CN114525365A - PRCP kit acting on SARS-Cov-2 induced acute lung injury - Google Patents

Abstract

The invention relates to the field of biotechnology, in particular to a PRCP kit acting on SARS-Cov-2 induced acute lung injury. The invention constructs a mouse model of SARS-CoV-2 induced ALI by Vero E6 cell strain capable of expressing SARS-CoV-2 protein (the sequence is shown as SEQ ID NO: 1), intervenes by injecting lentiviral particles with high expression PRCP and atomizing PRCP protein with different concentrations through tail vein, finds out a better administration mode of PRCP for inhibiting SARS-CoV-2 induced ALI by observing the difference of lung elasticity change, lung injury score, lung tissue dry and wet weight and lung tissue pathology of mice of a control group and different intervened groups, and prepares a corresponding kit.

Description

PRCP kit acting on SARS-Cov-2 induced acute lung injury

Technical Field

The invention relates to the field of biotechnology, in particular to a PRCP kit acting on SARS-Cov-2 induced acute lung injury.

Background

Acute Lung Injury (ALI) is a clinically common and Acute severe condition, and is characterized by progressively aggravated dyspnea and intractable hypoxemia, and the severe stage of ALI can be developed into Adult Respiratory Distress Syndrome (ARDS), and ARDS is a main cause of Acute respiratory failure in clinical severe condition, and the fatality rate is up to 50%. The main common pathogenic factors are: infection, inhalation of harmful substances, trauma, shock, poisoning, etc.

It has been shown that SARS-Cov-2 induced Acute Lung Injury (ALI) is the leading cause of death in patients with novel coronavirus pneumonia (COVID-19) by the mechanism that SARS-Cov-2 binds to the cellular receptor angiotensin converting enzyme 2(ACE2), resulting in the loss of ACE2 and its feedback induces acute inflammatory responses mediated by angiotensin II (Ang II) and angiotensin type I receptor (AT 1R). Therefore, inhibition of Ang II/AT 1R-mediated acute inflammation is an important target for the treatment of ALI. However, the use of ACEI/ARB increases ACE2 expression, accelerating viral entry into cells and replication.

Proline carboxypeptidase (PRCP) is an important angiotensin II degrading enzyme. First discovered by Yang HYT, equal to 1968. PRCP decomposes Ang II into Ang1-7, feedback regulates ACE/ACE2 Ang II/Ang 1-7 dynamic balance, alleviates Ang II/AT 1R mediated inflammatory reaction without increasing ACE2 expression.

The PRCP in the prior art is up-regulated in the inflammatory reaction of endothelial cells, and no PRCP kit acting on SARS-Cov-2 induced acute lung injury exists at present.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a PRCP kit acting on SARS-Cov-2 induced acute lung injury. The invention screens out the administration mode (high-expression PRCP slow virus particles) with better effectiveness of PRCP in inhibiting SARS-CoV-2 induced ALI, and prepares a corresponding kit, thereby improving the effectiveness and safety of clinical medication.

In order to achieve the purpose, the invention adopts the technical scheme that:

in a first object, the present invention provides an oligonucleotide comprising a primer pair for detecting a gene of SARS-Cov-2 protein.

As a preferred embodiment of the oligonucleotide of the present invention, the oligonucleotide further comprises a primer pair for detecting PRCP.

As a preferred embodiment of the oligonucleotide of the invention, the sequence of the SARS-Cov-2 protein is shown as SEQ ID NO. 1.

As a preferred embodiment of the oligonucleotide of the invention, the sequence of a primer pair for detecting SARS-Cov-2 protein gene is shown as SEQ ID NO. 2-5.

As a preferred embodiment of the oligonucleotide, the sequence of a primer pair for detecting PRCP is shown as SEQ ID NO. 6-7.

In a second aspect, the present invention provides a PRCP kit for acute lung injury induced by SARS-Cov-2, the kit comprising the above-mentioned oligonucleotides.

