CN111363782A - Kit for identifying enzymatic activity of X-linked rickets PHEX gene wild type and mutant proteosome - Google Patents

Abstract

The invention discloses a kit for identifying X-linked rickets PHEX gene wild type and mutant proteosome enzyme activities, which comprises a carrier containing wild type and mutant secretory PHEX genes and host cells. The kit can rapidly identify the functions of PHEX gene mutation, provide a target point for designing a medicament for further treating the disease, and provide a theoretical basis for clarifying a pathogenic mechanism of the disease.

Description

Kit for identifying enzymatic activity of X-linked rickets PHEX gene wild type and mutant proteosome

Technical Field

The invention belongs to the field of medicine. In particular to a kit for identifying the enzymatic activities of wild type and mutant proteosome of X-linked rickets PHEX gene and a method for detecting by using the kit.

Background

Rickets with low blood phosphorus are common metabolic bone diseases of children, and are a group of diseases of poor bone mineralization and osteomalacia caused by low blood phosphorus level and insufficient generation of active vitamin D. Wherein X-linked rickets with low blood phosphorus are the most common hereditary rickets with low blood phosphorus, and the incidence rate is about 1: 20000, accounts for over 80% of hereditary rickets with low blood phosphorus, and is clinically characterized by retarded growth and development, short stature, hypophosphatemia, skeletal calcification defect, proximal tubular reabsorption phosphorus disorder, insufficient production of 1,25- (OH)2-D3, etc. The international cooperative research group in 1995 established that the pathogenic gene XLH is the PHEX gene located on the X chromosome, a phosphorus regulatory gene present on the X chromosome that is homologous to endopeptidase.

According to the HGMD database, nearly 500 PHEX gene mutations are currently recorded, wherein missense/nonsense mutation is 206, splice site mutation is 98, small insertion is 68, small deletion is 116, and large fragment insertion/deletion is 76. However, only less than 20 PHEX mutations are subjected to function evaluation, and the function evaluation is currently a research property, and the experiment is complicated, so that rapid function identification of patient-derived PHEX gene mutations cannot be carried out. The invention mainly aims to construct an enzyme activity detection for rapidly detecting PHEX gene mutation, and provides an experimental basis for evaluating PHEX mutation and treating a drug target in the future.

Disclosure of Invention

The invention aims to provide a kit for identifying X-linked rickets PHEX gene wild type and mutant proteosome enzyme activities.

The purpose of the invention is realized by the following modes:

a kit for identifying the enzymatic activity of wild-type and mutant proteosome of X-linked rickets with low blood phosphorus PHEX gene is characterized by comprising a carrier containing wild-type and mutant secretory PHEX genes and a host cell.

The construction of the recombinant expression vector comprises the construction of a secretory PHEX recombinant expression vector and the construction of a secretory PHEX plasmid.

The secretory PHEX mutation is constructed, and the primers are designed as follows:

Sec-F: cagtgatcgctcaacaaacaaccAGTCAAGGTCTCTTAAGTCTCCAAGC

Sec-R:ttgttgagcgatcactgtcagGACAAACACGACCAGGGCAA

the construction of the secretory PHEX recombinant expression vector is to synthesize a PHEX-cDNA template and carry out double enzyme digestion on the target vector pcDNA-3.1 by using XhoI and BamHI.

The secretory sec-PHEX-G553E point mutation primer is designed as follows:

G553E-F: TTCCAGCAGaAGAGCTCCAGAAGCCTTTCTTTT

G553E-R: GAGCTCTtCTGCTGGAAATCGGATCTGGTTGG

freshly extracted pcDNA-3.1-sec-PHEX-WT plasmid DNA is used as a template.

Compared with the prior art, the invention has the beneficial effects that: the kit comprises vectors and host cells of wild type and mutant secretory PHEX genes, so that the rapid functional identification of the PHEX gene mutation is carried out, a target point for designing a medicament is provided for further treating the disease, and a theoretical basis is provided for clarifying a pathogenic mechanism of the disease.

Drawings

FIG. 1 PHEX gene mutation G553E sequencing and conservation analysis diagram.

FIG. 2 schematic diagram of PHEX transmembrane domain engineering.

FIG. 3 PHEX protein assay shows SEC-PHEX^WT、SEC-PHEX^G553ECan be successfully secreted into cell supernatants.

FIG. 4 SEC-PHEX^WT、SEC-PHEX^G553EAnd (4) detecting enzyme activity.

Detailed Description

For the convenience of understanding the technical scheme of the invention, the technical scheme of the invention is described in detail in a specific use mode as follows:

experimental reagents and materials

(1) Cell: human embryonic kidney epithelial cells (293T cells) were provided by Shanghai Life sciences research institute.

