Schistosoma japonicum Hsc20 gene expressed siRNA and application thereof

Abstract

The invention relates to the field of molecular biology and biological medicine, and discloses a schistosoma japonicum Hsc20 gene expressed siRNA and application thereof, the siRNA contains a nucleotide sequence hybridized with a target sequence of the schistosoma japonicum Hsc20 gene, and the target sequence is selected from sequences shown as SEQ ID NO.1-SEQ ID NO.4. The invention also discloses a preparation method of the schistosoma japonicum Hsc20 gene expressed siRNA. The siRNA for specifically inhibiting the expression of the schistosoma japonicum Hsc20 gene, provided by the invention, can be used for interfering the transcription and expression of the schistosoma japonicum Hsc20 gene and the growth and development of schistosoma japonicum; in-vitro experiments prove that the siRNA provided by the invention can be used for efficiently silencing the SjHsc20 gene; in-vivo RNA interference test results of mice show that the siRNA can induce the mice to obtain 50.62% (Plt; 0.05) and an insect reduction rate of 44.29% (Plt; 0.01), and is suitable for preparing the medicine for treating the schistosomiasis.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of molecular biology and biological medicine, and particularly relates to a Schistosoma japonicum Hsc20 gene expressed siRNA and application thereof. BACKGROUND

[0002] Schistosomiasis japonica is a serious human and animal parasitic disease. In addition to humans, Schistosoma japonicum can also infect more than 40 species of mammals such as cattle, sheep and pigs, causing the diseased animals to lose weight and anemia, and reducing the service ability of livestock. In economic animals, the milk yield of dairy cows may decrease, and cows may not be pregnant or may have miscarriage, and in severe cases, death, which is one of the important epidemic diseases hindering the development of cattle and sheep breeding industry. Epidemiological investigations show that domestic animals such as buffaloes, cattle and sheep are the main sources of Schistosoma japonica, and the prevalence of animal Schistosoma japonica seriously threatens people's health, and effectively blocking the transmission of animal Schistosoma japonica helps to eliminate Schistosomiasis japonica.

[0003] Heat shock cognate protein 20 (Hsc20) is a J-type chaperone, also known as heat shock cognate B (HscB), which can bind and regulate the ATPase and peptide binding activity of Hsp70. Hsp70 has the biological function of assisting the folding of nascent proteins and correcting misfolded proteins, and has strong immunogenicity, and heterologous HSP70 can cause a strong Th1-type immune response in the host. In Escherichia coli, Hsc20 participates in the biosynthesis of iron-sulfur proteins together with Hsc66 (a Hsp70-type molecular chaperone). Iron-sulfur proteins are essential for many key cellular biological processes and play an important role in various cellular biological processes such as DNA replication and repair, oxidative respiration and photosynthesis. In addition, it has been found that the deletion of Hsc20 can cause damage to Fe-S cluster biogenesis, defects in hemoglobinization of red blood cells and cell development, and widespread interference with hematopoiesis in the body.

[0004] RNA interference (RNAi) is an evolutionarily conserved molecular regulatory mechanism, which is characterized by sequence-specific silencing of target genes in vivo or in vitro by introducing exogenous double-stranded RNA (dsRNA). This process mainly occurs at the post-transcriptional level, involving the synergistic action of multiple conserved protein factors, and can achieve epigenetic regulation of gene expression through mRNA degradation or translation inhibition pathway. The core effector molecule of this process is siRNA, which is generated by Dicer enzyme (RNase III family endonuclease) cleavage of the initial dsRNA, forming a double-stranded small molecule (21-23 nucleotides in size). After the siRNA double strand is unwound, the antisense strand is integrated into the RNA-induced silencing complex (RISC), which recognizes the open reading frame or untranslated region of the target mRNA through strict base complementary pairing, and activates the endonuclease activity of the Argonaute (AGO) protein, ultimately leading to specific degradation of the target gene mRNA. This mechanism requires that siRNA has high sequence specificity, and its silencing efficiency is also closely related to the GC content, thermodynamic stability and end modification of siRNA, and the prior art lacks siRNA that can significantly inhibit the transcription of Hsc20 gene of Schistosoma japonicum.

