Application of worm-derived protein in the treatment of autism

Abstract

The present invention relates to the use of worm-derived protein in the preparation of autism drugs. Through targeted research on the pathogenesis of autism and regulatory T cells, it is found that injecting worm-derived protein into pregnant mice with maternal immune activation or adult mice with autism can effectively eliminate or alleviate the core symptoms of autistic mice, which is expected to solve the current problem of autism being untreatable in the future.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to application of worm-derived protein Sjp90α in the preparation of autism drugs. Background Art

[0002] Autism spectrum disorder (ASD) is a neurodevelopmental spectrum disorder and a mental illness. Its core symptoms are social communication disorders, language communication disorders, and repetitive stereotyped behaviors. ASD has been defined by the World Health Organization as one of the most rapidly growing serious diseases in the world. Its treatment is mainly behavioral intervention and special education. There is currently no specific therapeutic drug for the core symptoms. Maternal immune activation (MIA) is an important cause of ASD in offspring. Although some prenatal intervention studies have proposed strategies such as injection of therapeutic IL-17a antibodies and anti-IL-6 antibodies during pregnancy, these strategies may have functional limitations such as insufficient pharmacokinetics and tissue accessibility, as well as interactions that damage the immune system.

[0003] Regulatory T (Treg) cells play a key role in pregnancy. Treg cells are more abundantly expressed in the decidua than in peripheral lymphoid organs and can prevent pregnancy-related diseases (including fetal growth restriction). Therefore, the specific induction of Treg cells at the fetal-maternal interface to improve the outcome of offspring has broad prospects for new therapeutic applications in autism spectrum disorders. ASD patients have a disordered immune system, and CD8 + T and CD4 + The number of T cells increases, Treg cells are suppressed, Th17 activity increases, and cytokines such as tumor growth factor β1 increase. The balance of Th17 / Treg cells may be a key factor in maintaining normal overall immune function and central nervous system function.

[0004] Helminth infection is an important theme of the "hygiene hypothesis", and there is a complex immune regulatory relationship between helminths and the host. When helminths are stimulated by different physical, chemical and pathological factors, they overexpress heat shock proteins (HSPs), especially some HSPs secreted by worm eggs, which may have immunoregulatory effects.

[0005] To date, there are no reports of worm-derived proteins in the treatment of autism spectrum disorders. Summary of the invention

[0006] The main purpose of the present invention is to address the above problems and provide an unreported worm-derived protein that can specifically induce mouse regulatory T (Treg) cells. Injection of this worm-derived protein during pregnancy can treat autism spectrum disorders in offspring caused by maternal immune activation, and direct injection of this worm-derived protein into offspring autistic mice can also alleviate autism symptoms.

[0007] The first object of the present invention is to provide a protein Sjp90α, the amino acid sequence of which is shown in SEQ ID NO.1:

[0008] MTSLKEYVSRMKPEQKDIYYITGETKQAVANSPFTEKLTQRGFEVLYMIDPIDEYSVTHLREYDGKKLVCVTKDGLQLPENEEDKKKFEELKASYEPLCKNVQEILGKSVEKVSISNRLTSSPCCVVTSE FGWSANMERIMKAQALRDSSTMGYMAAKKQLELNPYHPMIKALKDQFESGDSIKLAKDLVQLLYDTALLSSGFSLTDPKIHAKSIHHLVCMCLDIPDEEMKGKCVAADNGPTVAPPAEAGDDAGMEEVD.

[0009] Furthermore, the protein Sjp90α is derived from Schistosoma japonicum.

[0010] Furthermore, the protein Sjp90α specifically induces mouse regulatory T (Treg) cells.

[0011] The second object of the present invention is to provide the use of the aforementioned protein Sjp90α in preparing a reagent for inducing an increase in Treg cells.

[0012] The third object of the present invention is to provide the use of the aforementioned protein Sjp90α in the preparation of drugs for treating autism.

[0013] Among them, treating autism includes eliminating or alleviating the core symptoms of autism.