(2) The recombinant expression vector construction, the expression vector pcDNA-3.1-PHEX is constructed by Changsha Youbao biosynthesis, the site-directed Mutagenesis Kit Mut Express II Fast Mutagenesis Kit V2, the PCR amplification Kit LAmp DNApolymerase and the chemically competent cell DH5 α are purchased from Nanjing Novozam company.

(3) Cell culture and transfection DMEM, FBS, 0.25% Trypsin-EDTA (1 ×), 1 × PBS, Penicillin-Streptomyces Liquid (Gibco, USA), DMSO (Sigma, USA), Polyjet.

(4) Protein extraction and detection: protein purification column 30KD (Milipore), PHEX antibody (Abcam), secondary antibody (Biyuntian), SDS-PAGE gel reagent (Kaiyi organism)

(5) Enzyme activity detection reagent: recombinant human PHEX protein (R & D), fluorescent polypeptide Substrate Mca-YVADAPK (Dnp) -OHF fluorescent Peptide Substrate (R & D), MES (Sigma)

Bioinformatic analysis of PHEX Gene mutation G553E

The PHEX missense mutation c.1658G > A/G553E is from one patient with phosphorus-deficient rickets, and the sequencing is shown in figure 1A, and c.1658G > A causes the mutation of the 553 th position of the PHEX coding amino acid sequence from glycine (Gly) to glutamic acid (Glu). The mutatantaster software predicted it to be pathogenic and protein conservation analysis showed that glycine 553 is a highly conserved amino acid in various species (fig. 1B). Suggesting that the mutation may cause the three-dimensional conformation change of the PHEX protein, thereby affecting the stability of the PHEX protein and the cellular localization of the protein.

Example 1

1. Construction of secretory PHEX recombinant expression vector

PHEX-cDNA template is synthesized by Changsha Youbao company, and target vector pcDNA-3.1 is subjected to double enzyme digestion (shown as figure 1, respectively is a pcDNA3.1 plasmid map and a multiple cloning site thereof) by using XhoI and BamHI, wherein the enzyme digestion system is as follows:

the reaction conditions were 37 ℃ for 2 h.

(3) And (2) agarose gel electrophoresis, namely adding 0.375g of agarose powder into 25ml of 0.5 × TBE buffer solution, heating the agarose powder in a microwave oven until the agarose is completely dissolved, cooling the agarose powder to about 50 ℃, adding 2 mul of GelStain, slowly mixing the mixture uniformly, pouring the mixture into a gel preparation tank, pulling out a comb after the agarose is completely solidified for 30min, putting the comb into an electrophoresis tank, adding a proper amount of 0.5 × TBE buffer solution, adding a proper amount of 10 × loading buffer into a PCR product and a product after double enzyme digestion, adding the mixture into a sample hole, regulating the voltage to 120V for electrophoresis, after about 30min, carrying out electrophoresis on the sample to the upper 2/3 position of the gel, turning off the power supply, and observing the electrophoresis result under an ultraviolet transilluminator.

(4) And (3) glue recovery: the Gel band of the 4.0kb PCR product fragment and the 4.7kb size pcDNA3.1-3XFlag vector was excised under UV light, Gel recovered using the Omega Gel Extraction Kit, and 20. mu.l was Gel recovered according to the instructions, and the recovery procedure was as follows:

a. weighing a 1.5ml centrifuge tube in advance, cutting the gel, putting the cut gel into the 1.5ml centrifuge tube, weighing, and adding xp2 buffer according to the proportion that the gel weight is 1 g/ml;

b, water bath at 55 ℃ for 7min until the glue is completely melted, turning upside down and mixing uniformly for a plurality of times, and taking out the vortex centrifuge tube for 3 min;

c. sleeving a column in a 2ml collecting pipe, adding a glue solution into the column, centrifuging at room temperature of 10000g for 1min, and discarding the solution;

d. adding 300 μ l xp2 buffer, centrifuging at 10000g for 1min at room temperature, and discarding the solution;

e. adding 700 μ l spw wash buffer, centrifuging at 10000g for 1min at room temperature, discarding the solution, and repeating once;

13000g the column was air-separated at room temperature for 2min and the column was dried;

g. the column was placed in a new 1.5ml centrifuge tube, 20. mu.l of Elution buffer was added to the center of the column, left to stand for 2min, centrifuged at 10000g at room temperature for 1min to elute DNA, and stored at-20 ℃ after the concentration and purity were determined.