The third objective of the invention provides a method for constructing a mouse model of SARS-Cov-2 induced ALI, which comprises the following steps:

s1, constructing Vero E6 cell strain expressing SARS-Cov-2 protein;

s2, constructing slow virus particles pEZ-Lv105-PRCP expressing PRCP;

s3, dividing into a control group, an ALI + empty vector group, an ALI model group, an ALI + PRCP slow virus particle group and an ALI + PRCP atomization administration group to be applied to a mouse;

s4, constructing an ALI mouse model and introducing slow virus particles of PRCP.

As a preferred embodiment of the method for constructing a mouse model of SARS-Cov-2 induced ALI according to the present invention, the PRCP concentration in step S3 is 0.05 to 0.2 mg/ml.

Preferably, the concentration of PRCP in said step S3 is 0.05mg/ml, 0.1mg/ml or 0.2 mg/ml.

The invention screens out proline carboxypeptidase (PRCP) with better effectiveness to prepare the PRCP kit by comparing the curative effects of proline carboxypeptidase (PRCP) with different administration modes on an ALI mouse model induced by SARS-COV-2.

Compared with the prior art, the invention has the following beneficial effects:

the invention constructs a mouse model of SARS-CoV-2 induced ALI by Vero E6 cell strain capable of expressing SARS-CoV-2 protein (the sequence is shown as SEQ ID NO: 1), injects lentivirus particles (pEZ-Lv105-PRCP) of high expression PRCP and atomizes PRCP protein with different concentrations through tail vein to intervene, finds out a better administration mode of PRCP to inhibit the effectiveness of SARS-CoV-2 induced ALI by observing the difference of lung elasticity change, lung injury score, lung tissue dry and wet weight and lung tissue pathology of mice of a control group and different intervention groups, and prepares a corresponding kit. The invention can guide doctors to implement the predicted medication, and improves the effectiveness and safety of clinical medication. Meanwhile, the research result also provides a basis for developing and applying a new medicine which is used for developing and contains PRCP components and inhibiting SARS-CoV-2 induced ALI.

Drawings

FIG. 1 is a graph showing the results of PCR of SASR-Cov-2 protein;

FIG. 2 is a map of a protein expression vector constructed in the present invention;

FIG. 3 is a diagram showing the result of identification of SASR-Cov-2 protein;

FIG. 4 is a chart showing the results of dry-wet reanalysis of lung tissues of groups of mice in example 3;

FIG. 5 is a graph of the results of qPCR detection of PRCP and ACE gene mRNA expression;

FIG. 6 is a graph showing the result of qPCR detection of expression of mRNA of Ang and ACE2 genes.

Detailed Description

To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific embodiments.

In the following examples, the experimental methods used were all conventional methods unless otherwise specified, and the materials, reagents and the like used were commercially available without otherwise specified.

SASR-Cov-2, primer information are shown in Table 1 and Table 2.

TABLE 1

Name of Gene	Gene ID	Transcript ID	Product of	Position of the primer
					SASR-Cov-2	43740568	NC-045512.2	3861bp	1-3861
SASR-Cov-2	43740568	NC-045512.2	1369bp	794-2162

TABLE 2

Example 1 construction of protein expression vector

A protein expression vector carrying a gene coding SASR-Cov-2 protein is constructed by a gene recombination technology, and the method specifically comprises the following steps:

1) and (3) PCR: PCR amplification system: taking 2 mu L of recombinant protein template; primers (F: P10015 and R: P10016) each 2. mu.L; high eff bottle Gold High-Fide1 μ L of lite DNA Polymerase (2U/. mu.L); 2 × cancer Gold PCR buffer (containing Mg)²⁺，dNTPs)25μL；ddH₂O 18μL；

PCR amplification procedure: pre-denaturation at 98 ℃/3 min; 30 cycles (denaturation 98 ℃/10s, annealing 65 ℃/20s, extension 72 ℃ 1kb/min), extension 72 ℃/5min, 12 ℃ storage. The PCR results are shown in FIG. 1.

