CN115779093B - Use of a stem cell protein PLK1 and its gene in the development of drug targets or vaccines for the treatment of echinococcosis - Google Patents

Abstract

The invention discloses a stem cell protein PLK1 and application of a gene thereof in the development of a echinococcosis therapeutic drug target or a vaccine. The present invention provides the use of a substance that inhibits or interferes with the expression of the PLK1 gene or its encoded protein in any of the following: 1) Preparing a product for treating echinococcosis; 2) Preparing a product for preventing echinococcosis; experiments prove that the stem cell development related gene PLK1 can be used as a new drug target for developing and treating the two-type echinococcosis vaccine, thereby providing a new way for treating and preventing the two-type echinococcosis. Furthermore, the use of siRNA interfering with PLK1 gene expression to transfect the protocercaria has been found to inhibit the survival of the protocercaria, so that the stem cell development related gene PLK1 can be used as a novel anti-echinococcosis drug, thereby providing a new way for the treatment of two types of echinococcosis.

Description

Use of a stem cell protein PLK1 and its gene in the development of drug targets or vaccines for the treatment of echinococcosis

Technical Field

The invention belongs to the field of biotechnology and relates to a stem cell protein PLK1 and an application of a gene thereof in the development of drug targets or vaccines for the treatment of echinococcosis.

Background technique

Echinococcosis is a disease that occurs worldwide. Its scientific name is echinococcosis. The pathogen is the tapeworm Echinococcus, whose larvae parasitize humans and livestock. It is a serious zoonosis, including cystic echinococcosis (CE) caused by Echinococcus granulosus (Eg) in dogs and alveolar echinococcosis (AE) caused by Echinococcus multilocularis (Em) in foxes. Other tapeworms that cause echinococcosis include Echinococcus oligarthtus (Eo) and Echinococcus vogeli (Ev).

Echinococcus is a small tapeworm that parasitizes the small intestine of carnivorous hosts. For example, the body of Echinococcus granulosus is 2 to 7 mm long, hermaphroditic, and consists of a head segment and four body segments (i.e., neck segment, juvenile segment, adult segment, and gravid segment). The adult worm originates from the everted protoscolex, which is the original state of the adult worm (pre-worm) after the protoscolex is everted, including the head segment, which has a vertex and two circles of small hooks of different sizes on the top, and has four suckers. The gravid segments of the mature worm body contain infectious eggs. After the intermediate host, such as sheep, cattle, pigs, camels and other livestock and humans, ingests water or food contaminated by gravid segments or eggs, the larvae hatch in the stomach and duodenum, and the six hooked larvae shed their shells, first attach to the small intestinal mucosa, then drill into the blood vessels of the intestinal wall, and flow through the portal vein with the blood to the liver, so hepatic echinococcosis is the most common. A small number can pass through the liver through the right atrium to the lungs, and a very small number can reach other organs through the pulmonary circulation. However, the hexahedral larvae can also invade the lymphatic vessels from the intestinal wall, enter the bloodstream directly through the thoracic duct and spread to all parts of the body. After several months of development, the larvae become cystic larvae, called echinococcosis or hydatid cysts. Echinococcus contains many protoscoleci (protoscoleci). If echinococcosis containing protoscoleci are swallowed by dogs, wolves, etc., each of the protoscoleci can develop into an adult in its small intestine. Later, the gravid segments and eggs are discharged one after another, causing pollution and infection.

Both Echinococcus granulosus and Echinococcus multilocularis require two mammalian hosts, including definitive hosts (dogs, foxes and wolves) and intermediate hosts (sheep, cattle and small mammals) to complete their life cycle. Carnivores such as dogs or foxes ingest infected livestock or small mammal viscera such as liver or lungs, and the protoscolex contained therein develop into adults in the intestines of the carnivores. Under natural conditions, Echinococcus granulosus can lay eggs in the host 45 days after infection, and Echinococcus multilocularis can lay eggs in the host 30 days after infection (the eggs are expelled from the host body by shedding of the gravid segments) and are released into the environment through the host's feces. The intermediate host accidentally ingests the eggs, which develop into cysts (or vesicles) and protoscolex in the intermediate host. The entire life cycle is completed when the protoscolex is ingested by the corresponding carnivore.