[0005] Therefore, the prior art still needs to be improved and developed. SUMMARY

[0006] In view of the deficiencies of the prior art described above, the purpose of the present application is to provide siRNA for Hsc20 gene expression of Schistosoma japonicum and its application, which can significantly inhibit the transcription of Hsc20 gene of Schistosoma japonicum and can be used for preparing a drug for treating schistosomiasis.

[0007] In order to achieve the above purpose, the present application adopts the following technical solutions: The siRNA for specifically inhibiting the expression of Hsc20 gene of Schistosoma japonicum, wherein it contains a nucleotide sequence hybridized with a target sequence of Hsc20 gene of Schistosoma japonicum, and the target sequence is selected from the sequences shown in SEQ ID NO. 1-SEQ ID NO. 4.

[0008] The siRNA for specifically inhibiting the expression of Hsc20 gene of Schistosoma japonicum, wherein the siRNA comprises a first strand and a second strand, the first strand and the second strand are complementary to form an RNA dimer, and the RNA sequence of the first strand is consistent with the target sequence of Hsc20 gene of Schistosoma japonicum, and the first strand and the second strand of the siRNA are selected from one pair or any two or more combinations of the following: The nucleotide sequences shown in SEQ ID NO.5 and SEQ ID NO.6; The nucleotide sequences shown in SEQ ID NO.7 and SEQ ID NO.8; The nucleotide sequences shown in SEQ ID NO.9 and SEQ ID NO.10; The nucleotide sequences shown in SEQ ID NO.11 and SEQ ID NO.12.

[0009] The siRNA that specifically inhibits the expression of the Hsc20 gene of Schistosoma japonicum, wherein the first and second strands of the siRNA are the nucleotide sequences shown in SEQ ID NO.7 and SEQ ID NO.8.

[0010] A drug for treating schistosomiasis, wherein the active ingredient of the drug is: siRNA that specifically inhibits the expression of the Schistosoma japonicum Hsc20 gene by RNA interference, the siRNA containing a nucleotide sequence that hybridizes with a target sequence of the Schistosoma japonicum Hsc20 gene, the target sequence being selected from the sequences shown in SEQ ID NO.1-SEQ ID NO.4.

[0011] The drug for treating schistosomiasis, wherein the first and second strands of the siRNA are selected from one or any combination of two or more of the following: The nucleotide sequences shown in SEQ ID NO.5 and SEQ ID NO.6; The nucleotide sequences shown in SEQ ID NO.7 and SEQ ID NO.8; The nucleotide sequences shown in SEQ ID NO.9 and SEQ ID NO.10; The nucleotide sequences shown in SEQ ID NO.11 and SEQ ID NO.12.

[0012] The use of an siRNA that specifically inhibits the expression of the Hsc20 gene of Schistosoma japonicum as described in this invention in the preparation of drugs for the treatment or prevention of schistosomiasis.

[0013] Beneficial effects: The siRNA specifically inhibiting the expression of the Hsc20 gene in Schistosoma japonicum provided by this invention can be used to interfere with the transcription and expression of the Hsc20 gene in Schistosoma japonicum and the growth and development of Schistosoma japonicum; in vitro experiments have confirmed that the siRNA provided by this invention can efficiently silence the SjHsc20 gene; and in vivo RNA interference experiments in mice show that this siRNA can induce mice to obtain 50.62% ( P The insect reduction rate was <0.05% and 44.29% (P <0.01) of the liver, suitable for preparing a drug for treating schistosomiasis. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 A comparison chart of the effect of RNA interference on the schistosome at different time points is shown in the real-time quantitative PCR analysis of the present application. DETAILED DESCRIPTION

[0015] The present application provides siRNA for the expression of Schistosoma japonicum Hsc20 gene and its application. In order to make the purpose, technical scheme and effect of the present application more clear and explicit, the following examples are used to further illustrate the present application. It should be understood that the specific examples described herein are only used to explain the present application and do not limit the protection scope of the present application.

