CN114763562B

CN114763562B - Glucagon-like peptide-1 receptor stable expression cell strain

Info

Publication number: CN114763562B
Application number: CN202110588286.XA
Authority: CN
Inventors: 刘剑峰; 范治然; 李豫华; 李文征
Original assignee: Huazhong University of Science and Technology; Bioisland Laboratory
Current assignee: Huazhong University of Science and Technology; Bioisland Laboratory
Priority date: 2021-05-28
Filing date: 2021-05-28
Publication date: 2023-04-11
Anticipated expiration: 2041-05-28
Also published as: CN114763562A

Abstract

The present invention relates to a recombinant expression vector for expressing a glucagon-like peptide-1 receptor, an expression system, and a cell having the recombinant expression vector or the expression system. The glucagon-like peptide-1 receptor stable expression cell strain utilizes a gene editing method to stably knock a target gene into a cell genome, can be stably replicated along with cell replication, cannot be lost when the cell strain is subcultured, and can be used for drug development and function detection with the glucagon-like peptide-1 receptor as a target.

Description

Glucagon-like peptide-1 receptor stable expression cell strain

Technical Field

The invention relates to the field of biological medicines, in particular to a glucagon-like peptide-1 receptor stable expression cell strain.

Background

Glucagon-like peptide-1 (GLP-1) is an endogenous incretin secretagogue and plays a very important role in stabilizing blood glucose. It consists of 30 amino acid residues, is secreted by neuroendocrine L cells of the jejunum terminal, ileum and colon under oral feeding stimulation, is produced by posttranslational processing of glucagon, exists in 2 bioequivalent forms, GLP-1 (7-36) -NH2 and GLP-1 (7-37), wherein GLP-1 (7-36) -NH2 accounts for a higher proportion in the human body. It binds to and activates the glucagon-like peptide-1 receptor (GLP-1R) of the G protein-coupled receptor (GPCRs) class B family to exert its regulatory function. The GLP-1R gene is located on chromosome 6P21, is a 7-time transmembrane helical G protein coupled receptor consisting of 463 amino acids, and is expressed in tissues such as small intestine, kidney, heart, pancreas, lung and the like. In addition, research shows that GLP-1R has high content in central nervous tissues such as hypothalamus, pituitary, cerebral cortex, hippocampus, and the like, and the wide distribution characteristics determine the diversity of the action, namely, the GLP-1R can generate wide biological effects, and the main effect is to promote the biosynthesis and secretion of insulin; inhibiting glucagon secretion; reducing body weight; reducing blood lipid and blood pressure; protecting the cardiovascular system; protecting islet beta cells and improving insulin sensitivity; in addition, the medicine acts on the central nervous system to strengthen the learning and memory function, protect nerve and inhibit inflammation, oxidation stress, cell death and other mechanisms to exert direct or indirect cerebral ischemia resisting effect.

Research on human GLP-1R shows that the main action mechanism is that insulin secretion is stimulated in a glucose-dependent mode; decrease glucagon secretion; inhibiting gastric emptying; reduce appetite and promote growth and resuscitation of pancreatic beta cells. When the blood sugar concentration exceeds a normal level, GLP-1 can stimulate the secretion of insulin through the above mechanism so as to play a role in reducing blood sugar, so GLP-1 is a high-efficiency blood sugar reducing medicament and has glucose dependence. Because of these features, GLP-1R is an important target for the treatment of T2DM (type 2 diabetes).

GLP-1R has application prospects in various diseases. Recent studies have found that GLP-1R can improve Insulin Resistance (IR); regulating lipid metabolism; the medicine can inhibit liver inflammation, improve intestinal flora imbalance and other mechanisms, reduce liver fat accumulation, delay the occurrence and the development of non-alcoholic fatty liver disease (NAFLD), and lay a foundation for the clinical test and the future application in NAFLD treatment. In addition, GLP-1 has specific biological effect on cardiovascular systems of healthy people and people with cardiovascular pathological changes, and has important significance on cardiovascular protection. In recent years, GLP-1RAs have been found to play an important role in bone metabolism by promoting bone formation, inhibiting bone resorption, improving trabecular volume, thickness and number, increasing bone density, and decreasing trabecular spacing. In addition, GLP-1RAs play an important role in the treatment of obesity.

Disclosure of Invention

In a first aspect, the invention provides recombinant expression vectors that express a glucagon-like peptide-1 receptor.