At present, surgical resection of lesions is the main treatment for echinococcosis. For postoperative prevention of secondary infection or CE patients who cannot undergo surgery, oral ABZ (albendazole) is given for treatment, but the treatment effect is very limited. At the same time, a variety of adverse toxic and side effects occur after the use of ABZ, which limits its use. Since there is no ideal prevention and drug treatment for echinococcosis, especially alveolar echinococcosis, it still seriously endangers the health of humans and livestock and causes serious economic losses. Therefore, it is urgent to find new vaccine candidate proteins and drug targets for the prevention and treatment of echinococcosis.

In recent years, the development of drugs for the treatment of echinococcosis has been approached from different angles, mainly from the perspective of discovering new drug targets. Polo-like kinases (PLKs) are serine/threonine kinases that play an important role in cell cycle regulation in all eukaryotic lineages. In the M phase, PLKs regulate the assembly of the spindle and the activation of cyclin-dependent protein kinases (CDCs). Five PLKs, Plk1-Plk5, are expressed in humans. Plk1, Plk2, and Plk3 are very similar in structure, and they include a conserved N-terminal STK domain (required for phosphorylation of downstream molecules) and two C-terminal Polo-box domains (PBDs) (controlling protein-protein interactions and subcellular localization). The best studied PLK at present is mammalian PLK1, which is mainly expressed in late G2 and M phases and regulates mitosis and meiosis. Plk1 is highly expressed in embryonic stem cells (including many cancer cells) and has been verified as an anticancer drug target. The development of Plk1 inhibitors is of great significance in the fields of cell biology and pharmacokinetic research. However, there are no reports on the expression of Plk1 during the development of type 2 echinococcosis and its use as a new drug target and vaccine in the treatment and prevention of type 2 echinococcosis.

Summary of the invention

The purpose of the present invention is to provide the use of stem cell protein PLK1 and its gene in the development of echinococcosis treatment drug targets or vaccines.

In a first aspect, the present invention provides the use of a substance for inhibiting or interfering with the expression of the PLK1 gene or its encoded protein in any of the following:

1) Preparation of products for the treatment of echinococcosis;

2) Preparation of products for the prevention of echinococcosis;

3) Preparation of products for inhibiting the growth of echinococcosis pathogens;

4) Preparation of products that reduce the survival rate of echinococcosis pathogens;

5) Treatment of echinococcosis;

6) Prevent echinococcosis;

7) Inhibit the growth of echinococcosis pathogens;

8) Reduce the survival rate of echinococcosis pathogens.

In the above, the product may specifically be an inhibitor or vaccine against larvae or adults of the echinococcosis pathogen;

Furthermore, the above inhibitor or vaccine against larvae of the pathogen of echinococcosis is an inhibitor or vaccine against protoscolecus of the pathogen of echinococcosis;

Furthermore, the above-mentioned inhibitor or vaccine against the protoscolex of the pathogen of echinococcosis is an inhibitor or vaccine against the germinal layer of the protoscolex of the pathogen of echinococcosis.

In the above, the inhibition or interference of the expression of the PLK1 gene or its encoded protein is to inhibit or interfere with the expression of the PLK1 gene or its encoded protein in the protoscolex, germinal layer or adult of the echinococcosis pathogen.

In the above application, the echinococcosis is cystic echinococcosis, alveolar echinococcosis or a combination of both.

In the above application, the cystic echinococcosis is echinococcosis caused by Echinococcus granulosus (such as adult or protoscolex);

The alveolar echinococcosis is echinococcosis caused by Echinococcus multilocularis (such as adults or protoscolex);

Or, the pathogen of echinococcosis is Echinococcus granulosus (such as adults or protoscolex) or Echinococcus multilocularis (such as adults or protoscolex).

In the above application, the substance that inhibits or interferes with the expression of PLK1 gene or its encoded protein is siRNA, shRNA, small interfering RNA or antisense nucleotide that inhibits or reduces the expression of PLK1 gene or its encoded protein.

In the above application, the nucleotide sequence of the siRNA is sequence 1 or sequence 2.

In the above application, the inhibition of the growth of the echinococcosis pathogen is to inhibit the growth of the pathogen in the larval or adult stage of the echinococcosis pathogen;

Or, the inhibiting of pathogen growth in the larvae of the echinococcosis pathogen is specifically inhibiting the growth of the pathogen in the cyst germinal layer of the protoscolex of the echinococcosis pathogen.

In a second aspect, the present invention provides the use of the PLK1 gene or the protein encoded by it as a target in the development of therapeutic drugs for echinococcosis.

Alternatively, the present invention provides the use of the PLK1 gene or the protein encoded by it as a target in the development of a echinococcosis vaccine.

