阅读说明：本技术 基于人突变蛋白为基础的gdh抑制剂高通量筛选方法 (GDH inhibitor high-flux screening method based on human mutant protein ) 是由 李长红 储春霞 袁月 于 2019-11-04 设计创作，主要内容包括：本发明公开了一种基于人突变蛋白为基础的GDH抑制剂高通量筛选方法,达到筛选的抑制剂接近生理状态,涉及药物筛选技术领域,其技术方案要点是：包括以下步骤,步骤一,突变基因的质粒构建和制备携带突变基因质粒的逆转录病毒,该质粒装有红色荧光蛋白；步骤二,细胞转染：步骤三,酶学检测：绘制GDH活性抑制曲线,若待筛选化合物能使GDH抑制曲线左移,则表明该化合物具有抑制GDH的活性。(The invention discloses a GDH inhibitor high-flux screening method based on human mutant protein, which achieves that the screened inhibitor is close to the physiological state, relates to the technical field of drug screening, and has the technical scheme that: the method comprises the following steps of firstly, constructing a mutant gene plasmid and preparing a retrovirus carrying the mutant gene plasmid, wherein the plasmid is filled with red fluorescent protein; step two, cell transfection: step three, enzymatic detection: a GDH activity inhibition curve is plotted, and if the compound to be screened can shift the GDH inhibition curve to the left, the compound is indicated to have GDH inhibitory activity.)

1. A high-throughput screening method of GDH inhibitor based on human mutant protein is characterized in that: comprises the following steps of (a) carrying out,

step one, constructing a plasmid of a mutant gene and preparing a retrovirus carrying the plasmid of the mutant gene, wherein the plasmid is filled with red fluorescent protein;

step two, cell transfection: adding retrovirus carrying mutant gene plasmid into the culture medium, continuously culturing the transfected cells for 72 to 96 hours, judging the ratio of red fluorescent protein positive cells under a fluorescence microscope, determining the expression efficiency of the transfected and mutant genes, collecting the cells when the expression efficiency reaches more than 60 percent, and freezing and storing the cells in a refrigerator at the temperature of minus 80 ℃;

step three, enzymatic detection: freezing and storing the cells, thawing, adding cell lysate, homogenizing the broken cells, determining the protein content, diluting the protein of the lysate with 0.1M phosphate buffer, and adding a compound to be screened into a reaction system after serial dilution, wherein the reaction system comprises glutamic acid and NAD +, and the enzymatic reaction is based on the following reactions:

glutamic acid + NAD⁺A-ketoglutaric acid + NH4⁺+ NADH

A GDH activity inhibition curve is plotted, and if the compound to be screened can shift the GDH inhibition curve to the left, the compound is indicated to have GDH inhibitory activity.

2. The high-throughput screening method for human mutein-based GDH inhibitors as claimed in claim 1, wherein: in the third step, the enzyme reaction was carried out in 0.1M phosphate buffer, pH 7.5, NAD + concentration was 0.2 mM, sodium glutamate concentration was 50 mM, protein concentration of the cell lysate added to the reaction was 1 mg/mL, 5 ul was added per reaction, all the reaction solutions were freshly prepared on the day of the experiment, the enzyme reaction was read with a microplate reader, the measurement reaction wavelength was 340nm, the reaction was carried out in 384 well plates, and the total volume of the reaction system was 25 ul.

3. The high-throughput screening method for human mutein-based GDH inhibitors as claimed in claim 2, wherein: in the third step, the compound to be screened is added into the reaction system after serial dilution, and each compound to be screened is diluted from the highest concentration for 11 times according to the dilution of 1 time.

4. The high-throughput screening method for human mutein-based GDH inhibitors as claimed in claim 1, wherein: 0.1M phosphate buffer pH 7.0, and 1 mM EDTA was added.

5. The high-throughput screening method for human mutein-based GDH inhibitors as claimed in claim 1, wherein: the concentration range of the compound to be screened in the reaction system is～mol/L。

6. The high-throughput screening method for human mutein-based GDH inhibitors as claimed in claim 1, wherein: the cells used in the transfection of the cells in step two were 239T cells.

7. The high-throughput screening method for human mutein-based GDH inhibitors as claimed in claim 1, wherein: in the first step, the mutant gene plasmid is constructed, firstly, the point mutation of GDH gene is made by the method of standard molecular biology, and the point mutation of GDH gene uses the mutant protein found from congenital hyperinsulinemia patient as the screening template.

8. The high-throughput screening method for human mutein-based GDH inhibitors as claimed in claim 7, wherein: the point mutations of the GDH gene were gene mutations at five points of H454Y, S445L, R221L, V453M and R269H, respectively.

