CN104962560A - Nucleic acid aptamer for detecting schistosoma japonicum eggs and application of nucleic acid aptamer to manufacturing detection preparations - Google Patents
Nucleic acid aptamer for detecting schistosoma japonicum eggs and application of nucleic acid aptamer to manufacturing detection preparations Download PDFInfo
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Abstract
The invention discloses nucleic acid aptamer for detecting schistosoma japonicum eggs and application of the nucleic acid aptamer to manufacturing detection preparations. Compared with the prior art, the nucleic acid aptamer and the application have the advantages that the nucleic acid aptamer which is obtained by means of screening is high in affinity and specificity as compared with protein antibodies, is free of immunogenicity, can be chemically synthesized in vitro, is low in molecular weight, can modify and substitute different positions, has stable sequences and is easy to store and convenient to mark; operation is simple and speedy when the nucleic acid aptamer is used for detecting the schistosoma japonicum eggs; the nucleic acid aptamer is low in synthesis cost as compared with the manufacturing costs of the antibodies, and is short in period and good in reproducibility.
Description
Technical field
The present invention relates to a kind of aptamer and application thereof, particularly relate to a kind of can be used for SCHISTOSOMA JAPONICUM and clinical sample tissue detection aptamer and the application method of preparation detection reagent.
Background technology
Schistosomicide is the healthy serious infectious diseases with hindering socio-economic development of a kind of serious harm human body always.Schistosoma japonicum adult Male-female worm pairing resides in host's mesenteric vein, and every bar female worm lays eggs about 300 ~ 3000 every day, and the worm's ovum produced is deposited in intestines wall thin vessels, and part enters liver with blood flow.Reaching maturity the schistosome ovum miracidium secretory product of (after output about 11 days) can through chorion, destroy vessel wall, and cause surrounding tissue inflammation downright bad, because of wriggling, the intra-abdominal pressure increase of intestines, cause necrotic tissue to enteric cavity diabrosis, a small amount of worm's ovum falls into enteric cavity with diabrosis tissue, excretes with ight soil.
The links such as schistosome ovum develops in schistosomicide, pathophoresis is popular are significant, be mainly manifested in: one is that schistosome ovum chorion and its secretory product all can excite host to produce strong immune response and form egg granulomas synergentic and send out and and should produce the clinical symptom such as Chronic Liver (intestines) fibrosis subsequently, and schistosome ovum is acknowledged as major virulent factor.Two is after schistosome ovum is discharged to the external world from ight soil, in the breeding environment having oncomelania, miracidium in schistosome ovum hatches out, pierce in Vector factors-oncomelania body, its monogony Life Stages is completed in oncomelania body, develop into cercaria, cercaria can grow adult and output worm's ovum again infect people and other Mammals in water after in human body (or other Mammals), and schistosome ovum is crucial in effect in schistosomiasis propagation Epidemic Links of maintaining.The main path of current diagnosis schistosoma japonicum infection comprise based on schistosome ovum antibody (or antigen) Serologic detection and from ight soil, detect the etiology method of schistosome ovum.
Because obtain the gold standard (Gold Standard) that schistosome ovum is acknowledged as schistosomiasis diagnosis from ight soil, the etiology method detecting schistosome ovum at present has Kato-katz Microscopical Method For Detection (WHO recommend method) and collection egg hatch method.Both shortcomings have length consuming time (the former is overnight, and the latter needs more than 4 hours), and loss is high.Present stage schistosomicide prevention and control need high short, succinct diagnostic method responsive, special, consuming time.
Aptamer (nucleic acid aptamer) be screen from the random single chain nucleic acid library of synthetic can the oligonucleotide of 20-50 base of high-affinity binding target molecule, comprise DNA aptamer and RNA is fit.Aptamers has the features such as target molecule is wide, avidity is high, high specificity because of the diversity of its structure, meanwhile, compare conventional antibodies, and little, the easy transformation of adaptor molecules amount is modified, easy to prepare and non-immunogenicity.Therefore, aptamers presents wide application prospect in fundamental research, clinical diagnosis, drug development etc.In recent years for the widely used SELEX aptamer triage techniques (CELL-SELEX) based on intact cell of investigators can not need to obtain in advance the relevant information of cell surface target molecules and the aptamer keeping surface of cell membrane albumen native physiological state directly to screen acquisition and Cell binding to be measured, and the target molecule of these aptamers to the cell surface that it combines can be utilized to characterize and detect, contribute to finding new biomarker.Aptamer has been widely used in identifying various target molecules, finds new biomarker.Aptamer target is in extensive range, also good avidity is had to parasite, trypanosome in aptamer energy specific recognition host blood, leishmania, the parasitic agent such as plasmodium, for opening up new parasite detection method and therapeutic strategy provides new approach.
By Cell-SELEX technology, we successfully filter out can the aptamers of high specific and schistosome ovum specific binding, because aptamer has the following advantages: in-vitro screening and cycle short, easy synthesis and carry out that difference between chemically modified, different batches is little, good stability, can normal temperature transport, the little and tissue penetration fast of immunogenicity.Therefore, the aptamer screening of schistosome ovum specific binding will set up the sick diagnostic method of schistosomiasis for next step provides foundation; And for development schistosomicide targeted therapy and Drug efficacy evaluation means provide new thinking in the future.
Summary of the invention
Object of the present invention provides a kind of high degree of specificity, stability, can be used for the application method of the aptamer of pattern detection in the detection of schistosome ovum and body thereof and preparation detection reagent thereof.
