CN108531480B - Micro RNA and application thereof in preparation of anti-schistosoma japonicum infection preparation - Google Patents
Micro RNA and application thereof in preparation of anti-schistosoma japonicum infection preparation Download PDFInfo
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Abstract
The invention provides a micro RNA and application thereof in preparing a preparation for resisting schistosoma japonicum infection. Specifically, the invention provides a nucleic acid inhibitory molecule, in particular a micro RNA nucleic acid molecule, which has a remarkable killing or inhibiting effect on schistosoma. Experiments show that the specific amiRNA has high killing or inhibiting capability on the schistosoma japonicum (especially early schistosoma japonicum). The invention also provides a preparation method, a pharmaceutical composition and application of the nucleic acid inhibiting molecule.
Description
Technical Field
The invention relates to biological medicine, in particular to micro RNA and application thereof in preparing a preparation for resisting schistosoma japonicum infection.
Background
Schistosomiasis is a parasitic disease that seriously harms human health. At present, the treatment of schistosomiasis still takes praziquantel as a main part, but is difficult to control schistosomiasis repeated infection by completely depending on single drug treatment, and researches and findings in recent years show that praziquantel chemotherapy has potential risk of inducing drug resistance, so that development of novel vaccines and novel drugs for schistosomiasis has important strategic significance for effectively controlling and even eliminating schistosomiasis. Non-coding small ribonucleic acid (RNA) is widely present in eukaryotes, generally has a length of 18-30 nucleotides (nt), mainly comprises 3 types of micro ribonucleic acids (miRNA), small interfering ribonucleic acids (siRNA), Piwi-interacting ribonucleic acids (piRNA) and the like, and is a key regulator for biological gene expression, genome stabilization and resistance of foreign genetic factors. In recent years, researchers find that specific non-coding RNA exists in different development stages of schistosoma, which provides a new idea for further research on development, parasitism and pathogenic mechanisms of schistosoma.
The identification and discovery of the micro RNA are beneficial to discovering potential anti-schistosoma japonicum drug targets, and provide new clues for further understanding the biological characteristics of schistosoma japonicum and further developing new schistosoma japonicum drug research and development.
Therefore, there is an urgent need in the art to develop a microRNA drug that can effectively kill or inhibit schistosomiasis (particularly Schistosoma japonicum) with little side effects.
Disclosure of Invention
The invention aims to develop a micro RNA medicament which can effectively kill or inhibit schistosoma (particularly schistosoma japonicum) and has small side effect.
The invention provides a nucleic acid inhibiting molecule, wherein the nucleic acid inhibiting molecule is single-stranded or double-stranded amiRNA, and the 5 'end to the 3' end of a sense strand of the nucleic acid inhibiting molecule has a structure shown in a formula I:
Z0-Z1-Z2 (I)
wherein,
Z0a, T, C, G or none;
Z1is as set forth in SEQ ID No.: 1. 3, or 5; and
Z2a, T, C, G or none.
In another preferred embodiment, the structure of formula I is selected from the group consisting of: SEQ ID No.: 1. 3, 5, or a combination thereof.
In another preferred embodiment, the nucleic acid inhibitory molecule comprises an siRNA.
In another preferred embodiment, the nucleic acid inhibiting molecule is 16 to 50nt, preferably 16 to 30nt, more preferably 16 to 25nt in length.
In another preferred embodiment, the nucleic acid inhibitory molecule is an isolated, recombinant, or synthetic nucleic acid molecule.
In a second aspect, the invention provides a precursor sequence for producing a nucleic acid inhibitory molecule according to the first aspect of the invention, the precursor sequence sense strand having a structure according to formula II at the 5 'to 3' end:
in the formula,
b1 is a desired first ribonucleotide sequence, wherein said first ribonucleotide sequence comprises the form of an amiRNA sequence;
b2 is a sequence substantially or fully complementary to B1, and B2 is not complementary to C;
c is a stem-loop structure.
In another preferred embodiment, said B1 or said amiRNA has the structure of formula I, or is as shown in formula I.
In another preferred embodiment, the sequence of B1 is selected from the group consisting of: SEQ ID No.: 1. 3, 5, or a combination thereof.
In a third aspect, the invention provides a nucleic acid molecule, wherein the sequence of the nucleic acid molecule is shown in any one of SEQ ID No. 1, 3 and 5.
In another preferred embodiment, the nucleic acid molecule is single-stranded or double-stranded.
In a fourth aspect, the present invention provides a polynucleotide which is transcribed by a host to form a precursor sequence according to the second aspect of the invention.
In a fifth aspect, the present invention provides an expression vector comprising a precursor sequence according to the second aspect of the present invention or a polynucleotide according to the fourth aspect of the present invention.
In another preferred embodiment, the expression vector is a viral vector or a non-viral vector.