[0014] Furthermore, the core symptoms of autism include one or more of social communication disorders, repetitive stereotyped behaviors, or anxiety symptoms associated with autism.

[0015] Furthermore, the drug is administered by injection.

[0016] Furthermore, the drug is administered to a pregnant mother or the patient himself. The injection administration described in the present invention refers to: administering the drug to a pregnant mother to treat or alleviate autism in her offspring, or applying the drug to the patient himself to treat or alleviate autism.

[0017] In a specific embodiment, Sjp90α protein can effectively treat or alleviate autism symptoms in offspring mice by intraperitoneal injection of MIA mother mice. In addition, Sjp90α protein can also effectively treat or alleviate autism symptoms in mice by intraperitoneal injection of autistic adult mice. Specifically, autism symptoms include social communication disorders, repetitive stereotyped behaviors and anxiety symptoms associated with autism, especially immune abnormality-type autism.

[0018] Furthermore, the autism is immune abnormality type autism.

[0019] The technical solution of the present invention has the following beneficial effects:

[0020] The schistosome-derived protein Sjp90α prepared by the present invention can significantly induce the increase of Treg cells and can be used for preparing drugs for treating autism. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The results of regulatory T (Treg) cell expression after 24h stimulation of mouse spleen lymphocytes by Sjp90α protein; Figure 1 A is the gating strategy for Treg cell flow cytometry; Figure 1 B-1C are the percentages of Treg cells and their surface molecules.

[0022] Figure 2 These are the behavioral test results of the offspring of pregnant mice injected with Sjp90α protein. Figure 2 A is the timeline of intraperitoneal injection of poly(I:C) and Sjp90α in pregnant mice; Figure 2 B to 2C are the results of the open field test and the elevated plus maze test, which are used to detect the anxiety level of mice. Due to the nature of mice to move towards darkness, the shorter the time spent in the center of the open field and the open arms of the elevated plus maze test, the higher the anxiety level.

[0023] Figure 2 D-2E show the results of social interaction behavior experiments, where Figure 2 D shows the results of a three-box social experiment, which is used to detect social tendencies. The test mouse is placed in the middle box of the three boxes, with an empty cage (E) on one side and a small cage (S1) containing a strange mouse on the other side. The test mouse can sniff the mice in the cage. The social tendencies of the mice can be determined by comparing the time the test mouse sniffs E and S1. Figure 2E is used to detect social novelty preference. The test mice are placed in the middle box of three boxes. One side (box) is the S1 cage, and the other side is a small cage (S2) containing a strange mouse. The test mice can sniff the mice in the cage. The novelty preference of the mice can be determined by comparing the time the test mice sniff S1 and S2.

[0024] Figure 2 F-2G show the results of behavioral tests used to detect repetitive stereotypy and anxiety behaviors in the test mice, among which, Figure 2 F is the number of marble buryings; Figure 2 G stands for the ability to build a nest, which are both recognized behavioral indicators reflecting repetitive, stereotyped and anxious behaviors.

[0025] Figure 3 Behavioral test results of autistic adult mice injected with Sjp90α protein, including Figure 3 A is the timeline of intraperitoneal injection of Sjp90α into adult offspring autistic mice induced by poly(I:C); Figure 3 B-2C are the results of the open field test (openfield) and the elevated plus maze (elevated plus maze), which are used to detect the anxiety level of mice; Figure 3 D-3E show the results of social interaction behavior experiments, where Figure 3 D shows the results of a three-box social experiment to detect social tendencies. Figure 3 E was used to detect social novelty preference; Figure 3 F-3G show the results of behavioral tests used to detect repetitive stereotypy and anxiety behaviors in the test mice. DETAILED DESCRIPTION

[0026] The present invention is further explained below with reference to the examples, but the examples do not limit the present invention in any form.

[0027] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be obtained commercially. The experimental methods used are conventional methods unless otherwise specified.

