CN109971762B - SiRNA of schistosoma japonicum eIF4A gene and application thereof - Google Patents
SiRNA of schistosoma japonicum eIF4A gene and application thereof Download PDFInfo
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Abstract
The invention discloses siRNA of a schistosoma japonicum eukaryotic translation initiation factor eIF4A gene, which contains a nucleotide sequence hybridized with a target sequence of the schistosoma japonicum eIF4A gene, wherein the target sequence is selected from a sequence shown in SEQ ID NO. 1-SEQ ID NO. 3. The invention also discloses an application of siRNA of the schistosoma japonicum eIF4A gene. The siRNA of the schistosoma japonicum eIF4A gene can obviously inhibit the transcription of the SjeIF4A gene, can induce a mouse to obtain a 20.63 percent (P < 0.05) insect reduction rate and a 31.21 percent (P < 0.01) liver egg reduction rate, and is suitable for preparing the medicine for treating schistosomiasis.
Description
Technical Field
The invention relates to the technical field of molecular biology and biomedicine, in particular to siRNA of schistosoma japonicum eIF4A (eukaryotic translation initiation factor 4A) gene and application thereof.
Background
Schistosomiasis japonica is a parasitic disease which is commonly suffered by people and animals and seriously harms human and animal health in southern areas of China to influence social and economic development. Since the last 50 century China has achieved tremendous success in schistosomiasis control, but the intermediate host oncomelania is difficult to destroy and the repeated infection of schistosomiasis is serious, so that the schistosomiasis control still remains one of important public health problems in the middle and lower reaches and south areas of Yangtze river in China. The praziquantel has the advantages of high efficiency, low toxicity, convenient use, low cost and the like, and can be widely applied to the clinical treatment and prevention and treatment of schistosomiasis. However, in recent years, foreign scholars have reported praziquantel-resistant strains of Schistosoma mansoni, and domestic scholars have paid attention to the phenomenon of the change in sensitivity of Schistosoma japonicum to praziquantel. This suggests a need to find new drugs for the treatment or prevention of schistosomiasis.
The eukaryotic translation initiation factor eIF4A (eukaryotic initiation factor 4, eIF 4A) protein belongs to the family of DEAD-box RNA helicases and has RNA-dependent ATPase activity and ATP-dependent RNA helicase activity. At the initiation of protein synthesis, eIF4A, with the aid of other initiation factors such as eIF-3, causes the ribosomal 40S small subunit to recognize mRNA and initiate the translation process. Research shows that eIF4A is a multifunctional protein, not only participates in translation initiation of protein, but also plays an important role in various life processes such as embryonic development, nonsense-mediated mRNA degradation, selenoprotein synthesis, vertebrate development and the like. Research shows that the eIF4A of drosophila can control the self-renewal of germ stem cells by directly inhibiting the function of differentiation factor BAM (Bag of marbles, BAM) in ovary. The nucleic acid vaccine pVAX-eIF4A constructed by the gene of the toxoplasma gondii eIF4A can induce a mouse to generate strong humoral immunity and Th1 type immune response, and prolong the survival time of the immunized mouse.
RNA interference (RNAi) technology has now made dramatic advances in the fields of functional genomics, gene therapy, microbiological research and drug development. RNA interference is an evolutionarily conserved defense mechanism against transgene or foreign virus invasion, a process of double-stranded RNA (dsRNA) -mediated, sequence-specific post-transcriptional gene silencing of target genes. Introduction into a cell of a double-stranded RNA having a sequence homologously complementary to the mRNA product of transcription of the target gene specifically degrades the mRNA, thereby producing a corresponding loss of functional phenotype. So-called small interfering RNAs (siRNAs) are short-segment double-stranded RNA molecules that target and degrade homologous complementary sequence mRNAs to mediate the RNA interference pathway, usually consisting of more than 20 nucleotides, and are capable of specific post-transcriptional gene silencing. siRNA has been widely used in the fields of gene expression regulation mechanism research and the like.