In another preferred embodiment, the viral vector is selected from the group consisting of: a lentiviral vector, an adenoviral vector, or a combination thereof.
The sixth aspect of the present invention provides a composition for killing or inhibiting schistosoma, which comprises;
(a) a nucleic acid inhibitory molecule according to the first aspect of the invention, and/or a precursor sequence according to the second aspect of the invention; and
(b) a pharmaceutically acceptable carrier.
In another preferred embodiment, the composition further comprises a viral vector.
In another preferred embodiment, the composition further comprises (c) other drugs for killing or inhibiting schistosoma.
In another preferred embodiment, the schistosome is selected from the group consisting of: schistosoma japonicum, Schistosoma mansoni, Schistosoma japonicum, or their combination.
In another preferred example, the schistosoma japonicum comprises schistosoma japonicum.
In another preferred embodiment, the pharmaceutically acceptable carrier is selected from the group consisting of: water, saline, liposomes, lipids, proteins, peptidic substances, cellulose, nanogels, or combinations thereof.
In another preferred embodiment, the dosage form of the composition is selected from the group consisting of: tablet, capsule, powder, pill, granule, solution, suspension, emulsion, suspension, injection, or powder for injection.
In another preferred embodiment, the dosage form of the composition further comprises spray, aerosol, powder spray, volatile liquid, external solution, lotion, pour-on, liniment, cataplasm, plaster, rubber plaster, ointment, gargle.
In another preferred embodiment, the dosage form is an injection, preferably an intravenous injection or an intraperitoneal injection.
In another preferred embodiment, the dosage form is an oral dosage form.
In another preferred embodiment, the composition is selected from the group consisting of: a pharmaceutical composition, a vaccine composition, or a combination thereof.
In a seventh aspect, the invention provides the use of a nucleic acid inhibitory molecule of the first aspect of the invention or a precursor sequence of the second aspect of the invention, for the preparation of a composition or formulation for (i) the prevention or treatment of schistosome infection; and/or (ii) killing or inhibiting schistosomiasis.
In another preferred embodiment, the composition or formulation is also used for killing or inhibiting schistosomulum.
In another preferred embodiment, the composition or preparation further comprises other drugs for killing or inhibiting schistosoma.
The eighth aspect of the invention provides a method for inhibiting in vitro killing or inhibiting schistosome, which comprises the following steps: the method comprises culturing schistosoma japonicum in the presence of the nucleic acid inhibitory molecule of the first aspect of the invention or the precursor sequence of the second aspect of the invention or the composition of the sixth aspect of the invention, thereby killing or inhibiting schistosoma japonicum.
In another preferred embodiment, the schistosome is selected from the group consisting of: schistosoma japonicum, Schistosoma mansoni, Schistosoma japonicum, or their combination.
In another preferred example, the schistosoma japonicum comprises schistosoma japonicum.
In another preferred embodiment, the step is contacting the schistosoma japonicum with a viral vector expressing the nucleic acid inhibitory molecule of the first aspect of the present invention or the precursor sequence of the second aspect of the present invention in a culture system, thereby killing or inhibiting schistosoma japonicum.
In another preferred embodiment, the concentration of the viral vector in the culture system is 104-1010One/ml, preferably, 104-107One per ml.
The ninth aspect of the present invention provides a method for screening a candidate compound for inhibiting or killing schistosoma, comprising the steps of:
(a) culturing cells expressing FTL protein and/or SJCL protein in a culture system for a time T1 in the presence of a test compound in a test group, and detecting the expression amount E1 and/or activity a1 of FTL protein and/or SJCL protein in the culture system of the test group;
and testing the expression level E2 and/or activity A2 of FTL protein and/or SJCL protein in the culture system of a control group in the absence of the test compound and under otherwise identical conditions;
(b) comparing the expression level E1 and/or activity A1 of the FTL protein and/or SJCL protein detected in the previous step with the expression level E2 and/or activity A2 of the FTL protein and/or SJCL protein, thereby determining whether the test compound is a candidate compound for inhibiting or killing schistosoma,
wherein if E1 is significantly lower than E2, and/or a1 is significantly lower than a2, then the test compound is a candidate compound for inhibiting or killing schistosoma.
In another preferred embodiment, the phrase "substantially less than" means E1/E2 ≦ 1/2, preferably ≦ 1/3, and more preferably ≦ 1/4.
In another preferred embodiment, the phrase "substantially less than" means A1/A2 ≦ 1/2, preferably ≦ 1/3, and more preferably ≦ 1/4.
In another preferred embodiment, the method comprises the steps of (c) applying the candidate compound determined in step (b) to the schistosoma japonicum and determining the killing or inhibiting effect on the schistosoma japonicum.
In another preferred embodiment, the method is non-diagnostic and therapeutic.