The recombinant expression vector provided by the invention is obtained by inserting a nucleotide sequence coding a human glucagon-like peptide-1 receptor into a pAAVS1-Puro-DNR expression vector.

The expression vector provided by the invention can ensure that a target gene (namely a gene encoding the human glucagon-like peptide-1 receptor) is stably knocked into a host cell. The pAAVS1-Puro-DNR expression vector adopted by the invention has an AAVS1 site (also called PPP1R2C site), and for a gene coding the human glucagon-like peptide-1 receptor, the insertion of the site can ensure that a gene fragment is normally and stably transcribed to realize the expected function. Compared with other common expression vectors, the insertion of the gene segment coding the human glucagon-like peptide-1 receptor into the pAAVS1-Puro-DNR expression vector does not disturb the functions of endogenous genes and/or other regulatory factors, and has no side effect on cells.

In some embodiments, the nucleotide sequence encoding the human glucagon-like peptide-1 receptor is set forth in SEQ ID No. 1.

In some embodiments, a signal peptide sequence is also inserted into the pAAVS1-Puro-DNR expression vector. As a specific embodiment, the signal peptide sequence is shown as SEQ ID NO. 2. The invention can ensure that the receptor obtained by expression is transferred to the cell membrane efficiently by inserting the signal peptide into the vector.

In some embodiments, a nucleotide sequence encoding a protein tag is also inserted into the pAAVS1-Puro-DNR expression vector.

In some embodiments, the protein tag is selected from one or more of a Flag tag, a Halo tag, a SNAP tag, a His6 tag, an eGFP tag, an eYFP tag, an eCFP tag.

In some embodiments, the protein tag comprises a Flag tag. As a specific embodiment, the nucleotide sequence encoding the Flag tag is shown in SEQ ID NO. 3. By expressing the Flag tag, the invention can conveniently carry out cell membrane expression detection on the receptor and carry out related research by utilizing the Flag tag.

In some embodiments, the protein tag comprises a SNAP tag. As a specific embodiment, the nucleotide sequence encoding the SNAP tag is shown in SEQ ID NO. 4. According to the invention, by expressing the SNAP label, the receptor can conveniently carry out cell membrane expression detection, ligand binding experiment and other related researches by utilizing the SNAP label.

In some embodiments, the recombinant expression vector is pAAVS1-Puro-DNR-Flag-SNAP-GLP1R, having the map shown in figure 1.

In a second aspect, the invention provides a CRISPR-Cas9 expression system expressing a glucagon-like peptide-1 receptor, comprising the recombinant expression vector of the first aspect.

In some embodiments, the expression system further comprises a pCas-Guide-AAVS1 donor plasmid.

In a third aspect, the invention provides a cell comprising said recombinant expression vector or said expression system.

In some embodiments, the cell is a human cell.

As a preferred embodiment of the invention, the cell is a HEK293 cell. The practice of the invention shows that for the recombinant expression vector adopted by the invention, especially pAAVS1-Puro-DNR-Flag-SNAP-GLP1R, the HEK293 cell can ensure that the recombinant expression vector or an expression system containing the recombinant expression vector is efficiently transferred into cells, and especially the HEK293 cell can still ensure the stable high expression of the target protein glucagon-like peptide-1 receptor after multiple passages. In addition, the adoption of the HEK293 cell is more suitable for subsequent drug screening aiming at the glucagon-like peptide-1 receptor.

In some embodiments, the cell is a glucagon-like peptide-1 receptor stably expressing cell line deposited at 31/3/2021 under the international collection of microorganisms designated by the budapest treaty: china Center for Type Culture Collection (CCTCC), address: the preservation number of the university of Wuhan 299 in eight paths in Wuchang district of Wuhan city, hubei province is CCTCC NO: C202186.

In a fourth aspect, the present invention provides a method for constructing a cell line stably expressing a glucagon-like peptide-1 receptor, the method comprising: transfecting the recombinant expression vector of the first aspect or the expression system of the second aspect into a cell.

In some embodiments, the cell is a human cell. As a preferred embodiment of the invention, the cell is a HEK293 cell.

In some embodiments, the transfection is by lipofection.

In some embodiments, the cells are screened for resistance using a purine toxin.

In some embodiments, the method comprises the steps of:

(1) Transfecting the recombinant expression vector or the expression system into HEK293 cells by a lipofection method;

(2) Performing resistance screening on the cells obtained in the step (1) by using puromycin, wherein the concentration of puromycin is preferably 1-3 mu g/ml;

(3) Suspending cells with puromycin resistance, and sorting by a flow cytometry sorter to obtain monoclonal cells;

(4) And screening and verifying the monoclonal cells to obtain a positive monoclonal cell line.