[0016] In the following examples, the experimental methods not specified in the specific conditions are usually carried out according to the conventional conditions, such as the methods described in the Guide to Molecular Cloning Experiments (M.R. Green and J. Sambrook, He Fu, Chen Wei and Yang Xiaoming, et al. Translated. 3rd edition, Beijing: Science Press, 2017).

[0017] Example 1 In vitro RNA interference 1.1 Design of siRNA molecules According to the sequence of Schistosoma japonicum Hsc20 gene (GeneBank Sjp_0008430), four target sequences (SEQ ID NO. 1-SEQ ID NO. 4, see Table 1 below) and four pairs of siRNA molecules (SEQ ID NO. 5-SEQ ID NO. 12, see Table 1 below) specifically hybridized with the target sequences were designed. Irrelevant siRNA molecules were irrelevant controls. The siRNA was designed and synthesized by Shanghai Jimma Pharmaceutical Technology Co., Ltd.

[0018] Table 1 siRNA sequences of Schistosoma japonicum Hsc20 gene and corresponding target gene sequences

[0019] 1.2 Collection and culture of schistosomes and in vitro RNA interference Fifteen 6-8 week old male BALB / c mice were infected with Schistosoma japonicum cercariae by abdominal patch method, and each mouse was infected with 40 cercariae. At 21 and 28 days after infection, five mice were killed at each time point, and the schistosomes were collected by portal vein perfusion. The schistosomes were washed three times with RPMI 1640 medium containing double antibiotics (1000 units of each of ampicillin and streptomycin); then transferred into a disposable culture dish and washed once with RPMI 1640 complete medium (containing 10% fetal bovine serum, 1000 units of ampicillin and streptomycin); and cultured in a carbon dioxide incubator (37℃, 5% CO2).

[0020] The siRNA was contacted with the in vitro cultured worms by soaking method. First, the dry powder siRNA was taken out from -20℃ refrigerator and centrifuged in a low-temperature high-speed centrifuge to make the dry powder sink to the bottom of the centrifuge tube. The siRNA was carefully taken out and diluted on an ice box. According to the recommended amount in the instruction manual, 125 μL DEPC water was added to 1 OD siRNA to prepare a solution with a concentration of 40 μM. Second, the preheated culture medium was placed in a 12-well plate, 1 mL RPMI 1640 complete medium and 1 OD siRNA were added to each well, 3 pairs of worms were added to each well, and the plate was gently shaken to mix the siRNA and the culture medium evenly. At the same time, blank control and irrelevant RNAi control were set.

[0021] 1.3 SjHsc20 specific siRNA screening and interference effect The worm RNA of each interference group and interference control group was extracted, and qRT-PCR detection was performed after reverse transcription to cDNA. The expression level of Sj-Hsc20 gene in the irrelevant RNA control group was taken as the reference, and the transcription level of SjHsc20 gene in different interference groups was analyzed by 2 -ΔΔCt

[0022] Sj-PSMD4 Upstream primer: 5'-CCTCACCAACAATTTCCACATCT-3'; Downstream primer: 5'-GATCACTTATAGCCTTGCGAACAT-3'.

[0023] SjHsc20 Upstream primer: 5'-AAAAGCTCGCGTTGGATGCTT-3'; Downstream primer: 5'-GAAGACTTGAAGTCCGAAATCGCG-3'.