In a tenth aspect, the present invention provides a method for the prevention and/or treatment of schistosomiasis, comprising administering to a subject in need thereof a nucleic acid inhibitory molecule according to the first aspect of the invention or a precursor sequence according to the second aspect of the invention or a composition according to the sixth aspect of the invention.
In another preferred embodiment, the subject in need thereof comprises a mammal, preferably a human, mouse, rat, rabbit.
The eleventh aspect of the invention provides the use of an amiRNA, or a precursor thereof, or an expression vector thereof, for the preparation of a medicament for (i) preventing or treating schistosome infection; and/or (ii) killing or inhibiting schistosomiasis; and the amiRNA is a nucleic acid molecule that specifically inhibits the FTL gene and/or the SJCL gene.
In another preferred embodiment, the amiRNA is single-stranded or double-stranded.
In another preferred embodiment, the amiRNA is a nucleic acid inhibiting molecule according to the first aspect of the invention.
In another preferred embodiment, the amiRNA specifically inhibits transcription and/or translation of the FTL gene and/or the SJCL gene.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
FIG. 1 shows the result of identifying the pSIF recombinant vector.
FIG. 2 shows the results of qRT-PCR. Wherein, A, pSIF-SJCL4-miR and pSIF-SJCL4-trans are found to be capable of effectively inhibiting the expression of pCMV-SJCL; pSIF-SJFTL4-trans can effectively inhibit the expression of pCMV-ftl.
FIG. 3 shows pCMV6-SjCL co-transfected with pSIF-SjCL4-miR, pSIF-SjCL4-trans for 48 hours and 72 hours, respectively, and Wertern blot for detecting DDK tag expression, with β -actin as a control.
FIG. 4 shows the in vitro effect of recombinant lentivirus-mediated miRNA on killing schistosoma japonicum schistosomulum. Wherein, A: a control group; b: pSIF-FTL4-trans lentivirus treated group; c: pSIF-SjCL4-miR lentivirus-treated group; d: pSIF-SjCL4-trans lentivirus treated group.
Detailed Description
After extensive and intensive research, a large number of nucleotide sequences are screened, and it is found for the first time that certain specific nucleic acid inhibiting molecules (amiRNA) naturally have certain or high killing or inhibiting capability on schistosoma japonicum (especially schistosoma japonicum). Therefore, the amiRNA introduced into the specific schistosoma japonicum of the present invention can effectively inhibit or kill the schistosoma japonicum (especially the schistosoma japonicum). On this basis, the inventors have completed the present invention.
Experiments show that a plurality of amiRNAs are designed aiming at FTL and/or SJCL genes of schistosome, and the amiRNAs designed aiming at the target genes are unexpectedly found to be capable of killing or inhibiting the schistosome efficiently, and the inhibition rate can be as high as 26%.
FTL and SJCL genes or proteins thereof
The valve structure protein FTL is a schistosome epilemma protein.
In the invention, the FTL protein can be used as a target protein of a schistosome vaccine through quantitative proteomics analysis. The secretory protein SJCL3 is an intestinal enzyme protein of the schistosoma japonicum, and the research of the invention finds that the secretory protein has close relation with the survival and reproduction of the schistosoma japonicum in a human body and can also be used as a potential vaccine target protein of the schistosoma japonicum.
Genebank accession number of schistosome FTL gene: FN318171.1, genebank accession number of the schistosome SJCL gene: FN 319073.1.
amiRNA and precursors thereof
The invention provides a amiRNA which can effectively inhibit or kill the schistosoma japonicum, in particular the schistosoma japonicum.
In the invention, a nucleic acid inhibiting molecule is provided, wherein the nucleic acid inhibiting molecule is single-stranded or double-stranded amiRNA, and the 5 'end to the 3' end of a sense strand of the nucleic acid inhibiting molecule has a structure shown in a formula I:
Z0-Z1-Z2(formula I)
Wherein,
Z0a, T, C, G or none;
Z1is as set forth in SEQ ID No.: 1. 3, or 5; and
Z2a, T, C, G or none.
In a preferred embodiment, the amiRNA sequence of formula I is selected from the group consisting of: SEQ ID No.: 1. 3, 5, or a combination thereof.
The invention also provides a single-stranded nucleic acid molecule, wherein the sequence of the nucleic acid molecule is shown as any one of SEQ ID No. 1, 3 and 5 or a sequence completely complementary with the sequence.