Disclosure of Invention
The invention aims to solve the technical problem of providing the siRNA of the schistosoma japonicum eIF4A gene, which can obviously inhibit the transcription of the SjeIF4A gene and can be used for preparing the medicament for treating schistosomiasis.
In addition, the application of the siRNA of the schistosoma japonicum eIF4A gene in preparing the medicine for treating schistosomiasis is also provided.
In order to solve the technical problems, the invention is realized by the following technical scheme:
in one aspect of the invention, the siRNA for specifically inhibiting the expression of the schistosoma japonicum eIF4A gene comprises a nucleotide sequence hybridized with a target sequence of the schistosoma japonicum eIF4A gene, wherein the target sequence is selected from a sequence shown in SEQ ID NO. 1-SEQ ID NO. 3.
The siRNA comprises a first strand and a second strand, wherein the first strand and the second strand are complementary to form an RNA dimer, the RNA sequence of the first strand is consistent with the target sequence of the schistosoma japonicum eIF4A gene, and the first strand and the second strand of the siRNA are selected from one or the combination of any two or more of the following pairs:
nucleotide sequences shown in SEQ ID NO.4 and SEQ ID NO. 5;
nucleotide sequences shown as SEQ ID NO.6 and SEQ ID NO. 7;
the nucleotide sequences shown in SEQ ID NO.8 and SEQ ID NO. 9.
Preferably, the first strand and the second strand of the siRNA are nucleotide sequences shown in SEQ ID NO.4 and SEQ ID NO. 5.
In another aspect of the present invention, there is provided a medicament for treating schistosomiasis, the active ingredients of the medicament being: the siRNA inhibits the expression of the schistosoma japonicum eIF4A gene through RNA interference, and the siRNA contains a nucleotide sequence hybridized with a target sequence of the schistosoma japonicum eIF4A gene, wherein the target sequence is selected from a sequence shown in SEQ ID NO. 1-SEQ ID NO. 3.
The siRNA comprises a first strand and a second strand, and the first strand and the second strand are selected from one or a combination of any two or more of the following:
nucleotide sequences shown in SEQ ID NO.4 and SEQ ID NO. 5;
nucleotide sequences shown as SEQ ID NO.6 and SEQ ID NO. 7;
the nucleotide sequences shown in SEQ ID NO.8 and SEQ ID NO. 9.
Preferably, the first strand and the second strand of the siRNA are nucleotide sequences shown in SEQ ID NO.4 and SEQ ID NO. 5.
In another aspect of the invention, the invention also provides the application of the siRNA of the schistosoma japonicum eIF4A gene in preparing a medicament for treating schistosomiasis.
In another aspect of the invention, the invention also provides application of the siRNA of the schistosoma japonicum eIF4A gene in preparation of a medicament for preventing schistosomiasis.
The siRNA of the schistosoma japonicum eIF4A gene can be used for interfering the transcription and expression of the schistosoma japonicum eIF4A gene and the growth and development of schistosomiasis, and the result of an RNA interference test in a mouse shows that the siRNA can induce the mouse to obtain the insect reducing rate of 20.63% (P < 0.05) and the liver egg reducing rate of 31.21% (P < 0.01), and is suitable for preparing the medicament for treating schistosomiasis.
Drawings
The invention is described in further detail below with reference to the drawings and the detailed description.
FIG. 1 is a graph showing the effect of real-time quantitative PCR analysis on in vitro interference in example 1 of the present invention;
FIG. 2 is a graph showing the results of real-time quantitative PCR analysis of RNAi-induced SjeIF4A gene expression silencing at different time points in example 1 of the present invention.
Detailed Description
In the following examples, the experimental procedures without specifying the specific conditions were generally carried out according to conventional conditions, for example, as described in molecular cloning, A laboratory Manual (M.R. Green and J. SammBrukee, haifeng, chenwei and Yang Xiao, et al, 3 rd edition, beijing: scientific Press, 2017).
