml, taking 10ml cells after 48 hours, measuring the cell infection status by counting, 3500rpm, centrifuging for 2min, collecting the cell pellet, adding 5ml of a buffer (100 mM Tris-HCl (pH 7.5), 150mM NaCl,5% glycerol,1% DDM,0.2% CHS, SMNE,1mM MgCl, 1% DDM, 1mM CHS, and the like ₂ 1mM PTU,1mM PMSF) were sonicated. The supernatant after centrifugationTransferred to a tube to which 200. Mu.l of the pretreated Flag filler was added, incubated at 4 ℃ for 2 hours, added with 100. Mu.l of an eluent (100 mM Tris-HCl (pH 7.5), 150mM NaCl,5% glycol, 0.05% DDM,0.005% CHS,200ug/ml Flag Peptide), incubated for 30 minutes, centrifuged at 4500rpm, and the supernatant was subjected to SDS-PAGE. The results of the experiment are shown in FIG. 4b, and from the SDS-PAGE results, the EDNRA five mutant protein expression level in insect cells is not high in silkworm, and the calculated yield is 1 mug/2X 10 ⁷ A cell. The yield is 100 times lower than that of silkworm system.

The results show that the invention increases the stability of the protein by carrying out 5 mutations on the protein sequence of the endothelin receptor A, successfully expresses the EDNRA protein by utilizing a silkworm larva expression system, does not express the protein in escherichia coli, has very low expression level in an insect cell and a mammal cell expression system, and has the expression level of about 1 mu g/2 multiplied by 10 in the insect cell ⁷ Cells, it is difficult to enrich in protein. The expression quantity of the protein in the silkworm larva expression system can reach 100 mu g/silkworm, is 100 times of that of insect cells, and can provide a material basis for subsequent drug development.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention.

Claims (10)

1. An endothelin receptor A fusion mutant protein, characterized in that the fusion mutant protein EDNRA is composed of the following (1) to (3):

(1) An endothelin receptor A mutant sequence, wherein the mutation in the mutant sequence is one of (a) or (b):

(a) C379A, C a and C388A;

(b) R103Y, D A, Q252A, S325 5A and I364A;

(2) Replacing the signal peptide sequence of the N end with an HA signal peptide sequence, and then sequentially adding a TEV enzyme cutting site and a Flag label;

(3) And adding a 3C enzyme cutting site at the C end, and then sequentially adding a GFP protein sequence and a 10His tag protein sequence.

2. The endothelin receptor A fusion mutant protein of claim 1, wherein when the mutation in the mutant sequence in the fusion mutant protein EDNRA is (a), the amino acid sequence is shown as SEQ ID No. 3; when the mutation in the mutant sequence in the fusion mutant protein EDNRA is (b), the amino acid sequence of the fusion mutant protein EDNRA is shown as SEQ ID NO. 1.

3. The endothelin receptor A fusion mutant protein of claim 1, wherein the N-terminal signal peptide sequence is specifically the 20 amino acids N-terminal of the wild-type amino acid sequence of endothelin receptor A.

4. A polynucleotide encoding the fusion mutant protein EDNRA according to any one of claims 1 to 3.

5. A polynucleotide according to claim 4, wherein the sequence of the polynucleotide is as shown in any one of SEQ ID No.2 or SEQ ID No. 4.

6. A recombinant plasmid containing the polynucleotide of any one of claims 4 to 5 and capable of translationally expressing the fusion mutant protein EDNRA of any one of claims 1 to 3.

7. A recombinant plasmid according to claim 6, wherein the plasmid is Bacmid.

8. An EDNRA expression system of fusion mutant protein is characterized in that the EDNRA expression system is a baculovirus which is transformed into the recombinant plasmid of any one of claims 6 to 7, namely a recombinant baculovirus.

9. The expression system of claim 8, wherein the baculovirus is bombyx mori baculovirus BmNPV.

10. An expression method of endothelin receptor A fusion mutant protein is characterized in that a silkworm larva expression system is used for expression, and comprises the following steps:

(1) Injecting the recombinant silkworm baculovirus of claim 9 into 5-instar silkworm larvae;

(2) The infected silkworm larva expresses the fusion mutant protein EDNRA efficiently.

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