As used herein, the terms "amiRNA" and "miRNA" are used interchangeably to refer to a class of artificial small RNA (amiRNA) that is designed based on the principle of generation and action of natural miRNA, and is a small RNA molecule targeting one or more specific genes, which can efficiently and specifically inhibit gene expression and can be processed from a transcript that can form an amiRNA precursor. Mature amiRNAs typically have 16-50 nucleotides (nt), preferably 16-30nt, more preferably 16-25nt, although amiRNA molecules with other numbers of nucleotides are not excluded. amiRNA is commonly detected by Northern blotting. The efficacy of RNA silencing depends not only on the nature of the amiRNA, but also on the local structure of the target mRNA. Therefore, the invention designs 4 pieces of amiRNA aiming at the schistosoma japonicum epidermal membrane protein FTL and the intestinal enzyme protein SJCL, and the amiRNA with the best inhibition effect is obtained by screening.
As used herein, "isolated" refers to a substance that is separated from its original environment (which, if it is a natural substance, is the natural environment). If the polynucleotide or polypeptide in its native state in a living cell is not isolated or purified, the same polynucleotide or polypeptide is isolated or purified if it is separated from other substances coexisting in its native state.
It is worth mentioning that amirnas are typically produced by mimicking the mechanism of miRNA production, and such amirnas can be processed from Precursor RNA (prefrsor RNA, Pre-RNA). The precursor RNA can fold into a stable stem-loop (hairpin) structure, which is typically between 50-100bp in length. The precursor RNA can be folded into a stable stem-loop structure comprising two substantially complementary sequences on both sides of the stem-loop structure. The precursor RNA may be natural or synthetic.
The precursor RNA can be cleaved to produce an amiRNA that can be substantially complementary to at least a portion of the sequence of the mRNA encoding the gene. As used herein, "substantially complementary" means that the sequences of nucleotides are sufficiently complementary to interact in a predictable manner, such as to form secondary structures (e.g., stem-loop structures). Typically, two "substantially complementary" nucleotide sequences are complementary to each other for at least 70% of the nucleotides; preferably, at least 80% of the nucleotides are complementary; more preferably, at least 90% of the nucleotides are complementary; further preferably, at least 95% of the nucleotides are complementary; such as 98%, 99% or 100%. Generally, two sufficiently complementary molecules may have up to 40 mismatched nucleotides between them; preferably, there are up to 30 mismatched nucleotides; more preferably, there are up to 20 mismatched nucleotides; further preferred, there are up to 10 mismatched nucleotides, such as 1, 2,3, 4, 5, 8, 11 mismatched nucleotides.
As used herein, a "stem-loop" structure, also referred to as a "hairpin" structure, refers to a nucleotide molecule that can form a secondary structure comprising a double-stranded region (stem) formed by two regions (on the same molecule) of the nucleotide molecule flanking a double-stranded portion; it also includes at least one "loop" structure comprising non-complementary nucleotide molecules, i.e., a single-stranded region. The double-stranded portion of the nucleotide remains double-stranded even if the two regions of the nucleotide molecule are not completely complementary. For example, an insertion, deletion, substitution, etc., can result in the non-complementarity of a small region or the small region itself forming a stem-loop structure or other form of secondary structure, however, the two regions can still be substantially complementary and interact in a predictable manner to form a double-stranded region of the stem-loop structure. The stem-loop structure is well known to those skilled in the art, and usually, after obtaining a nucleic acid having a nucleotide sequence of a primary structure, those skilled in the art can determine whether the nucleic acid can form a stem-loop structure.
The amiRNA of the invention refers to: the microRNA for inhibiting or killing schistosoma amiRNA family comprises: amiRNA for inhibiting or killing schistosoma or modified amiRNA derivatives for inhibiting or killing schistosoma.
In a preferred embodiment of the invention, the nucleotide sequence of the double-stranded amiRNA for inhibiting or killing schistosoma is shown in SEQ ID NO. 1-6. Particularly preferred is any one of the sequences of SEQ ID No. 1, 3, 5 or a combination thereof.
The invention also includes amiRNA variants and derivatives. In addition, the amiRNA derivatives in the broad sense may also include amiRNA variants. One of ordinary skill in the art can modify the amirnas of the present invention using general methods, including but not limited to: methylation modification, alkyl modification, glycosylation modification (such as 2-methoxy-glycosyl modification, alkyl-glycosyl modification, sugar ring modification and the like), nucleic acid modification, peptide segment modification, lipid modification, halogen modification, nucleic acid modification (such as 'TT' modification) and the like.
Polynucleotide constructs
According to the amiRNA sequences provided by the invention, polynucleotide constructs can be designed which, after introduction, can be processed to amirnas which affect the expression of the corresponding mrnas, i.e. the polynucleotide constructs are capable of up-regulating the amount of the corresponding amirnas in vivo. Thus, the present invention provides an isolated polynucleotide (construct) that can be transcribed by human cells into a precursor amiRNA that can be cleaved by human cells and expressed into the amiRNA.