Example 1 in vitro RNA interference
1.1 Design of siRNA molecules
Three pairs of SjeIF4A specific siRNA molecules (S1, S2 and S3, see Table 1 below) are designed according to the Japanese blood fluke eIF4A gene sequence (GeneBank access: FN315265.1, SEQ ID NO. 12), irrelevant siRNA molecules are Irrelevant controls, and siRNA is designed and synthesized by Shanghai Jima pharmaceutical technology Limited.
TABLE 1 siRNA sequence of Schistosoma japonicum eIF4A gene and corresponding target gene sequence
1.2 Collection and culture of polypide and in vitro RNA interference
10 male BALB/c mice 7 weeks old were infected with schistosoma japonicum cercaria by the abdominal patch method, and each mouse was infected with 40 cercaria. The larvae were killed after 42d infection and collected by portal vein perfusion. Washing three times by using DMEM medium containing double antibodies (1000 units of each streptomycin penicillin); then transferred to a disposable petri dish, washed once with RPMI 1640 complete medium (containing 10% fetal bovine serum, 1000 units of streptomycin) and cultured in a carbon dioxide incubator (37 ℃,5% CO2).
siRNA is transfected into the in vitro cultured worm body by adopting an electrotransfer method. First, 125. Mu.L DEPC water was used to resuspend 1OD siRNA dry powder to make the dissolved siRNA concentration 20uM; after being shaken and dissolved, the mixture is placed in an ice box to be stored in dark. And secondly, after the electric rotor is rinsed by DMEM without serum and antibiotics, 70 mu L of DMEM culture medium without serum and antibiotics is added into the electric rotor, 30 mu L of diluted siRNA stock solution is added, the DMEM culture medium and the diluted siRNA stock solution are mixed gently and uniformly and then are kept still to avoid generating bubbles. 6 pairs of the armful insects with good state and similar size are added into each electric rotor cup, and the electric rotor cups are put into an electric rotor instrument for electric rotation (125V 24 mu s. After the electrotransformation is finished, adding proper amount of DMEM to suck out the schistosome and transferring the schistosome to a complete culture medium containing preheating for culturing for 5d. Blank control and irrelevant RNAi control are set at the same time.
1.3 SjeIF4A specific siRNA screening and interference effect
And extracting the RNA of the polypide of each interference group and each interference control group, inverting the RNA into cDNA, and performing qRT-PCR detection. Taking the NADH of the schistosoma japonicum as an internal reference gene and the expression quantity of the Sj-eIF4A gene in an unrelated RNA control group as a reference, adopting 2 -ΔΔCt The method analyzes the transcription level of SjeIF4A gene in the polypide of different interference groups. Sj-upstream primer of NADH: 5 'CGAGGACCTAACAGCAAGAGG-3'; downstream primer 5 'TCCGAACGAACTTTGAATCC-3'. Upstream primer of SjeIF 4A: 5 'ATCGCTCAAGCACAAATCAGG + 3'; downstream primer of 5 'AGTTCACGGTTGGCACA-3'. As a result, for three siRNAS1, S2 and S3 of the SjeIF4A gene, the transcription of the SjeIF4A gene in the polypide can be interfered to different degrees, and the transcription level is respectively reduced by 81.8 percent, 65.2 percent and 80.3 percent (see figure 1).
After obtaining the siRNA sequence with the best interference effect, the time for the interference of the best siRNA S1 in vitro is analyzed. And (3) interfering the cultured polypide in vitro according to the method, collecting polypides at 3d, 4d, 5d, 6d and 7d after interference, extracting polypide RNA of each interference group and interference control group, inverting the RNA into cDNA, and performing qRT-PCR detection. As a result, the transcription levels of the SjeIF4A gene were decreased at 3d, 4d and 5d after the interference, the transcription level at 5d was the lowest, and the transcription levels were increased at 6d and 7d after the interference, but the interference effect was still partially observed (FIG. 2).