Technical Field

The invention relates to the technical field of drug screening, in particular to a GDH inhibitor high-throughput screening method based on human mutant protein.

Background

The hypoglycemia of the newborn is a serious disease threatening the health and even the life of the newborn, the repeated severe hypoglycemia threatens the life and can cause permanent and irreversible brain function damage, the proportion of the brain function damage reaches 60-70 percent, and the serious hypoglycemia causes disability for life and brings serious damage and burden to families and society. Because the causes of hypoglycemia are complex (at least 11 pathogenic genes have been found), the major pathogenic genes include: amino acid metabolism key enzyme, Glutamate Dehydrogenase (GDH) gain functional mutation, etc.

Glutamate Dehydrogenase (GDH) is a key enzyme of amino acid metabolism, GDH gain function mutation is a subtype of neonatal hypoglycemia, a patient has hypoglycemia, hyperammonemia caused by liver and kidney function damage, epilepsy and brain development delay caused by brain function damage, learning ability disorder and the like, the GDH gain function mutation is a systemic disease, at present, the hypoglycemia is controlled only by diazoxide, no treatment method is provided for brain function disorder and liver and kidney damage, and therefore a systemic inhibitor for the mutant GDH is urgently needed.

Currently, high-throughput screening is an important mode for the discovery of current innovative drugs, and the development of GDH inhibitors requires finding inhibitors of GDH enzymes in a compound library by a method of high-throughput drug screening. The existing screening method is based on the bovine liver GDH, and because the existing screening method is based on the bovine liver GDH, the GDH expressed in cells cannot maintain the microenvironment in human cells, and cannot have complete related protein regulation and small molecule allosteric regulation environments, so that the screening system cannot approach the physiological state.

Disclosure of Invention

The invention aims to provide a high-throughput screening method of a GDH inhibitor based on human mutant protein, which can achieve that the screened inhibitor is close to the physiological state.

The technical purpose of the invention is realized by the following technical scheme: a high-throughput screening method of GDH inhibitor based on human mutant protein comprises the following steps,

step one, constructing a plasmid of a mutant gene and preparing a retrovirus carrying the plasmid of the mutant gene, wherein the plasmid is filled with red fluorescent protein;

step two, cell transfection: adding retrovirus carrying mutant gene plasmid into the culture medium, continuously culturing the transfected cells for 72 to 96 hours, judging the ratio of red fluorescent protein positive cells under a fluorescence microscope, determining the expression efficiency of the transfected and mutant genes, collecting the cells when the expression efficiency reaches more than 60 percent, and freezing and storing the cells in a refrigerator at the temperature of minus 80 ℃;

step three, enzymatic detection: freezing and storing the cells, thawing, adding cell lysate, homogenizing the broken cells, determining the protein content, diluting the protein of the lysate with 0.1M phosphate buffer, and adding a compound to be screened into a reaction system after serial dilution, wherein the reaction system comprises glutamic acid and NAD +, and the enzymatic reaction is based on the following reactions:

glutamic acid + NAD⁺A-ketoglutaric acid + NH4⁺+ NADH

A GDH activity inhibition curve is plotted, and if the compound to be screened can shift the GDH inhibition curve to the left, the compound is indicated to have GDH inhibitory activity.

By adopting the technical scheme, the plasmid is filled with red fluorescent protein and can directly test the cell transfection efficiency, retrovirus carrying mutant gene plasmid is added into a culture medium, the transfected cells are continuously cultured for 72 to 96 hours, the ratio of the red fluorescent protein positive cells is judged under a fluorescence microscope, so that the transfection and mutant gene expression efficiency is determined, GTP is a natural intracellular inhibitor of GDH, the mutant GDH has the characteristics of reduced sensitivity to GTP and obviously right-shifted inhibition curve, in the enzymological detection, the compound to be screened can make the GDH inhibition curve left-shifted, so that the compound has the activity of inhibiting GDH, the GDH expressed in cells maintains the microenvironment in the cells, has complete related protein regulation and small molecule allosteric regulation environments, the screening system is closer to the physiological state, in addition, the plasmid of the mutant gene is constructed into GDH mutation discovered from a patient with congenital hyperinsulinemia, the mutant protein found from the patient is used as a screening template, and the screened inhibitor has direct clinical guiding significance.

Preferably, in step three, the enzyme reaction is carried out in 0.1M phosphate buffer solution with pH 7.5, the concentration of NAD + is 0.2 mM, the concentration of sodium glutamate is 50 mM, the concentration of protein in the cell lysate added to the reaction is 1 mg/mL, 5 ul of each reaction is added, all the reaction solutions are freshly prepared on the day of the experiment, the enzyme reaction is read by a microplate reader, the measurement reaction wavelength is 340nm, the reaction is carried out in 384-well plates, and the total volume of the reaction system is 25 ul.