In a preferred embodiment of the present invention, the polynucleotide construct comprises a structure of formula II:
Seqforward direction-X-SeqReverse direction
Formula II
In the formula II, the reaction mixture is shown in the specification,
Seqforward directionIs a nucleotide sequence capable of expressing the amiRNA in cells, SeqReverse directionIs and SeqForward directionA substantially complementary nucleotide sequence; alternatively, SeqReverse directionA nucleotide sequence capable of expressing the miRNA in cells, SeqForward directionIs and SeqForward directionA substantially complementary nucleotide sequence; x is at SeqForward directionAnd SeqReverse directionA spacer sequence therebetween, and the spacer sequence and SeqForward directionAnd SeqReverse directionAre not complementary;
the structure of formula I, when transferred into a cell, forms a secondary structure of formula III:
in formula III, SeqForward direction、SeqReverse directionAnd X is as defined above;
i is expressed in SeqForward directionAnd SeqReverse directionThe base complementary pairing relationship is formed between the two.
Typically, the polynucleotide construct is located on an expression vector. Thus, the invention also includes a vector comprising said amiRNA, or said polynucleotide construct. The expression vector usually further contains a promoter, an origin of replication, and/or a marker gene. Methods well known to those skilled in the art can be used to construct the expression vectors required by the present invention. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The expression vector preferably comprises one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells, such as kanamycin, gentamicin, hygromycin, ampicillin resistance.
Pharmaceutical compositions and methods of administration
The invention provides a composition for killing or inhibiting schistosome, which comprises a pharmaceutical composition or an immune composition.
The composition is further illustrated by way of example as a pharmaceutical composition comprising;
(a) a nucleic acid inhibitory molecule according to the first aspect of the invention, and/or a precursor sequence according to the second aspect of the invention; and
(b) a pharmaceutically acceptable carrier.
In a preferred embodiment, the composition further comprises (c) other drugs for killing or inhibiting schistosoma.
In a preferred embodiment, the composition further comprises a viral vector.
As used herein, the term "effective amount" or "effective dose" refers to an amount that produces a function or activity in, and is acceptable to, a human and/or an animal.
As used herein, an ingredient of the term "pharmaceutically acceptable" is one that is suitable for use in humans and/or mammals without undue adverse side effects (such as toxicity, irritation, and allergic response), i.e., at a reasonable benefit/risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents.
The pharmaceutical composition of the present invention contains a safe and effective amount of the active ingredient of the present invention and a pharmaceutically acceptable carrier. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical composition of the invention can be prepared into injections, oral preparations (tablets, capsules, oral liquids), transdermal agents and sustained-release agents. For example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition is preferably manufactured under sterile conditions.
The effective amount of the active ingredient of the present invention may vary depending on the mode of administration and the severity of the disease to be treated, etc. The selection of a preferred effective amount can be determined by one of ordinary skill in the art based on a variety of factors (e.g., by clinical trials). Such factors include, but are not limited to: pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the weight of the patient, the immune status of the patient, the route of administration, and the like. In general, satisfactory results are obtained when the active ingredient of the invention is administered at a daily dose of about 0.00001mg to 50mg per kg of animal body weight (preferably 0.0001mg to 10mg per kg of animal body weight). For example, divided doses may be administered several times per day, or the dose may be proportionally reduced, as may be required by the urgency of the condition being treated.
The pharmaceutically acceptable carrier of the present invention includes (but is not limited to): water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptidic substances, cellulose, nanogels, or combinations thereof. The choice of carrier should be matched with the mode of administration, which is well known to those skilled in the art.
Typically, in the present invention, the nucleic acid inhibiting molecule of the present invention or a precursor thereof may be used to prepare a pharmaceutical composition for (i) preventing or treating schistosome infection; and/or (ii) killing or inhibiting schistosoma (preferably schistosomulum). In addition, the medicine composition of the present invention may also contain other medicine for killing or inhibiting schistosome.
Method for screening candidate compound for inhibiting or killing schistosome
In the present invention, there is also provided a method for screening a candidate compound for inhibiting or killing schistosoma, comprising the steps of:
(a) culturing cells expressing FTL protein and/or SJCL protein in a culture system for a time T1 in the presence of a test compound in a test group, and detecting the expression amount E1 and/or activity a1 of FTL protein and/or SJCL protein in the culture system of the test group;
and testing the expression level E2 and/or activity A2 of FTL protein and/or SJCL protein in the culture system of a control group in the absence of the test compound and under otherwise identical conditions; and
(b) comparing the expression level E1 and/or activity A1 of the FTL protein and/or SJCL protein detected in the previous step with the expression level E2 and/or activity A2 of the FTL protein and/or SJCL protein, thereby determining whether the test compound is a candidate compound for inhibiting or killing schistosoma,
wherein if E1 is significantly lower than E2, and/or a1 is significantly lower than a2, then the test compound is a candidate compound for inhibiting or killing schistosoma.