Example 2 in vivo RNA interference
1. Method step
15 6-week-old BALB/c mice were randomized into three groups: sjeIF4A siRNA interference group, irrelevant siRNA control group and DEPC blank control group, each group has 5. Each mouse was challenged with 40. + -.2 Schistosoma japonicum cercaria by the abdominal skin patch method. Starting at 21d post-infection, three groups of mice were injected with SjeIF4A siRNA (1 OD/100. Mu.L/mouse), irrelevant siRNA (1 OD/100. Mu.L/mouse) or PBS (100. Mu.L/mouse), respectively, via the tail vein. The injection is given every 7 days for 3 times. When infected for 42 days, mice are killed, and the insect bodies are collected by a portal vein infusion method and the insect charge amount is calculated. Meanwhile, the liver of the mouse is picked up to count the eggs of the liver worms.
Reduction rate = (1-mean insect charge number in immune group/mean insect charge number in control group) × 100%;
liver egg reduction rate = (1-immune group EPG/control group EPG) × 100%;
2. as a result: the results of the in vivo interference experiments showed that some of the reduction in parasites and egg drop rates were induced after intravenous injection of siRNA numbered S1 at the tail of mice, as shown in table 2. The SjeIF4A siRNA induced mice to achieve a 20.63% (P < 0.05) reduction in worm and 31.21% (P < 0.01) reduction in liver ova compared to the PBS control group (table 2).
TABLE 2 SjeIF4A Gene silencing Induction of changes in insect load number and liver egg number in mice
The above-mentioned embodiments only express the embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
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<110> Shanghai animal doctor institute of Chinese academy of agricultural sciences (Shanghai center of Chinese centers of animal health and epidemiology)
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gctattcaac agcgagcta 19
<210> 2
<211> 19
<212> DNA
<213> Schistosoma japonicum (Schistosoma japonica)
<400> 2
gcatcggttc ttatctaaa 19
<210> 3
<211> 19
<212> DNA
<213> Schistosoma japonicum (Schistosoma japonicum)
<400> 3
gcgatttgca gacatatta 19
<210> 4
<211> 21
<212> DNA/RNA
<213> Artificial sequence (Artificial)
<400> 4
gcuauucaac agcgagcuat t 21
<210> 5
<211> 21
<212> DNA/RNA
<213> Artificial sequence (Artificial)
<400> 5
uagcucgcug uugaauagct t 21
<210> 6
<211> 21
<212> DNA/RNA
<213> Artificial sequence (Artificial)
<400> 6
gcaucgguuc uuaucuaaat t 21
<210> 7
<211> 21
<212> DNA/RNA
<213> Artificial sequence (Artificial)
<400> 7
uuuagauaag aaccgaugct t 21
<210> 8
<211> 21
<212> DNA/RNA
<213> Artificial sequence (Artificial)
<400> 8
gcgauuugca gacauauuat t 21
<210> 9
<211> 21
<212> DNA/RNA
<213> Artificial sequence (Artificial)
<400> 9
uaauaugucu gcaaaucgct t 21
<210> 10
<211> 21
<212> DNA/RNA
<213> Artificial sequence (Artificial)
<400> 10
uucuccgaac gugucacgut t 21
<210> 11
<211> 21
<212> DNA/RNA
<213> Artificial sequence (Artificial)
<400> 11
acgugacacg uucggagaat t 21
<210> 12
<211> 1292
<212> DNA
<213> Schistosoma japonicum (Schistosoma japonica)
<400> 12
gaaatagcgc actttggatt cctgtcacgc ttattatgag caattccaca gaagattctg 60
agatcgaaac gaattattat gaggttgtcg atggcttcga gaagttagga ctaaaatctg 120
aacttcttcg cggtatatac agttacggat atgaaaaacc gtctgctatt caacagcgag 180
ctattaaacc atccgtcgag ggtcgtgatg ttatcgctca agcacaatca ggtacaggaa 240
agacggcaac atttgccatc agcattttac aaagaataga tgtttcgtca aacacatgtc 300
aagcccttgt ccttgtgcca acccgtgaac ttgcgagaca aattcaaacg gttgtgcaac 360
gcatcggttc ttatctaaat gtcaggtgtc atacgtgtat tggtgggaca aggatgtctg 420
aagacgtggc gtgccttcag cagggtcaac atgttgttgt aggtactcct ggccgtgtca 480
tcgatatgat gaatcgcagt attctggcaa cttctaatat caaaatcttt gtgctggacg 540