[0024] The results are shown in the following table, which includes two subgraphs, corresponding to 21d Schistosoma japonicum male-female copulatory worms (A) and 28d Schistosoma japonicum male-female copulatory worms (B), respectively; the horizontal coordinate is different treatment groups, including 4 siRNAs targeting Sj-Hsc20 gene (siRNA-33, siRNA-205, siRNA-474, siRNA-718), irrelevant control siRNA (siRNA NC) and blank control (Blank); the vertical coordinate is the relative expression amount, reflecting the transcription level of Sj-Hsc20 gene, and the lower the value, the more significant the gene silencing effect. Figure 1 Figure 1 Figure 1 ​​​​

[0025] From Figure 1 A can be seen from the blank control (Blank) group: Sj-Hsc20 gene relative expression amount is about 1.0, as a reference, reflecting the normal culture conditions under the transcription level of the gene in 21d worm body basis. Irrelevant control (siRNA NC) group: the relative expression of the gene is close to the blank control group, about 0.95-1.0, no significant statistical difference (p>0.05), indicating that the irrelevant siRNA does not produce specific interference on Sj-Hsc20 gene transcription, excluding nonspecific silencing effect. siRNA-718 group: the relative expression of the gene is about 0.55-0.6, compared with the control group decreased significantly, reaching a statistically significant level (p<0.05), indicating that the siRNA can effectively inhibit Sj-Hsc20 gene transcription in 21d worm body, the silencing efficiency is about 40%-45%. P P P

[0026] From Figure 1 B can be seen from the siRNA-718 group: the relative expression of the gene is about 0.03, compared with the control group decreased significantly, the statistical difference is extremely significant (p<0.0001), the silencing efficiency is improved to 97%, the silencing effect is greatly enhanced than 21d worm body. siRNA-33 group: the relative expression of the gene is about 0.02, the largest decrease than 21d worm body, and the statistical difference is extremely significant (p<0.001), indicating that the siRNA on the mature worm (28d) silencing effect is significantly better than the young worm (21d). siRNA-205 group: the relative expression of the gene is only 0.01-0.02, more than 98% lower than the control group, the statistical difference is extremely significant (p<0.0001). P P P ​​​​​<0.0001), with a high silencing efficiency of up to 98.37%, showing a strong and persistent silencing ability, and being the most stable and significant molecule in all tested siRNAs. In the siRNA-474 group, the relative expression of the gene was about 0.6-0.7, which was lower than that in the 21d worm body, and the silencing efficiency was increased to 30%-40%, indicating that the silencing effect of the siRNA on the mature worm body (28d) was better than that on the young worm body (21d).

[0027] In summary Figure 1 Based on the above results, the following conclusions can be drawn: Target specificity: All siRNAs targeting Sj-Hsc20 gene provided by the present application can inhibit the transcription of the gene to varying degrees, while the control siRNA has no significant effect, proving that the silencing effect is sequence-specific, due to the base complementary pairing between siRNA and target gene, which is consistent with the molecular mechanism of RNAi.

[0028] Time dependence: The silencing effect of most siRNAs is enhanced with the extension of the culture time of the worm body, especially siRNA-205, siRNA-33 and siRNA-718, which have significantly higher silencing efficiency in 28d worm body than in 21d worm body. This may be because as the worm body matures, the cell metabolic activity increases, the uptake efficiency of siRNA improves, or the expression of RNAi pathway related proteins (such as Dicer, AGO, etc.) increases, thereby strengthening the silencing effect.

[0029] Molecular diversity: There are significant differences in the silencing efficiency of different siRNAs. siRNA-205 shows the best silencing activity, which can efficiently inhibit the transcription of Sj-Hsc20 gene in 21d and 28d worm bodies, and the silencing effect is stable and persistent. siRNA-718 is the second, which shows strong silencing ability in mature worm body. The silencing efficiency of siRNA-33 and siRNA-474 is relatively weak. This difference may be related to the sequence characteristics of siRNA, such as GC content, thermodynamic stability, and the position of the target sequence in the gene, etc., which will affect the binding efficiency of siRNA and target mRNA and the assembly activity of RISC complex.