The main advantages of the invention include:
(1) before the present invention, nucleic acids which kill or inhibit schistosoma were never found. The invention discovers for the first time that the amiRNA designed aiming at the FTL and/or SJCL gene of the schistosome can efficiently inhibit or kill the schistosome, and the inhibition rate of the schistosome can reach 26 percent.
(2) The invention discovers that the FTL and/or SJCL may play an important role in physiological processes of growth, development and the like of the schistosoma japonicum for the first time.
(3) The invention takes FTL and/or SJCL as targets for the first time, screens and identifies a plurality of miRNAs related to the 2 genes, and discusses the possible biological functions of the miRNAs.
The invention is further illustrated with reference to specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight. The materials and equipment used in the examples of the present invention are commercially available unless otherwise specified.
Example 1 design of schistosome amiRNA
The SjCL gene and the FTL gene can play important roles in physiological processes such as the growth and the development of schistosoma japonicum by screening a large number of genes, so that the invention designs four amiRNAs aiming at the SjCL gene (accession number: FN319073.1) and the FTL gene (accession number: FN318171.1) of the schistosoma japonicum, and the four amiRNAs of the SjCL gene are named as amiSJCL1, amiSJCL2, amiSJCL3 and amiSJCL4 respectively, and the amiSJCL1, amiSJCL2, amiSJCL3 and amiSJCL4 are respectively located in 123bp, 283bp, 455bp and 674bp segments of the SJCL gene. The four amiRNAs of the FTL gene are named as amiFTL1, amiFTL2, amiFTL3 and amiFTL4 respectively, and the amiFTL1, amiFTL2, amiFTL3 and amiFTL4 are located in 211bp, 276bp, 354bp and 454bp segments of the FTL gene respectively. amiRNA is double-stranded by annealing two single strands, the top strand and the bottom strand.
The annealing system is as follows
The reagents are mixed evenly and treated for 4min at 95 ℃, and cooled for 10min at room temperature. The double strand formed by annealing is successfully obtained.
Through a large amount of screening, amiRNA with excellent inhibition effect is unexpectedly obtained.
SjCL gene screening obtains SJCL2 and SJCL4 with good inhibition effect. The results of qPCR and Wertern blot showed that among the four amirnas SJCL1-4, SJCL2 and SJCL4 resulted in a decrease in the mRNA and protein levels of SJCL, with statistical significance. SJCL2 is annealed and synthesized by SJCL2-TOP and SJCL2-bottom two chains respectively. SJCL4 was annealed and synthesized from SJCL4-TOP, SJCL4-Bottom two strands, respectively.
In addition, the qPCR result shows that only FTL4 of FTL1-4 can cause the mRNA level of FTL gene to be reduced, and the result has statistical significance. Wertern blot detects the effect of FTL4 recombinant plasmid on expression of FTL at protein level, and expression of FTL is obviously inhibited. FTL4 was synthesized by two strand annealing of FTL4-TOP, FTL 4-bottom.
Specific information of the screened amiRNAs is shown in Table 1.
TABLE 1
| Name of amiRNA | Sequence information (5'-3') | SEQ ID NO.: |
| FTL4-Top (sense strand) | TAGGACGAGGGAATTGAAA | 1 |
| FTL4-Bottom (antisense strand) | TTTCAATTCCCTCGTCCTA | 2 |
| SJCL2-Top (sense chain) | GAGCATAAGTGCCTATCAA | 3 |
| SJCL2-Bottom (antisense strand) | TTGATAGGCACTTATGCTC | 4 |
| SJCL4–Top(sense chain) | ATGTTCGTCAACAAGGGTA | 5 |
| SJCL4-Bottom (antisense strand) | TACCCTTGTTGACGAACAT | 6 |
Example 2 construction of recombinant lentiviral vector pSIF-H1 corresponding to amiRNA for Schistosoma japonicum ftl and sjcl Gene
Since only one fragment was designed, there was a possibility that the recombinant vector did not function, amiFTL4, amiSJCL2 and amiSJCL4 were each designed to have two fragments with different lengths. The short fragment FTL4-miR in the amiFTL4 fragments only contains short amiRNA fragments, the long fragment contains amiRNA, and also contains a trans element named FTL4-trans, wherein the trans element represents that a transcription initiation element (so as to drive transcription) is added at the upstream, the miR represents that the short fragment only contains amiRNA, the short fragment of the amiSJCL2 is SJCL2-miR, the long fragment is SJCL2-trans, the short fragment of SJCL4 is SJCL4-miR, the long fragment is SJCL4-trans, the FTL4, the SJCL2 and the SJCL4 fragments contain BamH I enzyme cutting sites at the upstream, and the EcoR I enzyme cutting sites at the downstream. After PCR amplification, a target band is recovered from 1% agarose gel, and after purification, double digestion is carried out to obtain a target fragment containing BamHI and EcoRI at the 5 'end and the 3' end respectively, meanwhile, a conventional vector pSIF1-H1 (sold in the market) is subjected to double digestion to obtain a linearized vector, and T4DNA ligase is used for connecting the sticky ends of the products. The ligation product was converted according to a conventional method. After the DNA of the small quality-improved particles is identified by methods such as enzyme digestion and PCR, six positive recombinant plasmids are obtained and named as pSIF-SjCL2-miR, pSIF-SjCL2-trans, pSIF-SjCL4-miR, pSIF-SjCL4-trans, pSIF-FTL4-miR and pSIF-FTL4-trans respectively, and the results are shown in figure 1 by double enzyme digestion identification of BamH I and Xho I.