[0028] Example 1

[0029] This example uses autism model mice to provide an implementation scheme for using Sjp90α protein to effectively alleviate autism symptoms. The specific steps are as follows:

[0030] (1) Extraction and expression of Sjp90α protein: The Sjp90α protein shown in SEQ ID NO.1 was synthesized, and Sjp90α was transformed into competent cells BL21. About 200 μL of bacterial solution was evenly spread on solid LB medium containing kanamycin, and the medium was placed in a 37°C constant temperature medium overnight. Positive colonies were picked for culture, and placed in a 37°C constant temperature shaker in a liquid LB medium containing kanamycin for culture, and shaken horizontally at 200 rpm overnight. The overnight cultured bacterial solution and the LB medium containing kanamycin were mixed at a ratio of 1:100, and cultured in a 37°C constant temperature shaker, shaking at 200 rpm for 3 hours, and the turbidity of the bacterial solution was observed at all times. When the OD value of the bacterial solution was 0.6-0.8, IPTG was added to a final concentration of 1 mM, and cultured again in a 37°C constant temperature shaker. After induction at 200 rpm for 4 hours, the bacterial solution was collected and ultrasonically disrupted, and three layers of whole bacteria, supernatant and precipitate were separated. First, use SDS-PAGE to confirm that the protein is expressed in the precipitate, and then repeat the above steps to put the bacterial solution into a large amount of LB medium, and culture it in a constant temperature shaker at 37°C, 200rpm. Then, the collected bacterial solution with an OD value of 0.6-0.8 is ultracentrifuged, and the final volume after centrifugation of 2L LB medium is about 30mL (30mL of lysate is added to a tube of bacterial precipitation, blown and mixed, and then added to another tube, and resuspended in sequence). After the collected bacterial solution is ultrasonically broken, centrifuge it at 4°C for 20-30min, 10000rpm. The ultrasonic liquid is resuspended with Lysis Buffer, and protein purification is performed according to the requirements of AKTA-pure-100. Samples are collected at different peak values ​​to obtain flow and target proteins of different peaks. Use SDS-PAGE to verify the flow and target protein, and use an ultrafiltration tube to concentrate the protein to about 2mL. After removing endotoxins using an endotoxin removal kit, the protein concentration is measured by the BCA method and placed in a -80°C refrigerator for standby use.

[0031] (2) Mouse spleen lymphocytes were stimulated with 40 μg / ml Sjp90α for 24 h, and Treg cells and their surface molecule expression were detected by flow cytometry. The results showed that Sjp90α could significantly induce the increase of Treg cells and their surface molecules ICOS and ST2 ( Figure 1 ).

[0032] (3) Poly(I:C) was injected intraperitoneally into pregnant mice to induce maternal immune activation (MIA), and the offspring mice developed autism phenotypes:

[0033] Pregnant mice were injected with 20 mg / kg poly(I:C) at 12.5 days of gestation.

[0034] Polyinosinic-cytidylic acid (poly(I:C)) is a synthetic double-stranded RNA (dsRNA) analog that, when injected into pregnant mice, causes autism phenotypes in offspring mice; the autistic mice have multiple immune abnormality phenotypes, such as cytokine imbalance and immune cell subset imbalance.

[0035] (4) Pregnant mice induced by poly(I:C) were intraperitoneally injected with 40 μg of Sjp90α protein at 14.5 and 16.5 days of pregnancy, and the offspring were raised for 8 weeks and then subjected to animal behavioral tests related to autism symptoms ( Figure 2 A).

[0036] (5) Poly(I:C)-induced autistic mice were intraperitoneally injected with 40 μg of Sjp90α protein twice at week 8, followed by animal behavioral tests related to autism symptoms ( Figure 3 A).

[0037] (6) Behavioral testing related to autism.

[0038] Experiment A: Intraperitoneal injection of MIA into female mice.