[0030] Application potential: The high silencing efficiency of siRNA-205 provides a key basis for subsequent in vivo experiments and drug development, which can effectively silence the target gene in different developmental stages of the worm body, laying a molecular foundation for interfering with the growth and development of schistosomes and reducing infectivity.

[0031] Example 2 In vivo RNA interference 1. Method steps Fifteen 6-week-old BALB / c mice were randomly divided into three groups: siRNA-205 interference group, irrelevant siRNA control group and PBS blank control group, 5 mice in each group. Each mouse was challenged with 40±2 Schistosoma japonicum cercariae by abdominal skin patch method. At 12 days post-infection, the three groups of mice were injected with siRNA-205 (1 OD / 125 μL / mouse), irrelevant siRNA (1 OD / 125 μL / mouse) or PBS (125 μL / mouse) through the tail vein, respectively. The injection was performed every 4 days, and a total of 7 injections were performed. At 42 days post-infection, the mice were killed, and the worms were collected by portal vein perfusion and the worm burden was calculated. At the same time, the mouse liver was removed for liver egg count.

[0032] Reduction rate of worms = (1-interference group average worm burden / control group average worm burden) x 100%; Liver egg reduction rate = (1-interference group EPG / control group EPG) x 100%; 2. Results: The results of the in vivo interference experiment showed that the injection of siRNA No. 205 into the tail vein of mice induced partial reduction of worm burden and egg count, as shown in Table 2.

[0033] Table 2 Changes in worm burden and liver egg count in mice induced by SjHsc20 gene silencing

[0034] As can be seen from Table 2, only siRNA-205 targeting SjHsc20 gene can produce significant and stable worm inhibition and egg inhibition effect, irrelevant siRNA has no such effect, which proves that the effect is a specific result of SjHsc20 gene silencing, and not a non-specific effect of exogenous RNA.

[0035] The data in Table 2 also show that silencing SjHsc20 gene can inhibit the survival and reproduction of schistosomes, which are key links in the transmission and pathogenesis of schistosomiasis (adult worm survival is the basis for persistent infection, and eggs are the core of pathogenesis). Therefore, SjHsc20 gene is an effective target for the treatment of schistosomiasis, providing a core target basis for subsequent drug development.

[0036] The in vitro experiment in Example 1 has proved that siRNA-205 can efficiently silence SjHsc20 gene, and the data in Table 2 further verify that it still has significant activity in the live animal model (worm reduction rate > 50%, egg reduction rate > 40%, and both have statistical significance), solving the key problem of whether in vitro activity can be converted into in vivo effect, and providing direct in vivo experimental evidence for siRNA-205 as an active ingredient of anti-schistosomiasis drug.

[0037] In summary, the present application first determines the Hsc20 gene as a target gene for the prevention and treatment of schistosomiasis japonica. In the prior art, schistosomiasis-related researches are mostly focused on traditional targets such as proteases and surface antigens. However, through the prediction and verification of the function of the Hsc20 gene, it is found that the Hsc20 gene is involved in the synthesis of iron-sulfur proteins, which are essential proteins for key life processes such as DNA replication and oxidative respiration of schistosomes. Silencing of the Hsc20 gene can lead to metabolic disorders and hinder the growth and reproduction of schistosomes. The selection of this target gene breaks through the traditional research ideas.

[0038] Further, the present application first confirms that the Hsc20 gene is a key gene for the growth and reproduction of schistosoma japonicum through in vitro and in vivo experiments. Example 1 shows that silencing of the gene can significantly inhibit gene transcription in the worm body; Example 2 shows that it can induce mice to obtain a worm reduction rate of up to 50.62% and an egg reduction rate of 44.29%, proving that the target gene has important application value. There is no report on the function of the Hsc20 gene of schistosoma japonicum in the prior art, and the research of the present application fills the gap in this field.