The enzyme digestion and sequencing identification result shows that pSIF-SjCL2-miR, pSIF-SjCL2-trans, pSIF-SjCL4-miR, pSIF-SjCL4-trans, pSIF-FTL4-miR and pSIF-FTL4-trans recombinant lentiviral vectors are successfully constructed.
EXAMPLE 3 screening of efficient pSIF-recombinant vectors
3.1 method
293T cell culture: the previously constructed SJCL gene overexpression vector pCMV6-SJCL (purchased from Origene Co.) was co-transfected into 293T cells with pSIF-SjCL4-miR and pSIF-SjCL4-trans, and cultured for 48 hours and 72 hours, respectively; the previously constructed FTL gene overexpression vectors pCMV-FTL (purchased from Origene Co.), pSIF-SJCL4-miR and pSIF-SJCL4-trans were co-transfected into 293T cells, and cultured for 48 hours and 72 hours, respectively.
3.2 transcript and protein level identification
3.2.1 results of real-time fluorescent quantitative PCR (qPCR)
qPCR (quantitative polymerase chain reaction) verification and selection of fragments with good effects, and statistics of PCR results by Graphad-prism5 software show that pSIF-SJCL4-miR and pSIF-SJCL4-trans can effectively interfere with expression of pCMV-SjCL, as shown in FIG. 2A, the P value is 0.0005, and the results have statistical significance. Moreover, pSIF-FTL4-trans can effectively interfere the expression of pCMV-FTL, the P value is less than 0.0001, and the result has statistical significance; however, pSIF-FTL4-miRNA may be due to the fact that the fragments are short and lack some necessary elements, thereby influencing the interference effect. (FIG. 2B).
3.2.2 Wertern blob detection results
The effect of pSIF-FTL4-trans lentivirus on the expression of FTL at the protein level and the effect of pSIF-SJCL4-miR and pSIF-SJCL4-trans lentivirus on the expression of SJCL at the protein level were examined separately, and Wertern blot examined the expression of DDK tag, beta-actin as a control. The results showed that after 48h, pSIF-FTL4-trans lentivirus inhibited FTL protein expression, and pSIF-SJCL4-miR and pSIF-SJCL4-trans lentivirus inhibited SJCL protein expression, as shown in FIG. 3.
Western blot experiment results show that the pSIF-FTL4-trans lentivirus has obvious inhibition effect on expression of Schistosoma japonicum epidermal FTL protein, and the pSIF-SJCL4-miR and pSIF-SJCL4-trans lentivirus have obvious inhibition effect on expression of Schistosoma japonicum SJCL protein.
Example 4 Effect of killing schistosoma japonicum schistosomulum in vitro
4.1 in vitro preparation of recombinant lentiviruses
packaging of pSIF1 lentivirus:
cells were plated at a.10cm to a confluency of 50% -70%.
b. 20uL (10ug) of the packaging plasmid was mixed with 2ug (2-25uL depending on concentration) and the diluent plasmid mixture was then added to 400uL of DMEM.
c. Adding 40uL of X-treme, and standing at room temperature for 20-30min
d. The 293T cell plates were washed with anti-free DMEM, and then 9mL of anti-free DMEM medium containing 2% serum was added.
e. Adding the mixed compound obtained in the steps 2 and 3, and culturing overnight.
f. The medium containing fresh 2% serum and antibiotics was replaced and incubated in a carbon dioxide incubator at 37 ℃ for 48 hours.
g. Supernatants were collected at 24, 48, and 62 hours of transfection. It may also be collected only once after 48 hours of transfection.
h. The entire 10mL of culture medium containing pseudovirus was collected in a 15mL sterile medium, centrifuged at 3000rpm for 5min at room temperature, after which the supernatant was filtered through a 0.45um PVDF membrane and dispensed into 1.5mL centrifuge tubes.