aagctgacca aatgttaggt cggggttttg agccacaaat caaagaaata tataaatatt 600
tacctgaatc cgcccaaatc atgttactat ctgctactat gccgaagcaa atgcttacta 660
ttgcacgcgg tataatgcaa gatcctgttc aaatattgat taagaaagaa gaattaacat 720
tggatgggat caaacagttt tatattaatg tatccaaaga agaatacaag ttggaaactt 780
taatggactt atataaagtg atgaatttaa gtcaagtcgt tattttcgtt aactctgtac 840
gaaaggcttc atacctttct gaggagctcg ccaacagaaa ttttcaagtt tcctgcatta 900
atagcgacat ggaacaagaa aaacgtgaca gagttatgga agaatatcga agtggaagat 960
cacgaatttt actgtccact gatgttctgg cgcgtggtat tgatgtacag caggtttccc 1020
tagtcgttaa ctatgattta cctggtgatc gtgagactta tattcacaga attggtagag 1080
gaggtcgttt cggtaggaaa ggaacagcaa tcaactttat cactgataca gaaaaagaag 1140
ctttacgcga tttgcagaca tattacaaca cagaaattct tgaaatgcct gatgatattg 1200
ttgactttct gtaactgatc ccaaactctg agcccactta tacatttgat atttgttcct 1260
agctgaatca atttcaattt ttcttggtgg at 1292
Claims (4)
1. A siRNA for specifically inhibiting the expression of a schistosoma japonicum eIF4A gene comprises a nucleotide sequence hybridized with a target sequence of the schistosoma japonicum eIF4A gene, wherein the target sequence is a sequence shown as SEQ ID NO.1, the siRNA comprises a first chain and a second chain, the first chain and the second chain are complementary to form an RNA dimer, the RNA sequence of the first chain is consistent with the target sequence of the schistosoma japonicum eIF4A gene, and the first chain and the second chain of the siRNA are nucleotide sequences shown as SEQ ID NO.4 and SEQ ID NO. 5.
2. The medicament for treating schistosomiasis is characterized by comprising the following active ingredients: the siRNA inhibits the expression of the schistosoma japonicum eIF4A gene through RNA interference, and comprises a nucleotide sequence hybridized with a target sequence of the schistosoma japonicum eIF4A gene, wherein the target sequence is a sequence shown as SEQ ID NO.1, the siRNA comprises a first chain and a second chain, and the first chain and the second chain are nucleotide sequences shown as SEQ ID NO.4 and SEQ ID NO. 5.
3. The use of the siRNA of claim 1 in the preparation of a medicament for the treatment of schistosomiasis.
4. The use of the siRNA of claim 1 in the preparation of a medicament for the prevention of schistosomiasis.
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| CN103667286A (en) * | 2012-09-14 | 2014-03-26 | 中国农业科学院上海兽医研究所 | Small interfering ribonucleic acid (siRNA) of schistosoma japonicum katsurada membrane-associated progesterone receptor component (PGMRC2) gene and application thereof |
| CN108795934A (en) * | 2018-05-23 | 2018-11-13 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | The siRNA of 2 genes of Schistosoma japonicum SjELAV-like and its application |
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| WO2011005566A2 (en) * | 2009-06-23 | 2011-01-13 | University Of Miami | Aptamer-targeted sirna to inhibit nonsense mediated decay |
| CN108601788B (en) * | 2015-11-25 | 2021-10-22 | 效应治疗股份有限公司 | EIF 4A-inhibiting compounds and methods relating thereto |
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Patent Citations (2)
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| CN103667286A (en) * | 2012-09-14 | 2014-03-26 | 中国农业科学院上海兽医研究所 | Small interfering ribonucleic acid (siRNA) of schistosoma japonicum katsurada membrane-associated progesterone receptor component (PGMRC2) gene and application thereof |
| CN108795934A (en) * | 2018-05-23 | 2018-11-13 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | The siRNA of 2 genes of Schistosoma japonicum SjELAV-like and its application |
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