2) Plasmid extraction: firstly, inoculating an unloaded colony in 5mL of LB culture medium (GL7002, Czech) containing corresponding resistance, placing the inoculated colony in a constant-temperature shaking table at 37 ℃, shaking at 220rpm and culturing overnight; ② extracting the plasmid by using a plasmid DNA micro-extraction kit.

3) Enzyme digestion: firstly, an enzyme digestion system: pLVX-AcGFP 1-N1900-1500 ng; fastigest EcoR I (cat No. FD0274, brand Thermo fisher) 1. mu.L; fastduest Xho I (FD0694, brand Thermo fisher) 1. mu.L; 10 × unisource Buffer 5 μ L; ddH₂O To 50μL；

Enzyme digestion program: and (3) at 37 ℃ for 10-30 min.

4) Homologous recombination: preparing a homologous recombination system: 120ng of digested plasmid, 100ng of PCR fragment, 5. mu.L of 2 × Assembly Mix, complement ddH₂O TO 10μL；

② reaction system: gently mixed, ligated at 50 ℃ for 15 minutes, and after the reaction is complete, the recombinant product (protein expression vector) can be stored at-20 ℃ or used directly for transformation after cooling the centrifuge tubes on ice for several seconds. The map of the vector is shown in FIG. 2.

4) Competent cell transformation:

mixing the connecting system with 50 mu L of competent cells, and incubating for 30min on ice; ② heat shock is carried out for 45s at 42 ℃; ③ instantaneously transferring to ice and incubating for 5 min; mixing the competent cells with 1mL of LB culture medium, and performing shake culture at 37 ℃ for 45 min; and coating the plate.

5) And (3) identification: picking single colony (5-10) on a culture plate, dissolving in 10 mu L of sterile water, and taking 4 mu L as a template to carry out PCR; ② adding the rest into 1mL of culture medium containing corresponding resistance to perform shake culture at 37 ℃. The identification result is shown in FIG. 3, and the identification primer is F: p10077 and R: p10078, the product length is 1369 bp.

6) Sequencing: PCR identifies positive colonies, and the positive colonies are sent to a sequencing company for first-generation sequencing by using sequencing primers. The sequence of SASR-Cov-2 protein is shown in SEQ ID NO:1, and the size of the recombinant protein is 142.9 kDa.

7) Extraction of endotoxin-free plasmids:

first, 5mL of overnight-cultured bacterial solution was collected, centrifuged at 12000Xg for 2min to collect cells, and the overnight-cultured bacterial solution was collected and the supernatant was removed (removed by aspiration).

If the amount of the bacterial liquid is too large, the bacterial liquid can be centrifugally collected for multiple times;

adding 250 mu L of colorless solution RB (containing RNase A), and oscillating the suspended bacteria without leaving small bacterium blocks;

adding 250 mu L of blue solution LB, turning and mixing for 4-6 times gently to ensure that the thalli are fully cracked to form a blue transparent solution, wherein the color is changed from semi-transparent to transparent blue, and the complete cracking is indicated (less than 5 minutes);

fifth, adding 350 mu L of yellow solution NB, mixing gently for 5-6 times (color is changed from blue to yellow completely, indicating mixing is uniform, neutralization is complete) until a compact yellow aggregate is formed, and standing for 2 minutes at room temperature;

sixthly, centrifuging for 5 minutes at 12000Xg, carefully sucking the supernatant and adding the supernatant into a high-heart column. Centrifuging at 12000Xg for 1 min, and discarding the effluent (the volume of supernatant is more than 800 μ L, and adding into the column for several times, and centrifuging above, and discarding the effluent);

seventhly, adding 250 mu L of solution TB, standing at room temperature for 10 minutes, centrifuging at 12000Xg for 1 minute, and discarding effluent;

adding 650 mu L of solution WB, centrifuging at 12000Xg for 1 minute, and discarding the effluent;

ninthly 12000Xg is centrifuged for 1-2 minutes to completely remove the residual WB;

r the column was placed in a clean centrifuge tube and 50-70 μ L EB or deionized water (pH >7.0) was added to the center of the column and allowed to stand at room temperature for 1 minute. (EB or deionized water is preheated in water bath at the temperature of 60-70 ℃, and the using effect is better). 10000g are centrifuged for 1 minute, DNA is eluted, and the eluted DNA is stored at-20 ℃.