The above-mentioned therapeutic drugs or vaccines are therapeutic drugs or vaccines for larvae or adults of the pathogen of echinococcosis;

Furthermore, the above-mentioned therapeutic drug or vaccine for the larvae of the pathogen of echinococcosis is a therapeutic drug or vaccine for the protoscolex of the pathogen of echinococcosis;

Furthermore, the above-mentioned therapeutic drug or vaccine for the protoscolex of the pathogen of echinococcosis is a therapeutic drug or vaccine for the germinal layer of the protoscolex of the pathogen of echinococcosis.

In a third aspect, the present invention provides a product for treating or preventing echinococcosis, comprising the substance for inhibiting or interfering with the expression of PLK1 gene or its encoded protein in the first aspect.

In the above products, the echinococcosis is cystic echinococcosis, alveolar echinococcosis or a combination of the two types of echinococcosis.

The above products are vaccines or medicines.

In the above, the product may specifically be an inhibitor or vaccine against larvae or adults of the echinococcosis pathogen;

Furthermore, the above inhibitor or vaccine against larvae of the pathogen of echinococcosis is an inhibitor or vaccine against protoscolecus of the pathogen of echinococcosis;

Furthermore, the above-mentioned inhibitor or vaccine against the protoscolex of the pathogen of echinococcosis is an inhibitor or vaccine against the germinal layer of the protoscolex of the pathogen of echinococcosis.

The present invention discloses for the first time the application of stem cell development-related gene PLK1 as a target for the development of vaccines and new drugs for the treatment of two types of echinococcosis. RT-PCR experimental data show that the PLK1 gene is highly expressed in the germinal layer of the cyst of Echinococcus granulosus, and is also highly expressed in the adult stage of Echinococcus granulosus. The expression difference is obvious compared with other stages of Echinococcus granulosus development (see Figure 1A-B) (P<0.001) . The immunohistochemical experimental data show that in the everted protoscolecere, the stem cells are obviously located at the bottom of the developing protoscolecere (below the sucker) (see Figure 3A), and in the adult development stage, the PLK1 gene is highly expressed in the head and neck segment area of the worm body (see Figure 2C, 3B), which is the only area where the adult segment proliferates. The experimental research of the present invention proves that PLK1 is highly expressed in the germinal layer and adult stage of Echinococcus granulosus, and proves that PLK1 plays an important role in the proliferation of embryonic stem cells during the development of Echinococcus granulosus. In the everted protoscolex, stem cells were clearly located in the conjoined growth area at the bottom of the developing scolex, and the PLK1 gene was also highly expressed in the head and neck segments of the worm body, proving that PLK1 is a highly expressed gene in the developmental stem cells of adult Echinococcus. The above results indicate that the stem cell development-related gene PLK1 can be used as a target for the development of vaccines and new drugs for the treatment of two types of echinococcosis, thereby providing a new approach for the treatment and prevention of two types of echinococcosis. Furthermore, protoscolex was transfected with siRNA that interfered with the expression of the PLK1 gene, and it was found that it could inhibit the survival of protoscolex. Therefore, the stem cell development-related gene PLK1 can be used as a new anti-echinococcosis drug, thereby providing a new approach for the treatment of two types of echinococcosis.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the mRNA expression levels of the PLK1 gene at different developmental stages of E.g.

Figure 2 shows the mRNA expression levels of the PLK1 gene in different parts of the adult worms of E.g.

Figure 3 shows the expression and localization of the PLK1 gene in different parts of the protoscolex (PSC) and adult (AW) of E.g.

Figure 4 shows the mRNA expression level of PLK1 gene at different developmental stages of E.g and E.m.

Figure 5 shows the mRNA expression levels of PLK1 gene and albendazole target β-tublin at different stages of E.g.

Figure 6 shows the expression effect of Cy3 after transfection of E.g. protoscolex.

FIG. 7 shows the survival rate of E. m protoscolex and the mRNA expression level of PLK1 after siRNA-PLK1 transfection.

Detailed ways

Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

Ethics statement for the following examples: This study was approved by the Ethics Committee of the First Affiliated Hospital of Xinjiang Medical University (approval number: IACUC-2015, IACUC-2013), and all animals were handled in accordance with the animal ethics procedures and guidelines of the People's Republic of China.