Preferably, in step three, the compound to be screened is added into the reaction system after being serially diluted, and each compound to be screened is diluted from the highest concentration for 11 times according to the dilution of 1 time.

Preferably, 0.1M phosphate buffer is pH 7.0 and 1 mM EDTA is added.

Preferably, the concentration range of the compound to be screened in the reaction system is～mol/L。

Preferably, the cells used in the transfection of the cells in step two are 239T cells.

Preferably, in the step one, plasmid construction of the mutant gene is performed by first performing point mutation of the GDH gene using a standard molecular biology method, and the point mutation of the GDH gene is performed using a mutant protein found from a patient with congenital hyperinsulinemia as a screening template.

Preferably, the point mutations of the GDH gene are gene mutations at five points of H454Y, S445L, R221L, V453M and R269H, respectively.

In conclusion, the invention has the following beneficial effects: the plasmid is filled with red fluorescent protein and can directly test the cell transfection efficiency, retrovirus carrying mutant gene plasmid is added into a culture medium, the transfected cells are continuously cultured for 72 to 96 hours, the red fluorescent protein positive cell ratio is judged under a fluorescence microscope, thereby determining the transfection and mutant gene expression efficiency, GTP is a natural intracellular inhibitor of GDH, the mutant GDH has the characteristics of reduced sensitivity to GTP and obvious right shift of an inhibition curve, in the enzymological detection, a compound to be screened can shift the GDH inhibition curve to the left, which indicates that the compound has the activity of inhibiting GDH, GDH expressed in cells maintains the microenvironment in the cells, has complete related protein regulation and small molecule allosteric regulation environment, a screening system is closer to the physiological state, in addition, the plasmid of the mutant gene is constructed into GDH mutation discovered from a patient with congenital hyperinsulinemia, the mutant protein found from the patient is used as a screening template, and the screened inhibitor has direct clinical guiding significance.

Drawings

FIG. 1 shows the effect of GTP concentration on the activity of wild-type human GDH (WT) and GDH with different gain-of-function mutations;

FIG. 2 is a graph of the inhibition of mutant R221L human GDH by three compounds to be screened, Comp-1, Comp-2 and Comp-3.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings.

A high-throughput screening method of GDH inhibitor based on human mutant protein comprises the following steps,

step one, constructing a plasmid of a mutant gene and preparing a retrovirus carrying the plasmid of the mutant gene, wherein the plasmid is filled with red fluorescent protein; five GDH mutations (H454Y, S445L, R221L, V453M and R269H) were all found in patients with idiopathic hyperinsulinemia, each expressed on a retroviral plasmid by point mutation.

First, point mutation of GDH gene was performed by standard molecular biology method, for example, H454Y, 1532 site "C" was mutated to "T".

(1) Designing a primer:

an upstream primer: CATCTGAGAAAGACATCGTGTACTCCTGGCTTGG

A downstream primer: ACACGATGTCTTTCTCAGATGCACCCGATATC

(2) PCR cloning:

carrying out PCR amplification (10 ul) by using the original plasmid as a template;

(3) taking 2-3 ul from the PCR system, verifying the size by nucleic acid electrophoresis, if the size is correct, adding 0.5 ul DpnI into the rest reaction system, and digesting at 37 ℃ for more than 3h (or overnight);

(4) sucking 5 ul of reaction liquid in the step three to transform DH5a into competence;

(5) selecting a monoclonal, and sequencing to verify whether mutation is successful;

(6) delivering to biological companies to prepare lenti virus carrying the mutant gene plasmid;

(7) the lenti virus carrying the S445L, R221L, V453M and R269H mutant gene plasmids was prepared in the same manner.

The "TC" at positions S445L, 1505 and 1506 was mutated to "CT", and the forward primer was designed to be CAGAGTTCCAAGACAGGATACTGGGTGCATCTGA and the reverse primer to be AGTATCCTGTCTTGGAACTCTGCCGTGGGTACA.

R221L, G at position 833 was mutated to T, the upstream primer was designed to be ACGCATCTCTGCTACTGGCCTTGGTGTCTTCCA, and the downstream primer was designed to be AGGCCAGTAGCAGAGATGCGTCCATGGATTCC.

V453M, mutation of 1529 site 'G' to 'A', design of upstream primer GTGCATCTGAGAAAGACATCATGCACTCTGGCT and downstream primer TGATGTCTTTCTCAGATGCACCCGATATCCTG.