The experimental results are as follows:

after PCR and identification steps, the size of the PCR band is determined to be the same as that of the target amplified fragment, and the clone is represented as a successful clone of vector recombination.

Example 2 transfection of nucleic acid and amplification culture

Plate preparation: the day before transfection, the Vero E6 cell strain is inoculated to a cell culture plate, and the cell confluency is controlled to be about 70-80% in the next day;

temperature balance: balancing Hieff TransTM, endotoxin-free plasmid and serum-free culture medium to 15-25 ℃;

configuring a transfection system: mu.g of protein expression vector was diluted with 50. mu.l of serum-free medium, and 6. mu.l of Hieff TransTM reagent was diluted with the same 50. mu.l of medium; standing for 5min, slightly swirling the carrier solution, dropwise adding diluted Hieff TransTM reagent, mixing well, and refining at room temperature for 10-25min to form DNA-Hieff TransTM compound;

transfection: changing the old culture medium in the plate to 1ml of fresh basal medium, and dropping the compound into the wells while shaking at 37 deg.C and 5% CO₂The medium was further cultured for 48 hours, and the cells (Vero E6 cell line expressing SASR-Cov-2 protein) were collected.

Example 3 construction of a mouse model of SASR-Cov-2 induced ALI

Group setting: 84 male mice of BALB/c, 3 months old, were randomly divided into a control group, an ALI + empty vector group (SASR-Cov-2 protein-induced ALI model + empty vector), an ALI model group (SASR-Cov-2 protein-induced ALI model), an ALI + PRCP lentiviral particle group (ALI + overexpression group), an ALI + low dose nebulization group (SASR-Cov-2 protein-induced ALI model + 5. mu.g/Kg PRCP protein), an ALI + medium dose nebulization group (SASR-Cov-2 protein-induced ALI model + 10. mu.g/Kg PRCP protein), and an ALI + high dose nebulization group (SASR-Cov-2 protein-induced ALI model + 20. mu.g/Kg PRCP protein). 12 mice per group.

The experimental steps are as follows:

1. and (3) injection: the day before model construction, 125 μ L of pEZ-Lv105-PRCP viral particles were injected via the tail vein in ALI + PRCP groups (low, medium, high dose), 125 μ L of empty vector was injected via the tail vein in ALI + empty groups, and 125 μ L of saline was injected via the tail vein in other groups, respectively.

2. Constructing a model: after anesthesia of the mice, the trachea was opened and 5.5nmol/kg of SASR-Cov-2 protein was injected intraperitoneally, followed by intratracheal infusion with hydrochloric acid solution (pH 1.5, 2ml/kg), and after 1h and 2h again 5.5nmol/kg of SASR-Cov-2 protein was injected intraperitoneally.

3. Atomizing administration: 1) dissolving 10mg PRCP protein in 1ml deionized water (preheated at 30 ℃) to prepare 10mg/ml stock solution for later use; 2) diluting PRCP stock solution with 30 deg.C preheated deionized water to 0.05mg/ml (solution I), 0.1mg/ml (solution II), and 0.2mg/ml (solution III), respectively; 3) the atomization drug delivery instrument special for mouse atomization is used for atomization drug delivery: ALI + low, medium and high dose nebulization groups were administered with respective solutions I, II, III according to body weight (100ml/kg body weight), and the remaining groups were administered with deionized water according to body weight (100ml/kg body weight).

Mice were sacrificed and the dry and wet weight of lung tissue was determined.