The worm strains in the following examples: the protoscolex (PSCs) of Echinococcus granulosus (Eg) were collected from the livers of fresh sheep naturally infected with Echinococcus granulosus slaughtered in Hualing Market, Urumqi, Xinjiang Uygur Autonomous Region; the protoscolex and middle tapeworm larvae of Echinococcus multilocularis ( E,m) were passaged, isolated and preserved by gerbils in our laboratory.

The main reagents of the following examples: PRMI-1640 culture medium, double antibody (Hyclone), fetal bovine serum (Hyclone), yeast extract, glucose, taurocholate, trypsin, pepsin, methylene blue (Sigma), hematoxylin stain (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.); PLK1 goat anti-rabbit antibody (abcon); RNA extraction kit (Beijing Tiangen Biochemical Technology Co., Ltd.); cDNA reverse transcription kit, 2×SG Fast qPCR Mix (Shanghai Sangon Biotechnology Co., Ltd.).

Canine bile was obtained from dogs dissected in the physiological experiment of Xinjiang Medical University. It was diluted 1:10 with normal saline and filtered through a 0.22 μM filter membrane to obtain a 5% canine bile solution. It was stored at -20°C with a shelf life of one month.

Some of the methods of the following embodiments are as follows:

1. The origin of protoscolex (PSC)

The protoscolex of Echinococcus granulosus (Eg-PSC) from sheep liver were collected from Hualing slaughterhouse in Urumqi, Xinjiang Uygur Autonomous Region; the protoscolex of Echinococcus multilocularis (Em-PSC) were collected from the long-clawed gerbils cultured in the animal room of the First Affiliated Hospital of Xinjiang Medical University.

2. Preparation of protoscoleces (PSC) and germinal layer (GL)

The surface of the liver of infected sheep with cysts obtained from Hualing Slaughterhouse in Urumqi, Xinjiang Uygur Autonomous Region was repeatedly rinsed with clean water. The surface was then disinfected with 75% alcohol and placed in a biosafety cabinet. The clear cyst fluid was extracted with a sterile syringe, the outer cyst was picked up with tweezers, the inner cyst was peeled off, and placed in a culture dish containing physiological saline. The inner cyst wall was cut open, the inner cyst skin was everted, and the Echinococcus granulosus protoscolecus (Eg-PSC) was separated. It was digested with 1% pepsin for 15 minutes, and rinsed with physiological saline for 5 times to obtain E.g protoscolecus (Eg-PSC). The inner cyst skin was cut into pieces, added with an appropriate amount of physiological saline, stirred rapidly for 15 minutes, and naturally settled for 2 minutes. The upper cell suspension was centrifuged at 3000 rpm for 15 minutes, and the precipitate was repeatedly washed with physiological saline for 3 times to obtain E.g germinal layer cells (Eg-GL).

Alternatively, the long-clawed gerbils with multilocular Echinococcus in the peritoneal passage from the animal room of the First Affiliated Hospital of Xinjiang Medical University were killed and placed in a biosafety cabinet, soaked in 75% alcohol for 1 minute, rinsed with saline 2-3 times, the peritoneal cavity was cut open, the cyst tissue was removed, the cyst wall was repeatedly washed with saline, and then the cyst was cut open, and the cyst contents such as cyst fluid, protoscolex and germinal layer cells were transferred to a centrifuge tube, shaken vigorously for 20 seconds, and naturally precipitated for 2 minutes. The natural precipitate contained protoscolex, which was digested with 1% pepsin for 15 minutes, and rinsed with saline for 5 times to obtain E.m protoscolex (Em-PSC). The upper cell suspension was centrifuged at 3000 rpm for 15 minutes, and the precipitate was repeatedly washed with saline for 3 times to obtain E.m germinal layer cells (Em-GL).

3. Detection of the vitality of protoscolex

Add 1% (mass volume percentage g: ml) pepsin solution (solvent is water, pH 2.0) to the collected protoscolex precipitate and digest at 37°C for 15-30 minutes. After digestion, wash 5 times with sterile PBS natural precipitate with double antibody to remove dead protoscolex and debris. Stain with 0.1% methylene blue for 5 minutes and discard the staining solution to detect vitality. Dead ones are stained blue. Protoscolex with vitality of more than 98% can be used for later experiments.

4. Collection of insects at different developmental stages

5000 protoscolex were added to a cell culture bottle, and then added to 68 ml of 0.02% canine bile medium. The culture was cultured in a 37°C, 5% CO2 incubator. On the third day, the medium was changed to observe whether there was any contamination. The original medium was discarded and fresh 0.02% canine bile medium was added to continue the culture. The medium was changed every 3 days, and the development of the worms was observed under a microscope every day.

