CN102220326B - SiRNA of SjLGL gene of schistosoma japonica and use thereof - Google Patents
SiRNA of SjLGL gene of schistosoma japonica and use thereof Download PDFInfo
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Abstract
The invention discloses the siRNA of SjLGL gene of schistosoma japonica. The siRNA may be one pair or any more than two pairs of nucleotide sequences represented by SEQ ID No.1 and SEQ ID No.2, nucleotide sequences represented by SEQ ID No.3 and SEQ ID No.4, and nucleotide sequences represented by SEQ ID No.5 and SEQ ID No.6. The invention also discloses the use of the siRNA of the SjLGL gene of schistosoma japonica. The siRNA of the SjLGL gene of schistosoma japonica can obviously inhibit the transcription of the SjLGL gene, obviously lower the hatchability of the eggs of insects in livers of mice infected with schistosomes and is suitable for preparing medicines for treating schistosomiasis.
Description
Technical field
The present invention relates to molecular biology and biological medicine technology field, relate in particular to a kind of siRNA and application thereof of Schistosoma japonicum SjLGL gene.
Background technology
Schistosomicide is that a kind of humans and animals all can receive infectious parasitosis, and popular scope is wide, and people and domestic animal are all produced serious harm.At present, mainly adopt medicine, for example PRAZIQUANTEL BP 98 is treated the human or animal who infects schistosomicide, but the PRAZIQUANTEL BP 98 medicine carries out when oral, and " first pass effect " is big, and bioavailability is poor, has influenced clinical application effect.Be necessary to seek the medicine of new treatment schistosomicide.
(Lethal Giant Larvael LGL), can cause that cell hyperproliferation causes the gene of hyperproliferative when being meant sudden change to the huge lethal gene of larva.The LGL transgenation can cause the forfeiture of fruit bat epithelial cell hyper-proliferative, polarity, can not break up and the epithelium monolayer phenotype that is similar to human cancerous tumour such as destructurized.Its mutant phenotype mainly shows larval stage, i.e. larva epithelial cell polarity forfeiture, and hyper-proliferative and can not normal differentiation, causing can not pupa, forms at last " huge larva " and dead.Fruit bat is not merely limited in the existence of LGL gene; The homologue of LGL gene is identified that the homologous gene of high conservative is being exercised the conservative function of regulating cell polarity and propagation probably equally between different plant species in the mankind, mouse, rat, ox, insect, worm, slime mould and yeast.
In schistosoma japonicum gene group examining order of delivering in 2009 and the work of Schistosoma mansoni gene order-checking, all reported the existence of LGL gene in the schistosomicide body.Schistosoma japonicum LGL gene (SjLGL), its gene order are numbered AY812588 in GenBank.This seminar successfully confirms to exist interaction between schistosomicide LGL albumen and the schistosomicide gynecophoric canal albumen in Yeast system and mammalian cell system in previous research work.And gynecophoric canal albumen be a kind of express at schistosomicide male worm gynecophoric canal position and fill the span of a man's arms through male and female pass to having of female worm and influence the schistosomicide male and female worm sex-specific material of function of filling the span of a man's arms, and have the growth regulatory function.Periostin albumen with the gynecophoric canal albumen homology in the research proof Mammals plays a significant role in heart development, bone and tooth generation and aspect keeping as important growth regulatory factor; Clinical study is also found generation, the development of Periostin albumen and human kinds of tumors and shifts relevantly that this point and the effect of LGL albumen in human tumor have certain dependency.Therefore interaction between LGL and the schistosomicide gynecophoric canal albumen is indicating the dependency on the two function, and function and LGL gene and other the intergenic interactions of studying the LGL gene have crucial meaning.
RNA disturbs (RNAi) technology in fields such as functional genomics, gene therapy, microbiological research and medicament research and development, to obtain the progress that attracts people's attention at present.It is that a kind of the evolution gone up the conservative defense mechanism of resisting transgenic or adventitious viruses infringement that RNA disturbs, and is double-stranded RNA (dsRNA) process mediation, sequence-specific target gene PTGS.After will having the double-stranded RNA transfered cell of homology complementary sequence with the product mRNA that target gene is transcribed, this mRNA that can degrade specifically, thus produce function corresponding phenotype disappearance.So-called small molecules interference RNA (small interfering RNAs; SiRNAs); Be for can being target and with homology complementary sequence mRNA with the short segments double stranded rna molecule of its degraded mediate rna interference channel; Usually form by more than 20 Nucleotide, can carry out specific PTGS.SiRNA has been widely used in fields such as gene expression regulation Mechanism Study.
Summary of the invention
The technical problem that the present invention will solve provides a kind of siRNA of Schistosoma japonicum SjLGL gene, and this siRNA can obviously suppress the SjLGL gene transcription, can be used for preparing the medicine of treating schistosomicide.