(5) Connecting: preparing the following reaction system in an ice water bath, wherein the reaction condition is 30min at 37 ℃, and cooling for 5min in the ice water bath after the reaction is finished.

(6) And (3) transformation:

a. adding 20 μ l of the ligation product into 200 μ l of competent cell DH5 α, mixing, ice-cooling for 30min, heat-shocking in 42 deg.C water bath for 90s, and ice-cooling for 3 min;

b. adding the mixture into 900 μ l LB liquid medium without antibiotics, and shaking at 37 deg.C and 200rpm for 60 min;

c. uniformly mixing 50 mu L of X-gal at a concentration of 50mg/ml and 50 mu L of IPTG at a concentration of 25mg/ml, uniformly coating the mixture on an LB solid culture medium plate containing kanamycin at a concentration of 50mg/L, and rightly standing the mixture at 37 ℃ for 60min until the liquid is completely absorbed;

d. centrifuging the bacteria solution after bacteria shaking at 12000rpm/s for 1min, discarding the supernatant, resuspending with non-resistant LB liquid culture medium 100 μ l, and uniformly coating on the prepared LB solid culture medium plate containing kanamycin, X-gal and IPTG;

e.37 ℃ upright culture medium until the bacteria liquid is completely absorbed, and then 37 ℃ inverted culture is carried out overnight;

(7) screening and identification of recombinant plasmid pcDNA-3.1-PHEX

a. Randomly selecting 5 white colonies from each sample on a solid culture medium plate with a single clone, inoculating the white colonies in 1ml of LB liquid culture medium containing 50mg/ml kanamycin, and shaking the colonies at 37 ℃ and 200rpm overnight;

b. taking 500 mu l of each bacterial liquid, sending to Huada Gene science and technology company for sequencing, and storing the rest at 4 ℃ for waiting for a sequencing result;

c. selecting bacterial liquid with correct sequencing, transferring the bacterial liquid into 5ml of new LB liquid culture medium with kanamycin resistance at 37 ℃, shaking the bacterial liquid at 200rpm overnight, adding sterilized glycerol to 15%, subpackaging and storing at-80 ℃.

Construction of secreted PHEX plasmid

(1) The secretory PHEX mutation construction is shown in FIG. 2, and the primers are designed as follows:

Sec-F: cagtgatcgctcaacaaacaaccAGTCAAGGTCTCTTAAGTCTCCAAGC

Sec-R:ttgttgagcgatcactgtcagGACAAACACGACCAGGGCAA

(2) the target plasmid amplification is carried out by taking freshly extracted pcDNA-3.1-PHEX plasmid DNA as a template, and the system and the reaction conditions are as follows:

reagent	Amount used (ul)
		2×Max Buffer	25.0
dNTP Mix (10 mM each)	1.0
		Plasmid DNA	1ng
Upstream primer (10uM)	2.0
		Downstream primer (10uM)	2.0
Phanta Max Super-Fidelity DNA Polymerase	1.0
		ddH2O	Up to 50

And (3) PCR reaction conditions:

（3）

(4) the amplification product, Dpnl, was digested as follows:

reagent	Amount used (ul)
		Dpnl	1
Amplification product	50

Gently blowing, beating and mixing, and keeping the temperature at 37 ℃ for 2 h.

(5) A recombination reaction system:

reagent	Amount used (ul)
		5×CE II Buffer	4.0
Dpnl digestion product	170ng
		ExnaseII	2.0
ddH2O	Up to 20

Blowing, beating and mixing uniformly (avoiding generating bubbles), reacting at 37 ℃ for 30min, immediately placing in an ice water bath for cooling for 5min, and then carrying out reaction product conversion, plate coating and clone identification with the same steps as 1.1.

(2) The secretory sec-PHEX-G553E point mutation primer was designed as follows:

G553E-F: TTCCAGCAGaAGAGCTCCAGAAGCCTTTCTTTT

G553E-R: GAGCTCTtCTGCTGGAAATCGGATCTGGTTGG

the plasmid construction process refers to the method by using freshly extracted pcDNA-3.1-sec-PHEX-WT plasmid DNA as a template.