On the 0th, 14th, 35th, and 50th days of culture, the worms were observed and photographed under a microscope, and samples were collected and stored in 4% paraformaldehyde at 4°C and RNAlater at -80°C for subsequent immunohistochemical staining, RNA extraction, and fluorescence quantitative PCR detection.

The formula of 0.02% canine bile medium per 100 ml is as follows: 68 mL PRMI-1640, 20 mL fetal bovine serum, 9 mL 5% yeast extract aqueous solution, 1.6 mL 30% glucose aqueous solution, 1 mL penicillin-streptomycin aqueous solution (0.4 μg/mL), and 0.4 mL 5% canine bile solution.

The PLK1 sequence is available from GenBank under accession number HG931729.1, submission date 2014-6-20.

Example 1. Expression of stem cell development-related gene PLK1 in different developmental stages of Echinococcus

1. mRNA expression levels of PLK1 gene at different developmental stages of E.g.

Primers were designed using Primer5.0 with reference to the housekeeping gene Eif3 (EGR-09912) mRNA gene and PLK1 gene sequences published in GenBank, and sent to Beijing Dingguo Changsheng Biotechnology Co., Ltd. for synthesis.

The primer sequence of Eif3 is

Upstream primer F: 5′-GGTTACATCCCTCCGACCTTG-3′,

Downstream primer R: 5′-AAGCAGCCTCCTCTTGAGTG-3′,

The primer sequence for PLK1 is

Upstream primer F: 5′-AGAGGGAGGTTTCTTGGA-3′,

Downstream primer R: 5′-TCTTCTTTCATTGGAGCA-3′,

Take 10μＬ E.g of protoscolex sediment (about 2000) or the corresponding volume of sediment cells or adult tissues of the worms at various developmental stages, extract worm RNA according to the RNA extraction kit operation steps, and use DNase to digest DNA to remove genomic DNA before synthesizing cDNA. Use Mighty Script First-Strand cDNA Synthesis Kit (Master Mix Kit) for reverse transcription to synthesize cDNA.

Fluorescence quantitative PCR (RT-PCR) was performed according to the following fluorescence quantitative PCR reaction system:

Fluorescence quantitative PCR reaction system 20μL: 2×SG Fast qPCR Mix 10μL, upstream and downstream primers 0.5μL (10 μmol/L), cDNA 1μL, add dd H2O to 20μL. Reaction program: 95℃ pre-denaturation for 5 minutes; 95℃ denaturation for 5s, 65℃ annealing/extension for 30s, a total of 38 cycles. Each sample was repeated 3 times, and the Ct value was automatically obtained after the program was completed.

The fluorescence value was counted, and the expression level of the target gene was calculated using 2 ^-ΔΔCt . The experimental results were expressed as (x±s). SPSS 26.0 was used for one-way analysis of variance, and P < 0.05 indicated that the difference was statistically significant.

The results are shown in Figure 1. Figure A shows the expression level of PLK1 in vivo after 0, 14, 35 and 50 days of culture of E.g. protoscolex. It can be seen that the expression level of PLK1 in the adults obtained on the 35th and 50th days of culture (all the adults above 30 days are AW) is higher than that in the larvae on the 0th and 14th days of culture. Figure B shows the results of E.g. protoscolex culture on the 0th day (protoscolex PSC), 50th day (adult AW) and germinal layer GL. It can be seen that the expression level of PLK1 gene is highest in the germinal layer of E. granulosus cysts, followed by adults.

2. mRNA expression levels of PLK1 gene in different parts of E.g. adults

Adult E.g. worms cultured for 56 days were cut (i.e., the head and neck segments and the worm body were cut) for sampling.

Extract RNA from the samples and perform RT-PCR detection according to the above method.

The results are shown in Figure 2, where A and B are the microscopic results of the head and body segments of Eg adults after culture for 56 days, respectively, where Aa is the microscopic result of the head and neck segments of Eg adults after culture for 56 days; Bc and Bd are the microscopic results of the body segments of Eg adults after culture for 56 days (c, mature segment, d, gravid segment), respectively; C is the expression level of the PLK1 gene in the head and neck segments and body segments of Eg adults after culture for 56 days; it can be seen that the mRNA expression level of the PLK1 gene in the head and neck segments of Eg adults is 1.9 times that of the mature body segments ( P < 0.01 ), indicating that the PLK1 gene is highly expressed in the head and neck segments of Eg adults.