4.2 in vitro killing Effect
The schistosoma japonicum schistosomulum was observed microscopically after adding the blank control group to freshly prepared schistosoma japonicum schistosomulum for 48 hours of co-culture in pSIF-FTL4-trans low dose group (200uL), high dose group (400uL), pSIF-SJCL4-miR, pSIF-SJCL4-trans low dose group (200uL), high dose group (400uL), 200uL pSIF-H1 empty vector negative control group (200uL), respectively. As shown in FIGS. 4A-4D and Table 2, the number of schistosoma japonicum schistosomulum deaths of the pSIF-FTL4-trans, pSIF-SjCL4-miR and pSIF-SjCL 4-trans-treated groups were significantly increased and dose-dependent, as compared with the schistosomulum blank control group and the negative control group. Furthermore, whether or not a transcription initiation element (trans) is added, there is no significant effect on correcting mortality.
TABLE 2
| Recombinant lentivirus | 48h in vitro schistosoma japonicum juvenile insect corrected mortality (%) |
| pSIF-FTL4-trans (Low dose group) | 9.4 |
| pSIF-FTL4-trans (high dose group) | 26 |
| pSIF-SjCL4-miR (Low dose group) | 11.5 |
| pSIF-SjCL4-miR (high dose group) | 22.8 |
| pSIF-SjCL4-trans (Low dose group) | 8.8 |
| pSIF-SjCL4-trans (high dose group) | 21.5 |
| Blank control group | 0 |
| Negative control group | 5.8 |
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Sequence listing
<110> Chinese disease prevention and control center parasitic disease prevention and control institute
<120> micro RNA and application thereof in preparation of anti-schistosoma japonicum infection preparation
<130> P2017-0109
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<170> PatentIn version 3.5
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<213> Artificial sequence
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<213> Artificial sequence
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gagcataagt gcctatcaa 19
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Claims (13)
1. A nucleic acid inhibiting molecule, wherein the nucleic acid inhibiting molecule is a single-stranded or double-stranded amiRNA, and the structure of the 5 'end to the 3' end of the sense strand of the nucleic acid inhibiting molecule is shown as formula I:
Z0-Z1-Z2 (I)
wherein,
Z0a, T, C, G or none;
Z1is as set forth in SEQ ID No.: 1. 3, or 5; and
Z2a, T, C, G or none;
the formula I structure is selected from the group consisting of: SEQ ID No.: 1. 3, 5, or a combination thereof.
2. A precursor sequence for producing the nucleic acid inhibitory molecule of claim 1, wherein the 5 'to 3' end of the sense strand of the precursor sequence has the structure of formula II:
in the formula,
b1 is a desired first ribonucleotide sequence, wherein said first ribonucleotide sequence comprises the form of an amiRNA sequence; the sequence of B1 is selected from the group consisting of: SEQ ID No.: 1. 3, 5, or a combination thereof;
b2 is a sequence complementary to B1, and B2 is not complementary to C;
c is a stem-loop structure.
3. A polynucleotide which is transcribed by a host to form a precursor sequence according to claim 2.
4. An expression vector comprising the precursor sequence of claim 2 or the polynucleotide of claim 3.
5. A composition for killing or inhibiting schistosomiasis, which comprises:
(a) the nucleic acid inhibitory molecule of claim 1, and/or the precursor sequence of claim 2; and
(b) a pharmaceutically acceptable carrier.
6. The composition of claim 5, wherein the composition further comprises a viral vector.
7. The composition of claim 5, wherein the composition further comprises (c) other agents that kill or inhibit schistosomiasis.
8. Use of the nucleic acid inhibitory molecule of claim 1 or the precursor sequence of claim 2, for the preparation of a composition or formulation for (i) the prevention or treatment of schistosome infection; and/or (ii) killing or inhibiting schistosomiasis.
9. The use of claim 8, wherein the composition or formulation is also for killing or inhibiting schistosomulum.
10. The use of claim 8, wherein the composition or formulation further comprises other agents that kill or inhibit schistosomiasis.
11. A method for killing or inhibiting schistosome in vitro is characterized by comprising the following steps: culturing schistosoma japonicum in the presence of the nucleic acid inhibitory molecule of claim 1 or the precursor sequence of claim 2 or the composition of claim 5 to kill or inhibit schistosoma japonicum.
12. The method of claim 11, wherein the schistosome is selected from the group consisting of: schistosoma japonicum, Schistosoma mansoni, Schistosoma japonicum, or their combination.
13. Use of an amiRNA, or a precursor thereof, or an expression vector thereof, for the preparation of a medicament for (i) preventing or treating schistosome infection; and/or (ii) killing or inhibiting schistosomiasis; and the amiRNA is a nucleic acid molecule that specifically inhibits the FTL gene and/or the SJCL gene, and the amiRNA is the nucleic acid inhibitory molecule of claim 1.
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