2. HEK293 cell culture and transfection wild type SEC-PHEX^WTAnd mutant SEC-SEC-PHEX^G553ERecombinant expression vector

After the HEK293 cells were recovered, the cells were inoculated into 10cm cell culture dishes at 37 ℃ with 5% CO₂When the cells are cultured under the condition that the density is close to 60 percent, the serum-free DMEM medium is used for plasmid transfection. According to the recommended operation process of the transfection reagent, 500 mu l DMEM and 30 mu l polyjet transfection reagent are mixed uniformly, and then 500 mu l DMEM culture medium and 10 mu g of no-load pcDNA-3.1 plasmid and wild type SEC-PHEX are taken^WTAnd mutant SEC-SEC-PHEX^G553EAnd respectively mixing the recombinant expression vectors uniformly, and incubating the mixture of the two tubes for 15 minutes at room temperature after mixing uniformly. 1ml of the mixture was added to each culture well at 37 ℃ with 5% CO₂After 4 hours of incubation under conditions, the complete medium (DMEM with 10% FBS) was replaced. 37 ℃ and 5% CO₂After 48h of culture, cell supernatants were collected for subsequent experiments.

3. Secretory SEC-PHEX^WT、SEC-PHEX^G553EConcentration and detection of supernatant protein

(1) Cell supernatant protein concentrate

1. Transferring the cell supernatant to a 15ml centrifuge tube, centrifuging at 12000rpm for 3min,

2. transferring the supernatant to a 30KD protein concentration tube (millicore), centrifuging at 5000rpm for 15min, collecting the concentrated protein, and subpackaging at-80 deg.C for use.

(2) Western Blot detection

The experimental results are as follows: western Blot for detecting PHEX protein expression secreted by cells, and the result shows that SEC-PHEX^WT、SEC-PHEX^G553ECan be successfully secreted into the cell supernatant. The mutant protein was significantly reduced compared to the wild type (figure 3).

4. Secretory PHEX-SEC^WT、PHEX-SEC^G553EEnzyme activity detection

The experimental steps are as follows:

1. buffer 50mM MES, 250mM NaCl, pH 6.0

2. The recombinant protein was formulated to 4 ng/. mu.l using Buffer, and the fluorogenic substrate was formulated to 100. mu.m

3. Sample preparation: SEC-PHEX^WT、SEC-PHEX^G553EThe supernatant was concentrated to 50. mu.l + substrate to 50. mu.l;

yang Shen: 50ul (200ng) of recombinant PHEX protein plus 50ul of substrate;

yin Shen: substrate 50ul +50 ul Buffer

4. Detection by a microplate reader (Molecular Devices) on a 96-well microplate, wherein the excitation light wavelength is as follows: 320nM, 405nM wavelength of emitted light, 30min each 5min for detection of enzyme activity.

The experimental results are as follows:

SEC-PHEX in comparison with the negative control^WT、SEC-PHEX^G553EAll can enzymolyze fluorescent polypeptide substrate, and is combined with wild type SEC-PHEX^WTIn contrast, mutant SEC-PHEX^G553EThe enzyme activity was significantly reduced, with only 13% of the activity present (figure 4).

Claims (6)

1. A kit for identifying the enzymatic activity of wild-type and mutant proteosome of X-linked rickets with low blood phosphorus PHEX gene is characterized by comprising a carrier containing wild-type and mutant secretory PHEX genes and a host cell.

2. The kit for identifying the enzymatic activities of wild type and mutant proteosome of X-linked rickets PHEX gene in low blood phosphorus content as claimed in claim 1, wherein the construction of recombinant expression vector comprises construction of secretory PHEX recombinant expression vector and construction of secretory PHEX plasmid.

3. The kit for identifying the enzymatic activities of wild type and mutant proteosome of X-linked rickets PHEX gene in low blood phosphorus level as claimed in claim 2, wherein said secreted PHEX mutation is constructed by the following primers:

Sec-F: cagtgatcgctcaacaaacaaccAGTCAAGGTCTCTTAAGTCTCCAAGC

Sec-R:ttgttgagcgatcactgtcagGACAAACACGACCAGGGCAA。

4. the kit for identifying PHEX gene wild type and mutant proteosome enzyme activities of X-linked rickets with low blood phosphorus content as claimed in claim 3, wherein said PHEX recombinant expression vector for secretion construction is constructed by first synthesizing PHEX-cDNA template, and simultaneously performing double enzyme digestion on the target vector pcDNA-3.1 with XhoI and BamHI.

5. The kit for identifying PHEX gene wild type and mutant proteosome enzyme activities of X-linked rickets with low blood phosphorus as claimed in claim 2, wherein said secretory sec-PHEX-G553E point mutation primer is designed as follows:

G553E-F: TTCCAGCAGaAGAGCTCCAGAAGCCTTTCTTTT

G553E-R: GAGCTCTtCTGCTGGAAATCGGATCTGGTTGG。

6. the kit for identifying PHEX gene wild type and mutant proteosome enzyme activities of X-linked rickets with low blood phosphorus content according to claim 5, wherein pcDNA-3.1-sec-PHEX-WT plasmid DNA is freshly extracted as a template.

Images