3. Immunohistochemical staining and expression of PLK1 gene in different parts of protoscoleces and adults of E.g.

The protoscolex and adult worms of E.g. (adults obtained by culturing for 50 days) stored in 4% paraformaldehyde were rinsed several times with distilled water, and 2% agarose and 4% paraformaldehyde were heated to 65°C. The protoscolex and adult worms were placed in the melted agarose solution, and then fixed in 4% paraformaldehyde overnight after cooling. Paraffin sections were prepared and baked at 70°C overnight. Dewaxing was performed step by step by soaking in anhydrous ethanol, 95%, 85%, and 75% ethanol for 2 minutes each. Antigen retrieval was performed by heating in a microwave oven with citric acid buffer for 15 minutes. Endogenous peroxidase was blocked by soaking in 3% hydrogen peroxide for 10 minutes in the dark, and blocked at room temperature for 1 hour with 10% goat serum. Rabbit anti-PLK1 antibody (1:500 dilution) was added at 4°C overnight, washed three times with PBS, goat anti-rabbit IgG enzyme-labeled secondary antibody (1:5000 dilution) was added, incubated at 37°C for 1 hour, washed three times with PBS, stained with hematoxylin for 1 minute, differentiated with hydrochloric acid alcohol for 10 seconds, transparent for 30 minutes, and sealed with neutral gum. Goat anti-rabbit IgG enzyme-labeled secondary antibody (1:5000 dilution) was used as the control group.

The results are shown in Figure 3, A is the expression and localization results of immunohistochemical staining of E.g protoscolex, where CU is the protoscolex capsule; SU is the protoscolex sucker; B is the expression and localization results of immunohistochemical staining of E.g adults, where a, scolex; b, larvae; c, adult segments; d, gravid segments; it can be seen that in the larval stage, PLK1 is lowly expressed in the protoscolex capsule and highly expressed at the bottom of the sucker; in the adult stage, PLK1 appears in a band-shaped positive area at the junction of the head and neck segments and the larvae of the adults, and PLK1 is highly expressed at the junction of the AW head and neck segments and the larvae, indicating that the primary site of adult segment development and differentiation is at the junction of the head and neck segments and the larvae.

4. mRNA expression levels of PLK1 gene in different developmental stages of E.g and E.m

According to the method of "a", the mRNA expression level of PLK1 gene in protoscolex PSC, adult AW and germinal layer GL of E.g and E.m at different developmental stages was detected.

The results are shown in Figure 4 , and it can be seen that, consistent with the results of “ 1 ”, the expression level of the PLK1 gene is highest in the germinal layer stage of E.g and E.m, followed by the adult.

5. mRNA expression levels of PLK1 gene and albendazole target β-tublin in different parasite stages

The target of albendazole is β-tublin. The present invention detects the expression levels of β-tublin and PLK1 genes in different developmental stages of the parasite.

The detection method is basically the same as that of "1", except that the internal reference gene is β-tublin.

The submission date of β-tublin (MK967342.1) published in GenBank was 2019-12-07. Primers were designed using Primer5.0 for the mRNA gene sequence and sent to Beijing Dingguo Changsheng Biotechnology Co., Ltd. for synthesis.

The primers for amplification of β-tublin are as follows:

Upstream primer F: 5′-ATGCGTGAAATTGTGCACATTCAAG-3′,

Downstream primer R: 5′- TCATCCGAAATCACCTCCC-3′,

The results are shown in Figure 5. It can be seen that, like the albendazole target β-tublin, the PLK1 gene is highly expressed in the protoscolex, adults and germinal layer of the parasite, and it is predicted that the PLK1 gene can also be used as a therapeutic target for echinococcosis.

Example 2: Application of interfering with PLK1 gene expression in inhibiting the growth of hydatid disease pathogen Echinococcus protoscolex

1. Transfection

The protoscolex PSCs of Echinococcus granulosus E.g and Echinococcus multilocularis E.m were cultured in 0.02% canine bile culture medium for 3 days, the culture medium was carefully aspirated, and the precipitate was collected to obtain the protoscolex PSCs of E.g and E.m cultured in vitro for 3 days.