A kind of application of siRNA in the medicine of preparation treatment schistosomicide of Schistosoma japonicum SjLGL gene also need be provided in addition.
In order to solve the problems of the technologies described above, the present invention realizes through following technical scheme:
In one aspect of the invention, a kind of siRNA of Schistosoma japonicum SjLGL gene is provided, said siRNA is selected from following a pair of or any combination more than two pairs:
Nucleotide sequence shown in SEQ ID NO.1 and the SEQ ID NO.2;
Nucleotide sequence shown in SEQ ID NO.3 and the SEQ ID NO.4;
Nucleotide sequence shown in SEQ ID NO.5 and the SEQ ID NO.6.
Preferably, said siRNA is the nucleotide sequence shown in SEQ ID NO.1 and the SEQ ID NO.2.
In another aspect of this invention, a kind of medicine of treating schistosomicide is provided, the activeconstituents of said medicine is: disturb the siRNA that suppresses Schistosoma japonicum SjLGL genetic expression through RNA.
Preferably, said siRNA is selected from following a pair of or any combination more than two pairs:
Nucleotide sequence shown in SEQ ID NO.1 and the SEQ ID NO.2;
Nucleotide sequence shown in SEQ ID NO.3 and the SEQ ID NO.4;
Nucleotide sequence shown in SEQ ID NO.5 and the SEQ ID NO.6.
Preferred, said siRNA is the nucleotide sequence shown in SEQ ID NO.1 and the SEQ ID NO.2.
A kind of application of siRNA in the medicine of preparation treatment schistosomicide of above-mentioned Schistosoma japonicum SjLGL gene also is provided in another aspect of this invention.
A kind of application of siRNA in the vaccine of preparation prevention schistosomicide of above-mentioned Schistosoma japonicum SjLGL gene also is provided in another aspect of this invention.
The siRNA of Schistosoma japonicum SjLGL gene of the present invention; Can be used for disturbing Schistosoma japonicum SjLGL genetic transcription, expression and bilharzial growing; The RNA interference experiment shows in the body of mouse; This siRNA can significantly reduce infecting mouse liver egg hatch rate, is suitable for preparing the medicine of treating schistosomicide.
Description of drawings
Below in conjunction with accompanying drawing and embodiment the present invention is done further detailed explanation.
Fig. 1 is that the embodiment of the invention 1 real-time quantitative PCR is analyzed each treatment group SjLGL gene transcription figure as a result;
Fig. 2 is that the embodiment of the invention 2 real-time quantitative PCRs are analyzed each treatment group SjLGL gene transcription figure as a result.
Embodiment
In the following example, the experimental technique of unreceipted actual conditions is usually by normal condition, like " molecular cloning experiment guide " (J. Sa nurse Brooker; D.W. the Russell is outstanding, Huang Peitang, Wang Jiaxi, Zhu Houchu; Deng translating. the 3rd edition, Beijing: Science Press, 2002) described in method carry out.
Embodiment 1 external RNA disturbs
1. method steps
1.1 virgin worm culture medium preparation
Be dissolved among the RPMI-1640, be settled to 1000ml, using the aperture is that 0.22 μ m filter filters 4 ℃ of preservations.
1.2 the preparation of rabbit erythrocyte
(1) in aseptic 1.5ml centrifuge tube, adds a little autoclaved PBS solution that contains 0.1% heparin sodium and concussion for several times, make its coating tube wall;
(2) new zealand white rabbit ear edge vein exploitating blood 1ml is transferred to rapidly in the centrifuge tube that contains heparin; Shake centrifuge tube gently, make the antithrombotics mixing;
(3) 4 ℃, the centrifugal 10min of 1500rpm abandons supernatant;
(4) Xiang Guanzhong adds 1ml sterilization PBS with a small amount of two anti-, with micropipet pressure-vaccum mixing gently;
(5) 4 ℃, the centrifugal 10min of 1500rpm abandons supernatant;
(6) repeating step (4), (5) three times sop up most of supernatant, then in 4 ℃ of preservations.
1.3siRNA the preparation of molecule and SjLGL gene real-time quantitative PCR primer
(http://www.ncbi.nlm.nih.gov/) finds out schistosomicide LGL gene order (AY812588) at the NCBI net.