R269H, the "G" at position 978 was mutated to "A", the upstream primer was designed to be CACTCTATGAGATATTTACATCATTTTGGTGCTAA, and the downstream primer was designed to be TGATGTAAATATCTCATAGAGTGTAGGCCCAC.

The rest of the processes are consistent with the above (2) - (5).

Step two, cell transfection:

239T cells are used as an expression system of mutant genes, and the specific transfection steps are as follows:

day 0: HEK 293 cells were digested with 0.25% pancreatin and the cell suspension concentration was maintained at 6X 10 after digestion and counting⁶about/mL, mixed well with 13mL DMEM medium (containing serum but no antibiotics), added to a 100mm dish, placed at 37 deg.C with 5% CO₂Overnight (about 20-24 hours);

day 1: 2 hours before infection, discardRemove the culture medium, add 8.5mL of fresh DMEM medium (serum free but antibiotic free), add 1.5mL of virus premix to a 100mm petri dish at 37 deg.C with 5% CO₂Culturing (16-18 hours, can prolong infection time to 24 hours). Shake several times during infection, if possible.

Day 2: the culture medium was discarded, and 15mL of fresh DMEM medium (containing serum without antibiotics) was added at 37 ℃ with 5% CO₂Culturing in an incubator. If the cells are nearly confluent, the cells can be separated into two to three new 100mm dishes;

day 4-5: after transfection, the cells were cultured for 72 to 96 hours, and the ratio of red fluorescent protein positive cells was observed under a fluorescence microscope to determine the efficiency of transfection and mutant gene expression to be above 60%, and then the cells were harvested (0.5% trypsin was used), washed 2 times with cold PBS, separated into 15-16 1.5mL EP tubes, centrifuged (13000 rpm 1 min), discarded in PBS, and stored in a refrigerator at-80 ℃.

Note: if the infection is not effective, the infection can be carried out again on day 3.

Five GDH mutations (H454Y, S445L, R221L, V453M and R269H) were all found in patients with idiopathic hyperinsulinemia, each expressed by point mutations on a lenti virus (retrovirus) plasmid, and by transfection into 293T cells. GTP is a natural intracellular inhibitor of GDH and mutant GDH gain of function is characterized by reduced sensitivity to GTP with a significant shift to the right of the inhibition curve (see FIG. 1).

Step three, enzymatic detection: selecting transfected cells after point mutation of GDH gene of R221L from the second step, freezing the cells, adding cell lysate into the cells, homogenizing the broken cells, determining the protein content, diluting the protein of the lysate to an ideal concentration (about 1 mg/mL) by using 0.1M phosphate buffer (pH 7.0, adding 1 mM EDTA), adding the compound to be screened into the reaction system after serial dilution, wherein the compound to be screened has the desired effect of inhibiting the activity of GDH, and the enzymatic reaction system comprises glutamic acid and NAD +, which is based on the following reactions:

glutamic acid + NAD⁺A-ketoglutaric acid + NH4⁺+ NADH

The enzyme reaction was carried out in 0.1M phosphate buffer pH 7.5 with a concentration of 0.2 mM NAD + and 50 mM sodium glutamate. The protein concentration of the cell lysate added to the reactions was 1 mg/mL, 5 ul was added per reaction, but fine-tuned according to the reaction rate. All reaction solutions were prepared fresh on the day of the experiment. The enzyme reaction is read by a Clariostar microplate reader of BMG company, the measurement reaction wavelength is 340nm, the reaction is carried out in a 384-well plate, the total capacity of a reaction system is 25 ul, three compounds to be screened are selected, the three compounds to be screened have the effect of inhibiting the activity of GDH, each compound to be screened is diluted from the highest concentration, and the compounds to be screened are diluted for 11 times according to the dilution of 1 time, if the lowest concentration is 1, the highest concentration is 211.

The absorbance light with the wavelength of 340nm is read by a microplate reader, and the reaction time is 1 hour. Calculation of IC50 values for GDH inhibitors: the IC50 for the GDH inhibitor was calculated using the software Prism 6.0.

And drawing an activity inhibition curve, as shown in figure 2, showing that all three compounds to be screened can shift the GDH inhibition curve to the left, indicating that the compounds have GDH inhibition activity, the GDH expressed in cells maintains the microenvironment in the cells, and has complete related protein regulation and small molecule allosteric regulation environments, and a screening system is closer to a physiological state.

The above description is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments, and all technical solutions belonging to the idea of the present invention belong to the protection scope of the present invention. It should be noted that modifications and embellishments within the scope of the invention may occur to those skilled in the art without departing from the principle of the invention, and are considered to be within the scope of the invention.