The images of E.g protoscolecus PSC and E.m protoscolecus PSC cultured in vitro for 3 days were collected by optical microscope observation, and the activity was detected after staining with 0.1% methylene blue. The cells were washed three times with PBS (pH 7.4), and then each group of PSCs was washed three times with electroporation buffer, and recorded as PSCs to be transfected.

The formula of the electrotransfer buffer is: 150 mM sucrose, 27 mM Na ₂ HPO ₄ , the balance is water, and the pH is adjusted to 7.5.

The transfection efficiency was first determined by laser confocal microscopy to observe the expression distribution of the red fluorescence (Cy3) transfection control. The results showed that the red fluorescence could be effectively introduced into the insect body using the American Bio-Rad electroporator at 125 V, a pulse of 20 ms, and a 4 mm electric shock cup (Figure 6).

Then, the three PLK1-siRNA interference sequences and the negative control sequence shown in Table 1 were respectively transferred into the worms, and the worms were collected after culturing for 3 days after transfection. The worm vitality was identified by methylene blue staining and the changes in the expression level of PLK1 gene mRNA in each group were detected by real-time fluorescence quantitative PCR.

PSCs are divided into three groups (2000 per group):

siRNA group (siRNA-PLK1-162): siRNA-PLK1-162 (sequence 1) was transferred;

siRNA group (siRNA-PLK1-1100): siRNA-PLK1-1100 (sequence 2) was transferred;

siRNA group (siRNA-PLK1-1798): siRNA-PLK1-1798 was transferred;

NC group transfected with irrelevant sequence (negative control sequence): negative control sequence was transfected;

Table 1 shows different siRNA sequences

Set up three repetitions per set.

Each group of PSCs to be transfected was transferred to a 4 mm electroporation cuvette.

The transfection system is as follows: add 100 ul of electroporation buffer, containing 2000 protoscolex to be transfected, and add 25 ul of each siRNA. The final concentration of each siRNA in the transfection system is 5 umol/L. Perform one square wave electric shock at 125 V, 20 ms, 1 pulse. After the electroporation, quickly place the electroporation cup incubated at 37°C for 10 minutes, and then transfer it to a 24-well plate containing 1 mL of 0.02% canine bile medium and continue to culture.

2. Survival rate detection

Three days after transfection, about 200 transfected protoscolex were collected from each group, rinsed with sterile PBS, stained with 0.1% methylene blue for 5 minutes, smeared, counted, and the activity and survival rate were observed under an inverted fluorescence microscope. Blue represents dead PSCs.

The results are shown in Figures 7A-7D, where A and B are the microscopic observation results of different transfection groups, and C and D are the survival rates of Eg protoscolex and Em protoscolex after transfection with different siRNAs, respectively; in the figure, siRNA-162 or siRNA-PLK1-162 are protoscolex transfected with siRNA-PLK1-162, siRNA-1100 or siRNA-PLK1-1100 are protoscolex transfected with siRNA-PLK1-1100, siRNA-1798 or siRNA-PLK1-1798 are protoscolex transfected with siRNA-PLK1-1798, NC or NC siRNA are protoscolex transfected with negative control, and Blank or Blank control are non-transfected protoscolex; it can be seen that siRNA-PLK1-162, siRNA-PLK1-1100, siRNA-PLK1-1100 and siRNA-PLK1-1798 are protoscolex transfected with negative control, and Blank or Blank control are non-transfected protoscolex. The survival rates of Eg protoscolex after PLK1-1798 transfection were (34.3±4.87)%, (25.4±2.98)% and (74.6±4.54)%, respectively. The survival rates of Em protoscolex after siRNA-PLK1-162, siRNA-PLK1-1100 and siRNA-PLK1-1798 transfection were (26.85±2.34)%, (24.53±4.68)% and (80.47±3.94)%, respectively. The optimal interference sequence was preliminarily screened as siRNA-PLK1-1100. The survival rate of protoscolex in the siRNA-PLK1-1100 interference group was significantly different from that in the negative control group NC [(88.3±1.23)%] and the blank control group Blank [(92.4±2.39)%] (P＜0.01) .

It shows that the survival rate of E.g protoscolex and E.m protoscolex decreased significantly after interfering with PLK1 gene expression.

3. RT-PCR detection

RNA was extracted from the above-collected protoscolex after transfection, and RT-PCR was used to detect the expression of PLK1 gene in the protoscolex after transfection according to the method of Example 1, and siRNA with good silencing efficiency was selected.