Design and synthesize three couples of dsRNA.Simultaneously, synthetic a pair of irrelevant contrast dsRNA (NC dsRNA) is:
sense：5’-UUCUCCGAACGUGUCACGUTT-3’(SEQ?ID?NO.7)，
Anti-sense：5’-ACGUGACACGUUCGGAGAATT-3’(SEQ?ID?NO.8)；
The dsRNA small molecules of three pairs of Schistosoma japonicum LGL genes is:
s1dsRNA：sense：5’-GGUCGACUUUAAAGUUCAATT-3’(SEQ?ID?NO.1)，
Anti-sense:5 '-UUGAACUUUAAAGUCGACCTT-3 ' (SEQ ID NO.2), the target sequence that this first couple of siRNA is directed against is GGUCGACUUUAAAGUUCAA;
s2dsRNA：sense：5’-GCUUGUUGCUGUUGAUUUATT-3’(SEQ?ID?NO.3)，
Anti-sense:5 '-UAAAUCAACAGCAACAAGCTT-3 ' (SEQ ID NO.4), the target sequence that this second couple of siRNA is directed against is GCUUGUUGCUGUUGAUUUA;
s3dsRNA：sense：5’-AGCUGUUAGUGAUCAACUATT-3’(SEQ?ID?NO.5)，
Anti-sense:5 '-UAGUUGAUCACUAACAGCUTT-3 ' (SEQ ID NO.6), the 3rd pair of target sequence that siRNA is directed against is AGCUGUUAGUGAUCAACUA.
According to the gene order of SjLGL, design real-time quantitative PCR primer send company (Invitrogen) synthetic then.
Sense：5’-CTGATTTTCCATTCGTTTG-3’(SEQ?ID?NO.9)，
Anti-sense：5’-GTTGTCTACCATGAAGGCA-3’(SEQ?ID?NO.10)。
1.4 collection and the cultivation of 12 days virgin worms of Schistosoma japonicum
The artificial paster of new zealand white rabbit belly infects schistosoma japonicum cercariae, and every is infected 8000, cuts open in metainfective 12 days and kills, and aseptic venous perfusion is collected polypide towards worm.With being added with two anti-PBS flushings three times, put into aseptic centrifuge tube, the lid upper tube cap; After 70% alcohol sprays; Change over to immediately in the Bechtop, wash once with the virgin worm substratum of 37 ℃ of preheatings again, add virgin worm substratum and fresh rabbit erythrocyte carries out vitro culture in 12 well culture plates.Every hole substratum is 3ml, and polypide is about 100.Experiment is divided into: blank control group, irrelevant control group (NC group), s1, s2, s3dsRNA treatment group.It is 5% that incubator is set gas concentration lwevel, changes substratum three times respectively at the 4h, 28h, the 76h that cultivate.
1.5 the processing of polypide
Every kind of siRNA molecule joins (final concentration is 100nM) in the substratum respectively, continues to disturb polypide 7 days.
1.6 the real-time quantitative PCR of external RNA interference effect checking
Extract total RNA of each treatment group Schistosoma japonicum polypide with the Trizol test kit, the digestion genome, reverse transcription is cDNA, carries out the real-time quantitative PCR analysis.
2. result
As shown in Figure 1, three kinds of different dsRNA molecules have all reduced SjLGL gene transcription level significantly, and wherein the transcriptional level of s1dsRNA group small molecules reduction is minimum, choose s1dsRNA group small molecules and use small molecules as RNA interference in the body.NC group and blank control group are more or less the same.
RNA disturbs in embodiment 2 bodies
1. method steps
(1) dsRNA injection
Control group, irrelevant control group (NC group) and s1dsRNA treatment group are established in test altogether, every group of totally three mouse.In metainfective 13 days, 14 days, 15 days and 16 days difference tail vein fast injection 1ml DEPC water, 1ml NC dsRNA (40 μ g) DEPC water and 1ml SjLGL gene s1dsRNA (40 μ g) DEPC water.
(2) liver egg hatch rate
Cut open the mouse after killing, get liver and weigh, add dechlorination water and be settled to 20ml; Behind the refiner mixing, get 4ml and blend in the liquid adding heart bottle (adding 3-5cm on dechlorination water to the bottleneck in the heart bottle), on heart bottle liquid level, fill in cotton; Add 8ml dechlorination water on the cotton, 26-27 ℃ of hatching 2h is transferred to cotton upper strata 8ml liquid in the 15ml conical centrifuge tube behind the 2h; Add 900 μ l formaldehyde fixed miracidiums, the centrifugal 5min of 4000rpm level siphons away supernatant with the 5ml liquid-transfering gun; Small amount of liquid is stayed at the pipe end, adds a tincture of iodine dye liquor, the microscopically counting.
(3) real-time quantitative PCR of RNA interference effect checking in the body
Extract total RNA of each treatment group Schistosoma japonicum polypide with the Trizol test kit, the digestion genome, reverse transcription is cDNA, carries out the real-time quantitative PCR analysis.