[0039] The experiment was divided into 4 sample groups, namely, the control group: injected with PBS; the poly(I:C) group: mice treated as described above (3); the Sjp90α group: pregnant mice were intraperitoneally injected with 40 μg of Sjp90α protein on days 14.5 and 16.5 of pregnancy; and the poly(I:C)+Sjp90α group: mice treated as described above (4).

[0040] (6-1) Figure 2 As shown in B, in the open field test, the time spent in the center of the open field by poly(I:C)-induced autistic mice was significantly lower than that of the healthy control mice (PBS group). This indicates that the offspring mice induced by poly(I:C) have anxiety symptoms. The time spent in the center of the open field by autistic mice whose mothers were treated with Sjp90α (poly(I:C)+Sjp90α group) was close to that of the healthy control group. This indicates that Sjp90α treatment of pregnant mice can effectively alleviate the anxiety symptoms associated with autism in adult offspring mice.

[0041] (6-2) Figure 2As shown in C, in the elevated plus maze experiment, the time spent in open arms by poly(I:C)-induced autistic mice was significantly lower than that by healthy control mice (PBS group). This indicates that poly(I:C)-induced offspring mice have anxiety symptoms. The time spent in open arms by autistic mice whose mothers were treated with Sjp90α (poly(I:C)+Sjp90α group) was close to that of the healthy control group. This indicates that Sjp90α treatment of pregnant mice can effectively alleviate the anxiety symptoms associated with autism in adult offspring mice.

[0042] (6-3) Figure 2 As shown in DE, there was no significant difference in the time that poly(I:C)-induced autistic mice spent sniffing the familiar mouse cage (S1) and the cage with strange mice (S2) in the three-box social test. However, the time that healthy control mice (PBS group) spent sniffing the cage with strange mice (S2) was significantly longer than the time spent sniffing the familiar mouse (S1). The time that autistic mice whose mothers were treated with Sjp90α (poly(I:C)+Sjp90α group) spent sniffing the familiar mouse cage (S1) and the cage with strange mice (S2) was close to that of the healthy control group. This indicates that Sjp90α treatment of pregnant mice can effectively alleviate the social interaction defects of autistic adult mice.

[0043] (6-4) Figure 2 As shown in F, the number of marble buryings in poly(I:C)-induced autistic mice was significantly higher than that in healthy control mice (PBS group). This indicates that the autistic mice have symptoms of repetitive stereotyped movements. The number of marble buryings in autistic mice whose mothers were treated with Sjp90α (poly(I:C)+Sjp90α group) was close to that in the healthy control group. This indicates that Sjp90α treatment of pregnant mice can effectively alleviate the repetitive stereotyped behavior symptoms of autism in adult offspring mice.

[0044] (6-5) Figure 2 As shown in G, the nest-building ability of the poly(I:C)-induced lonely mice was significantly impaired compared with that of the healthy control mice (PBS group). This indicates that the autistic mice have anxiety symptoms. The nest-building ability of the autistic offspring mice whose mothers were treated with Sjp90α (poly(I:C)+Sjp90α group) was close to that of the healthy control group. This indicates that Sjp90α treatment of pregnant mice can effectively alleviate the anxiety symptoms of autism in adult offspring mice.

[0045] Experiment B: Intraperitoneal injection of adult offspring autistic mice

[0046] The experiment was divided into 4 sample groups, namely, control group: PBS injection; poly(I:C) group: mice treated as described above (3); Sjp90α group: adult mice intraperitoneally injected with 40 μg Sjp90α protein twice; poly(I:C)+Sjp90α group: mice treated as described above (5).

[0047] (6-6) Figure 3 As shown in B, in the open field test, the time spent in the center of the open field by poly(I:C)-induced autistic mice was significantly lower than that of the healthy control mice (PBS group). This indicates that poly(I:C)-induced mice have anxiety symptoms. The time spent in the center of the open field by autistic mice treated with Sjp90α (poly(I:C)+Sjp90α group) was close to that of the healthy control group. This indicates that Sjp90α treatment can effectively alleviate the anxiety symptoms associated with autism.