The fifth aspect of the invention provides applications of the cell or the cell strain constructed by the method in receptor protein preparation, receptor protein expression detection, receptor protein function detection, receptor protein labeling, receptor protein imaging and/or drug screening.

In some embodiments, the drug binds to and/or activates a receptor on a cell line, and then the function of the drug can be detected by using experimental means such as calcium flux detection, cAMP detection and the like in the process of receptor drug development.

In some embodiments, the medicament is for treating diabetes, obesity, dyslipidemia, fatty liver disease, non-alcoholic steatohepatitis, and/or metabolic syndrome. Wherein the diabetes is preferably type 2 diabetes.

Compared with the prior art, the glucagon-like peptide-1 receptor stable expression cell strain provided by the invention is used for drug development and function detection with the glucagon-like peptide-1 receptor as a target spot, and has the main advantages that: the gene editing method is utilized to stably knock the target gene into the cell genome, so that the target gene can be stably copied along with cell replication, and the cell strain cannot be lost due to passage; preferably, GPCR upper membrane signal peptide is adopted, so that the receptor can be stably transported to a cell membrane after being expressed in a cell, and the function of the receptor is ensured; preferably, a Flag label and an SNAP label are adopted, so that various different function detection experiments can be conveniently carried out on the glucagon-like peptide-1 receptor, the functions of binding and activating the receptor by the drug can be further detected, and meanwhile, the second messenger such as downstream calcium signals and cAMP can be detected, and the activation capability of the drug to be detected on the receptor can be detected.

Drawings

FIG. 1 is a map of a recombinant expression vector;

FIG. 2 shows the results of the detection of the expression of the receptor Flag tag;

FIG. 3 shows the result of detection of SNAP-labeled TB;

FIG. 4 shows the results of fluorescent ligand-bound receptor detection;

FIG. 5 shows the results of the measurement of cAMP function activated by receptor;

FIG. 6 shows the results of the function test of the receptor-activated calcium signaling;

FIG. 7 shows the results of ELISA assay; wherein, panel A is the detection result of the cell line passed to the 5 th generation; FIG. B shows the results of the detection of the cell line passed to the 7 th generation; FIG. C shows the results of the detection of the cell line passed to the 10 th generation;

FIG. 8 shows the results of cAMP activation detection; wherein, panel A is the detection result of the cell line passed to the 5 th generation; FIG. B shows the results of the detection of the cell line passed to the 7 th generation; FIG. C shows the results of the measurement of the cell line passed to the 10 th generation.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

Example 1: construction of target, host and expression plasmid

1. And (3) target spot:

gene ID	Name of Gene	Species (II)
			2740	GLP-1R	Human being

2. Host cell: HEK293

3. Resistance: puromycin

4. Construction of expression plasmid:

constructing a target gene (shown in table 1) on an expression vector by using a restriction enzyme digestion and T4 DNA ligase connection method to obtain a plasmid: pAAVS1-Puro-DNR-Flag-SNAP-GLP1R (recombinant expression vector map as shown in FIG. 1). The vector is a specific gene knock-in vector, and can stably knock a target gene into a cell genome AAVS1 site by cotransformation with a donor pCas-Guide-AAVS1 plasmid.

TABLE 1

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Example 2: cell culture

1. Cell resuscitation

The cells stored in liquid nitrogen or-80 ℃ refrigerator were removed and rapidly moved to 37 ℃ water bath for thawing. A15 ml centrifuge tube was prepared in advance and 3ml of fresh medium was added. 1ml of the cell suspension in the cryopreserved tube was added to a previously prepared medium, and the mixture was centrifuged at 1000rpm/min for 3 minutes. During this period, 100mm cell culture dishes were prepared and 10ml of fresh medium was added. After completion of centrifugation, the supernatant was discarded, the cells were resuspended in 1ml of fresh culture medium, the cell suspension was uniformly added to a previously prepared petri dish and placed at 37 ℃ and 5% CO ₂ Culturing in an incubator.

2. Cell passage

1) The state of the cells was first observed under a microscope to determine whether or not cell passaging was performed. After the passage was established, the old medium was discarded first, and then the dead cells and cell debris were washed away with PBS (5 mL PBS/100mm dish), and the number of washing was determined depending on the cell condition, and is preferably 1 to 2.