The results of RT-PCR detection are shown in Figures 7E and 7F, where E and F are the expression levels of PLK1 gene in Eg protoscolex and Em protoscolex after transfection with different siRNAs, respectively; siRNA-162 or siRNA-PLK1-162 are protoscolex transfected with siRNA-PLK1-162, siRNA-1100 or siRNA-PLK1-1100 are protoscolex transfected with siRNA-PLK1-1100, siRNA-1798 or siRNA-PLK1-1798 are protoscolex transfected with siRNA-PLK1-1798, NC or NC siRNA are protoscolex transfected with negative control, and Blank or Blank control are protoscolex not transfected; it can be seen that siRNA-PLK1-162, siRNA-PLK1-1100, The expression levels of PLK1-mRNA in Eg protoscolex after siRNA-PLK1-1798 transfection were 0.46±0.023, 0.26±0.019 and 0.84±0.027, respectively. The expression levels of PLK1-mRNA in Em protoscolex after siRNA-PLK1-162, siRNA-PLK1-1100 and siRNA-PLK1-1798 transfection were 0.499±0.022, 0.281±0.049 and 0.862±0.018, respectively. The expression level of PLK1-mRNA in transfected protoscolex obtained after siRNA-PLK1-1100 sequence interference was the most different from that in the irrelevant sequence control group NC (0.95±0.032) and the blank control group blank (0.98±0.012), and the differences were statistically significant (all P＜0.01 ), thus determining the best interference sequence to be siRNA-PLK-1100.

The above technical features constitute the embodiments of the present invention, which have strong adaptability and implementation effect. Non-essential technical features can be added or reduced according to actual needs to meet the requirements of different situations.

Claims (9)

1. Use of substances that inhibit or interfere with the expression of PLK1 gene or its encoded protein in any of the following: 1) Preparation of products for treating echinococcosis; 2) Preparation of products for preventing echinococcosis; The echinococcosis is cystic echinococcosis, alveolar echinococcosis or a combination of both. The substance that inhibits or interferes with the expression of PLK1 gene or its encoded protein is siRNA that inhibits or reduces the expression of PLK1 gene or its encoded protein, and the nucleotide sequence of the siRNA is sequence 1 or sequence 2. 2. The use according to claim 1, characterized in that: The cystic echinococcosis is echinococcosis caused by Echinococcus granulosus tapeworm; The alveolar echinococcosis is echinococcosis caused by Echinococcus multilocularis tapeworm. 3. Use of substances that inhibit or interfere with the expression of PLK1 gene or its encoded protein in the preparation of products that inhibit the growth of echinococcosis pathogens: The echinococcosis is cystic echinococcosis, alveolar echinococcosis or a combination of both. The substance that inhibits or interferes with the expression of PLK1 gene or its encoded protein is siRNA that inhibits or reduces the expression of PLK1 gene or its encoded protein, and the nucleotide sequence of the siRNA is sequence 1 or sequence 2. 4. The use according to claim 3, characterized in that: The inhibiting of the growth of the echinococcosis pathogen is to inhibit the growth of the pathogen in the larvae or adult stages of the echinococcosis pathogen. 5. The use according to claim 4, characterized in that: the inhibiting the growth of the pathogen in the larval stage of the echinococcosis pathogen is inhibiting the growth of the pathogen in the cyst germinal layer of the protoscolex of the echinococcosis pathogen. 6. Use of substances that inhibit or interfere with the expression of PLK1 gene or its encoded protein in the preparation of products that reduce the survival rate of echinococcosis pathogens: The echinococcosis is cystic echinococcosis, alveolar echinococcosis or a combination of both. The substance that inhibits or interferes with the expression of PLK1 gene or its encoded protein is siRNA that inhibits or reduces the expression of PLK1 gene or its encoded protein, and the nucleotide sequence of the siRNA is sequence 1 or sequence 2. 7. The use according to any one of claims 3 to 6, characterized in that the pathogen of echinococcosis is Echinococcus granulosus or Echinococcus multilocularis. 8. A product for treating or preventing echinococcosis, comprising a substance that inhibits or interferes with the expression of the PLK1 gene or its encoded protein; The substance that inhibits or interferes with the expression of the PLK1 gene or its encoded protein is an siRNA that inhibits or reduces the expression of the PLK1 gene or its encoded protein, and the nucleotide sequence of the siRNA is sequence 1 or sequence 2; The echinococcosis is cystic echinococcosis, alveolar echinococcosis or a combination of both. 9. The product according to claim 8, characterized in that: The product is a vaccine or a medicine.