2. result and analysis
(1) RT-PCR of RNA interference effect analyzes in the body
As shown in Figure 2, control group and treatment group mouse polypide RT-PCR analysis revealed treatment group SjLGL gene transcription level significantly reduce, and the control group of the irrelevant dsRNA molecule of injection is not seen influence.
(2) RNA disturbs the influence of back to liver egg hatch rate
The Balb/c mouse was cutd open mouse extremely in 22 days in infecting 13,14,15,16 days tail vein injection dsRNA of Schistosoma japonicum, collected mouse liver, weighed egg count, 26-27 ℃ of hatching 2h, counting miracidium number.Subtract hatching rate after calculating RNA and disturbing, the result shows that RNA disturbs the egg hatch rate of aftertreatment group obviously to reduce than control group, subtracts hatching rate and reaches about 80%, explain that the RNA interference has bigger influence (seeing the following form 1) to the hatching rate of Schistosoma japonicum.
After disturbing, table 1RNA subtracts hatching rate
Test-results for the first time
Test-results for the second time
N=3, * * are P＜0.01
The above embodiment has only expressed embodiment of the present invention, and it describes comparatively concrete and detailed, but can not therefore be interpreted as the restriction to claim of the present invention.Should be pointed out that for the person of ordinary skill of the art under the prerequisite that does not break away from the present invention's design, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with accompanying claims.
Sequence table
< 110>China Agriculture Academe Shanghai Veterinary Institute
< 120>siRNA of Schistosoma japonicum SjLGL gene and application thereof
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Claims (5)
1. the siRNA of a Schistosoma japonicum SjLGL gene is characterized in that, said siRNA is selected from following a pair of or any combination more than two pairs:
Nucleotide sequence shown in SEQ ID NO.1 and the SEQ ID NO.2;
Nucleotide sequence shown in SEQ ID NO.3 and the SEQ ID NO.4;
Nucleotide sequence shown in SEQ ID NO.5 and the SEQ ID NO.6.
2. according to the siRNA of the said Schistosoma japonicum SjLGL of claim 1 gene, it is characterized in that said siRNA is the nucleotide sequence shown in SEQ ID NO.1 and the SEQ ID NO.2.
3. a medicine of treating schistosomiasis japanica is characterized in that, the activeconstituents of said medicine is: disturb the siRNA that suppresses Schistosoma japonicum SjLGL genetic expression through RNA, this siRNA is the nucleotide sequence shown in SEQ ID NO.1 and the SEQ ID NO.2.
4. the application of the siRNA of the said Schistosoma japonicum SjLGL of claim 1 gene in the medicine of preparation treatment schistosomiasis japanica, said siRNA is the nucleotide sequence shown in SEQ ID NO.1 and the SEQ ID NO.2.
5. the application of the siRNA of the said Schistosoma japonicum SjLGL of claim 1 gene in the vaccine of preparation prevention schistosomiasis japanica, said siRNA is the nucleotide sequence shown in SEQ ID NO.1 and the SEQ ID NO.2.
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| CN103667286B (en) * | 2012-09-14 | 2015-09-23 | 中国农业科学院上海兽医研究所 | The siRNA of Schistosoma japonicum PGMRC2 gene and application thereof |
| CN108531480B (en) * | 2017-03-01 | 2022-01-18 | 中国疾病预防控制中心寄生虫病预防控制所 | Micro RNA and application thereof in preparation of anti-schistosoma japonicum infection preparation |
| CN108795934B (en) * | 2018-05-23 | 2022-06-21 | 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) | SiRNA of schistosoma japonicum SjELAV-like 2 gene and application thereof |
| CN108795935B (en) * | 2018-05-23 | 2022-06-21 | 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) | SiRNA of SjELAV-like 1 gene of schistosoma japonicum and application thereof |
| CN110016477B (en) * | 2019-03-18 | 2022-04-12 | 中国农业科学院上海兽医研究所 | SiRNA of schistosoma japonicum NAT13 gene and application thereof |
| CN110564724B (en) * | 2019-06-11 | 2023-01-06 | 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) | SiRNA of Schistosoma japonicum SjFrzb2 gene and application thereof |
| CN111705057A (en) * | 2020-05-31 | 2020-09-25 | 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) | SiRNA of schistosoma japonicum SjGST gene and application thereof |
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| CN1974591A (en) * | 2006-11-14 | 2007-06-06 | 苏先狮 | SiRNA expression template for treating liver necrosis and liver fibrosis |
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| CN1974591A (en) * | 2006-11-14 | 2007-06-06 | 苏先狮 | SiRNA expression template for treating liver necrosis and liver fibrosis |
| CN101892240A (en) * | 2010-07-28 | 2010-11-24 | 中国农业科学院上海兽医研究所 | Japanese blood fluke beta-catenin gene, protein and application |
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