Dry and wet reanalysis of lung tissue: 1) the right lung tissue of the mice was blotted with filter paper to dry the surface water, accurately weighed and recorded (wet weight); 2) baking in a constant-temperature oven at 60 ℃ for 24 hours to constant weight, accurately weighing and recording (wet weight); 3) the W/D was calculated and the degree of edema of the lung tissue was evaluated (the higher the value, the more severe the edema of the lung tissue).

The results show, with reference to fig. 4, that the lung wet/dry weight ratio of mice of the ALI model group, ALI + empty vector group, ALI + low dose nebulization group, and ALI + medium dose nebulization group was significantly increased, and there was no significant difference between the ALI + PRCP lentiviral particle group and the ALI + high dose nebulization group, compared to the control group (ALI induced without SASR-Cov-2) mice. The ALI + PRCP slow virus particle group can obviously improve pulmonary tissue edema; the nebulization dose presents a dose-dependent relationship, the higher the drug concentration, the more significant the effect (compared with the control group, 1) t test, P < 0.001, the very significant difference (×); p < 0.001 < 0.01, with significant variability (. +); p is more than 0.01 and less than 0.05, and the difference is obvious (; 0.05 < P, no significant difference (ns).

4. Reverse transcription

Sample pretreatment: the lung tissue samples of the mice which are treated by each group are ground into powder in a mortar by liquid nitrogen; transferred to a 1.5ml centrifuge tube and 1ml Magzol added to the tissue as 0.1 g.

Total RNA extraction: oscillating a 1.5ml centrifuge tube for 10 times, and incubating for 5min at room temperature; adding chloroform into 0.2ml of chloroform/1 ml of Magzol and fully shaking for 20 s; ③ 12000g, centrifuging for 10min at 4 ℃; fourthly, sucking the supernatant (0.5ml of supernatant/1 ml of Magzol) and transferring the supernatant into a new centrifugal tube, and preventing the supernatant from being sucked into the middle layer; adding isopropanol according to the volume of the same time, and fully and uniformly mixing; standing on ice for 15-20 min; 12000g, centrifuging for 10min at 4 ℃, and carefully discarding the supernatant; adding 1ml of pre-cooled 75% ethanol (prepared by non-enzyme water), 7500g, centrifuging at 4 ℃ for 5min, and carefully discarding the supernatant; ninthly, opening the EP pipe cover, and naturally volatilizing the ethanol for 10min at room temperature; ri add 30-50 μ l DEPC solution to dissolve RNA for 5min at room temperature.

And (3) quality detection: firstly, 2 mul RNA sample is taken to detect RNA OD260 and OD280, and the total RNA concentration and the impurity pollution degree are calculated according to OD260/OD 280; ② the residual RNA sample is preserved at-80 ℃.

cDNA Synthesis: reverse transcription system: 4 XgDNA wiper Mix: 4. mu.l, total RNA: 0.5-1 μ l, adding RNase free ddH₂Supplementing O to 16 μ l, and keeping at 42 deg.C for 2 min; ② adding 5 XHiScript II qRT Supermix II: stopping at 4 μ l, 15min at 37 deg.C, 5s at 85 deg.C, and 12 deg.C; ③ preserving the cDNA at-20 ℃ for later use.

3、qPCR

qPCR: firstly, preparing a reaction system: ChamQ Universal SYBR qPCR (2 ×): 10 μ l, Forward Primer: 1 μ L, Reverse Primer: 1 μ L, cDNA template: 2 mu L, ddH₂Supplementing O to 20 μ L; denaturalization at 95 ℃ for 5 min; ③ 40 circulation: denaturation at 95 ℃ for 15s, annealing at 60 ℃ for 20s, and extension at 72 ℃ for 20 s; collecting a fluorescence signal; 70-95 ℃ denaturation and dissolution curve.