2) Adding pancreatin according to the proportion of 1mL of pancreatin to 100mm of culture medium for digestion, and placing the culture dish in an incubator at 37 ℃ for digestion for 1-3 min after adding the pancreatin. The extent of digestion of the cells can be observed with the aid of a microscope, and the termination of digestion can be taken into account when the cells become round and bright.

3) Complete medium was added to the digested dishes to stop cell digestion. And blowing the cells for many times by using a pipettor to form a cell suspension, and transferring the cell suspension into a 15mL centrifuge tube after uniformly mixing the cell suspension.

4) The 15ml centrifuge tube was placed in a centrifuge and centrifuged at 1000rpm/min for 3min. During centrifugation, the culture dish may be prepared in advance or a desired plate may be added with the culture medium. Cell supernatants were discarded after centrifugation was complete. Resuspend the cells in complete medium and add them to a new cell culture dish or plate prepared in advance in a certain ratio.

5) Finally, the cells were incubated at 37 ℃ and 5% CO ₂ Culturing in a cell culture box.

3. Cell conservation

The steps are similar to cell passage, firstly, the cells are cleaned by PBS, then 1ml of pancreatin is added for cell digestion, after digestion is finished, a fresh culture medium is added to stop cell digestion, a pipette is used for blowing and beating to enable the cells to be completely suspended in the culture medium, and cell suspension is transferred to a centrifugal tube for centrifugation. Suspending the cells with 1ml of cell freezing solution after centrifugation, transferring the cells into a cell freezing tube, injecting information such as cell names, seed preservation time, seed maintainer names and the like, storing the cells in a refrigerator at the temperature of 80 ℃ below zero, and transferring the cells to a liquid nitrogen tank for storage the next day. The culture medium was 10% DMSO-containing fetal bovine serum.

Example 3: cell line construction

1. Determination of puromycin screening concentration

1) HEK293 cells were seeded in 6-well plates.

2) Designing puromycin drug concentration gradients which are respectively 5 concentration gradients of 1 mug/ml, 2 mug/ml, 4 mug/ml, 6 mug/ml and 8 mug/ml; and adding the medicines with different concentrations respectively after the cell density in the 6-hole plate is 80 percent.

3) Then, the fresh culture medium containing puromycin is replaced every 24h, the survival condition and the growth state of the cells are observed, and the lowest puromycin concentration at which the cells die in a large batch after 2 days and all the cells die after 4 days is selected as the optimal screening concentration. Therefore, 2. Mu.g/ml was finally selected as puromycin screening concentration.

2. Liposome transfection

1) HEK293 cells were seeded into 35mm dishes one day in advance, and the cell status was observed the next day, and lipofection was performed when the cell density was about 80% and the fine state was good.

2) The medium was aspirated off, 0.8ml OMEM was added, and the mixture was placed in an incubator at 37 ℃.

3) Solution A, 100 mul OMEM and 2 mul Lipofectamine 2000 are prepared, mixed evenly and kept stand for 5min at room temperature.

4) The prepared solution B comprises 100 mul OMEM, 0.5 mug pCas-Guide-AAVS1 plasmid and 0.5 mug pAAVS1-Puro-DNR-flag-SNAP-GLP1R plasmid.

5) And fully and uniformly mixing the solution A and the solution B, and standing at room temperature for 20min.

6) The transfection solution was added drop wise to the cells, gently shaken and mixed well, placed in an incubator at 37 ℃ for 20min, then 1.5ml of complete medium was added and transfected for 48h.

3. Drug screening

Puromycin selection was started 48 hours after transfection and drug concentration was 2 μ g/ml, after which puromycin-containing fresh medium was replaced at 24h intervals until all remaining cells contained puromycin resistance for approximately 5-7 days.

4. Sorting monoclonal cell lines by flow cytometry

1) And (4) inoculating the polyclonal cells into a 100mm culture dish, checking the growth condition of the cells after 48 hours, and preparing to start collecting the cells when the culture dish is full of the cells and the growth state of the cells is good.

2) Discarding the old culture solution, washing the cells twice by using PBS, adding 1ml of pancreatin for cell digestion, adding 3ml of fresh culture medium after about 2-3 min to stop digestion, repeatedly blowing and beating the cells by using a micropipettor to obtain cell suspension, transferring the cell suspension into a 15ml centrifugal tube, and centrifuging the cell suspension for 3min in a centrifuge with the speed of 1000 rpm/min.