[0039] The present application screens the optimal siRNA molecule (siRNA-205, SEQ ID NO. 7 and SEQ ID NO. 8) through in vitro experiments, which can efficiently silence the target gene in 21d and 28d worm bodies, with a silencing efficiency of up to 98.37%, and the in vivo experiment effect is significant. This screening method based on different developmental stage worm bodies takes into account the life history characteristics of schistosomes, ensuring the sustained effectiveness of siRNA in vivo, which is superior to the screening strategy in the prior art which only targets a single stage worm body.

[0040] The present application applies RNAi technology to silencing of the Hsc20 gene of schistosoma japonicum, and constructs a complete technical system of target gene selection-siRNA design-in vitro screening-in vivo verification. The present application also provides a new type of anti-schistosomiasis drug by taking siRNA as an active ingredient of the drug for treating schistosomiasis, which has the advantages of high sequence specificity, small side effects and difficulty in developing drug resistance, enriches the technical means for treating schistosomiasis, and provides a new idea for the development of new drugs.

[0041] It should be understood that the application of the present application is not limited to the above examples, and those skilled in the art can make improvements or changes according to the above description, and all these improvements and changes shall belong to the protection scope of the appended claims of the present application.

Claims

1. An siRNA that specifically inhibits the expression of Hsc20 gene of Schistosoma japonicum, characterized in that, The siRNA contains a nucleotide sequence hybridizing with a target sequence of the Hsc20 gene of Schistosoma japonicum, and the target sequence is selected from the sequences shown in SEQ ID NO. 1-4.

2. The siRNA according to claim 1, wherein the siRNA specifically inhibits the expression of the Hsc20 gene of Schistosoma japonicum. The siRNA contains a first strand and a second strand, which are complementary to form an RNA duplex, and the RNA sequence of the first strand is identical to a target sequence of the Hsc20 gene of Schistosoma japonicum, and the first strand and the second strand of the siRNA are selected from one pair or any two or more combinations of the following pairs: the nucleotide sequence shown in SEQ ID NO. 5 and SEQ ID NO. 6; the nucleotide sequence shown in SEQ ID NO. 7 and SEQ ID NO. 8; the nucleotide sequence shown in SEQ ID NO. 9 and SEQ ID NO. 10; the nucleotide sequence shown in SEQ ID NO. 11 and SEQ ID NO.

12.

3. The siRNA according to claim 1, wherein the siRNA specifically inhibits the expression of Hsc20 gene of Schistosoma japonicum. The first strand and the second strand of the siRNA are the nucleotide sequences shown in SEQ ID NO. 7 and SEQ ID NO.

8.

4. A medicament for treating schistosomiasis, characterized by, The active ingredient of the drug is siRNA for specifically inhibiting the expression of the Hsc20 gene of Schistosoma japonicum by RNA interference, and the siRNA contains a nucleotide sequence hybridizing with a target sequence of the Hsc20 gene of Schistosoma japonicum, and the target sequence is selected from the sequences shown in SEQ ID NO. 1-4.

5. The medicament for treating schistosomiasis according to claim 4, wherein the medicament is a medicament for treating schistosomiasis caused by Schistosoma japonicum. The first strand and the second strand of the siRNA are selected from one pair or any two or more combinations of the following pairs: the nucleotide sequence shown in SEQ ID NO. 5 and SEQ ID NO. 6; the nucleotide sequence shown in SEQ ID NO. 7 and SEQ ID NO. 8; the nucleotide sequence shown in SEQ ID NO. 9 and SEQ ID NO. 10; the nucleotide sequence shown in SEQ ID NO. 11 and SEQ ID NO.

12.

6. Use of the siRNA for specifically inhibiting the expression of the Hsc20 gene of Schistosoma japonicum according to any one of claims 1-3 in the preparation of a drug for treating or preventing schistosomiasis.

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