And (3) data analysis:

the formula of delta Ct is as follows: Δ Ct ═ Ct (target gene) -Ct (reference gene, GAPDH); the formula of delta and delta Ct is as follows: Δ Ct ═ Δ Ct (target sample) - Δct (control sample); 3 e (- Δ Ct): the results show fold differences with base 2 and power-delta-Ct.

The results of qPCR detection of the expression of PRCP, AngII, ACE2 gene mRNAs are shown in FIGS. 5-6 (t test: P < 0.001, marked difference (.;) P < 0.01, marked difference (;) P < 0.05, marked difference (; P < 0.05, marked difference).

Relative to a control group, the genes PRCP, ACE and Ang are up-regulated in an ALI model group, an ALI + PRCP slow virus particle group, an ALI + empty vector group, an ALI + low dose atomization group, an ALI + medium dose atomization group and an ALI + high dose atomization group; the gene ACE2 was down-regulated in the ALI model group, ALI + PRCP lentiviral particle group, ALI + empty vector group, ALI + low dose nebulization group, ALI + medium dose nebulization group, and ALI + high dose nebulization group.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

SEQUENCE LISTING

<110> Songshan lake Central Hospital, Dongguan City (Shilong people Hospital, Dongguan city, third people Hospital, Dongguan city, cardiovascular disease institute)

<120> PRCP kit acting on SARS-Cov-2 induced acute lung injury

<130> 2021-12-22

<160> 15

<170> PatentIn version 3.5

<210> 1

<211> 3861

<212> DNA

<213> Artificial Synthesis

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tcctaggctg gccagagaat 20

<210> 9

<211> 20

<212> DNA

<213> R:8896

<400> 9

tcttccacga acctgtcagc 20

<210> 10

<211> 20

<212> DNA

<213> F:8897

<400> 10

cctgctaaga aacggagcca 20

<210> 11

<211> 20

<212> DNA

<213> R:8898

<400> 11

cgcttcatct cccaccactt 20

<210> 12

<211> 20

<212> DNA

<213> F:8899

<400> 12

ctggttgtga tccctcccac 20

<210> 13

<211> 20

<212> DNA

<213> R:8900

<400> 13

cacagtatct gtcgtcccgg 20

<210> 14

<211> 20

<212> DNA

<213> F:P185

<400> 14

tgtgtccgtc gtggatctga 20

<210> 15

<211> 21

<212> DNA

<213> R:P186

<400> 15

ttgctgttga agtcgcagga g 21

Claims (9)

1. An oligonucleotide comprising a primer pair for detecting a gene of a SARS-Cov-2 protein.

2. The oligonucleotide of claim 1, further comprising a primer pair for detecting PRCP.

3. The oligonucleotide of claim 1, wherein the sequence of SARS-Cov-2 protein is set forth in SEQ ID NO 1.

4. The oligonucleotide according to claim 1, wherein the sequence of the primer pair for detecting the SARS-Cov-2 protein gene is shown in SEQ ID NO 2-5.

5. The oligonucleotide of claim 2, wherein the primer pair for detecting PRCP has a sequence as shown in SEQ ID NO 6-7.

6. A PRCP kit acting on SARS-Cov-2 induced acute lung injury, comprising the oligonucleotide according to any one of claims 1 to 5.

7. A method for constructing a mouse model of SARS-Cov-2 induced ALI, which is characterized by comprising the following steps:

s1, constructing Vero E6 cell strain expressing SARS-Cov-2 protein;

s2, constructing slow virus particles pEZ-Lv105-PRCP expressing PRCP;

s3, dividing into a control group, an ALI + empty vector group, an ALI model group, an ALI + PRCP slow virus particle group and an ALI + PRCP atomization administration group to be applied to a mouse;

s4, constructing an ALI mouse model and introducing slow virus particles of PRCP.

8. The method according to claim 7, wherein the concentration of PRCP in step S3 is 0.05-0.2 mg/ml.

9. The method of claim 8, wherein the PRCP concentration in step S3 is 0.05mg/ml, 0.1mg/ml, or 0.2 mg/ml.

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