3) Discard the upper medium and resuspend the cells using 1ml PBS (optionally add 1% serum or not).

4) The cell suspension was simply filtered and transferred to a flow cytometer analysis tube.

5) 5 plates of 96 wells were prepared in advance and 100. Mu.l of complete medium was added to each well.

6) The flow tube to which the cell suspension is added is fixed on a sorter to start sorting, one cell per well.

5. Establishment of monoclonal cell lines

After the cells are overgrown in a 96-well plate, the cells are transferred to a 24-well plate, a 6-well plate, and the like in order for scale-up culture. And subsequently, screening and verifying a plurality of monoclonal cell lines to obtain a positive monoclonal cell line.

Example 4: target gene expression and functional verification

1. The ELISA method detects the expression of flag on the cell membrane surface, and the result is shown in FIG. 2.

2. The Snap label is marked by using a fluorescent molecule Snap-Tb of Cisbio company, and a fluorescent signal after marking is detected, and the result is shown in figure 3.

3. Based on HTRF principle, the combination condition of ligand and receptor is detected, the concentration of fluorescent ligand GLP1-red is fixed, and the non-fluorescent ligand Extendin-4 with concentration gradient is added in turn, so that the combination of GLP1-red and receptor can be obviously competed, and the result is shown in figure 4.

4. The activation of the downstream second messenger cAMP by the receptor was detected using the Cisbio assay cAMP kit, with significant cAMP activation, and the results are shown in FIG. 5.

5. The receptor was tested using flipr to induce activation of downstream calcium signaling upon stimulation with the specific agonist extensin-4, with significant activation of calcium signaling, as shown in figure 6.

From the above results, it is clear that the cell line provided by the present invention can stably express glucagon-like peptide-1 receptor and successfully induce activation of downstream calcium signaling.

Example 5: detection of expression stability of target Gene

The cell line constructed in the example 4 is transmitted to the tenth generation, the 5 th generation, the 7 th generation and the 10 th generation are selected, and the membrane expression level of the receptor and the activation level of cAMP in the cell line are respectively detected, so that the stability of the glucagon-like peptide-1 receptor expressed by the cell line is detected.

The detection results are shown in fig. 7 and 8. As can be seen from the results, the glucagon-like peptide-1 receptor expression and function in the cell line provided by the present invention are very stable when the cell reaches the 10 th generation.

The EC50 values for cAMP activation of cell lines of different generations are shown in Table 2 below.

TABLE 2

Cell algebra	EC50
		P5	2.173e-011
P7	1.992e-011
		P10	1.278e-011

From the above results, it was found that the EC50 value of the agonist was relatively stable when the cell line activated downstream cAMP.

Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

SEQUENCE LISTING

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Claims

1. A cell is a glucagon-like peptide-1 receptor stable expression cell strain, which is preserved in China Center for Type Culture Collection (CCTCC) at 31 months 3 in 2021, and the preservation number is CCTCC NO: C202186.

2. The cell of claim 1, wherein the nucleotide sequence encoding the human glucagon-like peptide-1 receptor is set forth in SEQ ID No. 1.

3. The cell of claim 1, wherein the nucleotide sequence encoding the human glucagon-like peptide-1 receptor has a signal peptide sequence; the sequence of the signal peptide is shown as SEQ ID NO. 2.

4. The cell of any one of claims 1-3, wherein the nucleotide sequence encoding the human glucagon-like peptide-1 receptor has a Flag tag and a SNAP tag.

5. The cell of claim 4, wherein the nucleotide sequence encoding the Flag tag is set forth in SEQ ID NO. 3 and/or the nucleotide sequence encoding the SNAP tag is set forth in SEQ ID NO. 4.

6. Use of a cell according to any of claims 1 to 5 for receptor protein production, receptor protein expression detection, receptor protein function detection, receptor protein labeling, receptor protein imaging and/or drug screening; the use is a non-disease diagnostic use.

7. The use of claim 6, wherein the drug binds to and/or activates a receptor on a cell line.

8. Use according to claim 6, wherein the medicament is for the treatment of diabetes, obesity, dyslipidemia and/or fatty liver disease.

9. The use according to claim 6, wherein the medicament is for the treatment of non-alcoholic fatty liver disease.

10. The use according to claim 6, wherein the medicament is for the treatment of non-alcoholic steatohepatitis.

11. The use according to claim 6, wherein the medicament is for the treatment of metabolic syndrome.