[0048] (6-7) Figure 3 As shown in C, in the elevated plus maze experiment, the time spent in open arms of poly(I:C)-induced autistic mice was significantly lower than that of healthy control mice (PBS group). This indicates that poly(I:C)-induced mice have anxiety symptoms. The time spent in open arms of autistic mice treated with Sjp90α (poly(I:C)+Sjp90α group) was close to that of the healthy control group. This indicates that Sjp90α treatment can effectively alleviate the anxiety symptoms associated with autism.

[0049] (6-8) Figure 3 As shown in Figures DE, there was no significant difference in the time that poly(I:C)-induced autistic mice spent sniffing the familiar mouse cage (S1) and the cage containing the strange mouse (S2) in the three-box social experiment. However, the time that healthy control mice (PBS group) spent sniffing the cage containing the strange mouse (S2) was significantly longer than the time spent sniffing the familiar mouse (S1). The time that autistic mice treated with Sjp90α (poly(I:C)+Sjp90α group) spent sniffing the familiar mouse cage (S1) and the cage containing the strange mouse (S2) was close to that of the healthy control group. This indicates that Sjp90α treatment can effectively alleviate the social interaction deficits of autism.

[0050] (6-9) Figure 3As shown in F, the number of marble buryings in poly(I:C)-induced autistic mice was significantly higher than that in healthy control mice (PBS group). This indicates that the autistic mice have symptoms of repetitive stereotyped movements. The number of marble buryings in autistic mice treated with Sjp90α (poly(I:C)+Sjp90α group) was close to that in the healthy control group. This indicates that Sjp90α treatment can effectively alleviate the repetitive stereotyped behavior symptoms of autism.

[0051] (6-10) Figure 3 As shown in G, the nest-building ability of the poly(I:C)-induced lonely mice was significantly impaired compared with the healthy control mice (PBS group). This indicates that the autistic mice have anxiety symptoms. The nest-building ability of the autistic offspring mice treated with Sjp90α (poly(I:C)+Sjp90α group) was close to that of the healthy control group. This indicates that Sjp90α treatment can effectively alleviate the anxiety symptoms of autism.

[0052] It has been verified that the autism mouse model constructed with poly(I:C) and having an immune abnormality phenotype shows that the Sjp90α protein of the present invention can effectively treat the social communication disorders, repetitive stereotyped behaviors and anxiety symptoms commonly associated with autism in the offspring of immune abnormality autistic mice; in addition, the Sjp90α protein of the present invention can also effectively treat the social communication disorders, repetitive stereotyped behaviors and anxiety symptoms commonly associated with autism in adult autistic mice.

[0053] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. The use of protein Sjp90α in the preparation of a reagent for inducing an increase in Treg cells, characterized in that: The amino acid sequence of the protein Sjp90α is as SEQ ID Shown in NO.1: MTSLKEYVSRMKPEQKDIYYITGETKQAVANSPFTEKLTQRGFEVLYMIDPIDEYSVTHLREYDGKKLVCVTKDGLQLPENEEDKKKFEELKASYEPLCKNVQEILGKSVEKVSISNRLTSSPCCV VTSEFGWSANMERIMKAQALRDSSTMGYMAAKKQLELNPYHPMIKALKDQFESGDSIKLAKDLVQLLYDTALLSSGFSLTDPKIHAKSIHHLVCMCLDIPDEEMKGKCVAADNGPTVAPPAEAGDDAGMEEVD.

2. The use of the protein Sjp90α described in claim 1 in the preparation of a drug for treating autism, characterized in that: The autism is autism caused by maternal immune activation.

3. The use according to claim 2, characterized in that: The autism treatment is to alleviate the core symptoms of autism.

4. The use according to claim 3, characterized in that: The core symptoms of autism include one or more of social communication disorders, repetitive stereotyped behaviors, or anxiety symptoms associated with autism.

5. The use according to claim 2, characterized in that: The drug is administered by injection.

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