Detect an aptamer for SCHISTOSOMA JAPONICUM, arbitrary in following sequence:
Sequence 1: aptamer LC6:
5’-ACGCTCGGATGCCACTACAG
TGATAGAATGGTAGTTGAGTAGTTTGTGTATATGTGGGGCCTCATGGACGTGCTGGTGAC-3’
Sequence 2: aptamer LC15:
5’-ACGCTCGGATGCCACTACAG
TGAGATATAAAGGGCAGAAATAAGTAGGGGCTCATGGACGTGCTGGTGAC-3’。
The aptamer of described detection SCHISTOSOMA JAPONICUM, can also be undertaken modifying and transforming by aptamer when keeping the conserved nucleotide sequence of LC6, LC15 underscore part constant.
Specifically comprise any one in following three kinds:
A) conserved nucleotide sequence of aptamer LC6, LC15 underscore part is constant, and the Nucleotide at constant sequence two ends is deleted;
B) conserved nucleotide sequence of aptamer LC6, LC15 underscore part is constant, and the base of constant sequence two terminal nucleotide carries out artificial bases's replacement;
C) fluorescent substance, radioactive substance, therapeutic substance, vitamin H or enzyme labelling in aptamer LC6, LC15 connection.
The application method of the aptamer of described detection SCHISTOSOMA JAPONICUM, by the aptamer of described detection SCHISTOSOMA JAPONICUM for the preparation of the preparation detecting SCHISTOSOMA JAPONICUM.
On the other hand, the present invention screens aptamer LC6, the method for LC15, comprises the steps: to screen nucleic acid library with schistosome ovum, obtains the aptamer with schistosome ovum specific combination; Described nucleic acid library is the library of the random nucleotides of strand;
(1) random single-stranded DNA banks shown in following sequence and primer is synthesized:
Random nucleic acid library:
5’-ACGCTCGGATGCCACTACAG(45N)CTCATGGACGTGCTGGTGAC-3’
Upstream primer: 5 '-fluorescein isothiocyanate-ACG CTC GGA TGC CAC TAC AG--3 ';
Downstream primer: 5 '-vitamin H-GTC ACCAGC ACG TCC ATG AG-3 ';
(2) just screen: above-mentioned random nucleic acid library and schistosome ovum are hatched; After having hatched, collect schistosome ovum; 95 DEG C of heat denatured are separated the aptamers be combined with schistosome ovum, are the nucleic acid library of first time screening;
(3) nucleic acid library of pcr amplification first time screening: carrying out standard PCR amplification by screening the library obtained in step (2), obtaining amplified production;
(4) DNA single chain library is prepared: with the pcr amplification product in Streptavidin dextran microspheres separating bio element markers step (3); Then utilize 2M sodium hydroxide that double-stranded DNA sex change is unwind, collect the DNA single chain library of marked by fluorescein isothiocyanate;
(5) instead sieve: the DNA library obtain step (4) and clonorchis egg are hatched, hatched rear collection clonorchis egg hatch after supernatant, namely exclude the nucleotide sequence be combined with clonorchis egg;
(6) just sieve: made supernatant liquor and schistosome ovum in step (5) are hatched, elution of bound, in the high-affinity aptamers storehouse on schistosome ovum surface, is the nucleic acid library of programmed screening;
(7) aptamer Cycle Screening: the amplified production obtained with the nucleic acid library step of replacing (3) of step (6) gained, and the screening process of repeating step (4) ~ (6), until screening obtains the nucleic acid library strong with schistosome ovum binding ability;
(8) the final nucleic acid library that screening obtains is carried out high-flux sequence, determine aptamer.
Compared with prior art, the invention has the advantages that: the present invention has the avidity higher than protein antibodies and specificity by screening the aptamer obtained; Non-immunogenicity; Can synthesize by iii vitro chemical, molecular weight is little, can modify different sites and replace, and sequence is stablized, and is easy to preserve; Be convenient to the advantages such as mark.Adopt aptamer of the present invention to carry out schistosome ovum when detecting, operate more simple, rapid, and the synthesis cost of aptamer of the present invention comparatively antibody preparation cost is low, and the cycle is short, favorable reproducibility.
Accompanying drawing explanation
Fig. 1 is the change procedure of Real-time PCR melting curve: peak shape represent primary libraries, the 1st take turns screening product, the 7th take turns screening product, the 9th take turns screening product by amplification after melting curve;
Fig. 2 is aptamer (LC15, LC6) and the schistosome ovum of mark fluorescein isothiocyanate and instead sieves clonorchis egg binding ability laser confocal microscope and analyze;
In fig. 2, first three width figure is respectively negative control aptamer (start library), LC6, LC15 and the displaying of schistosome ovum in conjunction with situation, and rear two width figure are the displaying in conjunction with situation of LC6, LC15 and clonorchis egg respectively; Obviously can find out that in laser co-focusing picture these two Aptamer of LC6 and LC15 can be good at being enriched in schistosome ovum surface, visible green fluorescence, image results shows, the fluorescence intensity of LC6 and LC15 is apparently higher than the fluorescence intensity of library group, and be not combined with clonorchis egg, there is no fluorescence (rear two width figure), illustrate that there is good specificity;
Fig. 3 is the laser confocal microscope analysis that the Aptamer that marks of indoles cyanine type dye (Cy5) combines schistosome ovum in the sub-hepatic tissue section of Rabbits Infected With Schistosoma Japonicum; A, c: negative control aptamer (start library) b:LC6, d:LC15;
Fig. 4 is the laser confocal microscope analysis that LC6, LC15 combine roundworm worm's ovum, and this figure is without any fluorescent appear.
Embodiment
Following embodiment is convenient to better understand the present invention, but is not limited to the present invention.Experimental technique in following examples if no special instructions, is ordinary method.Experiment material used in subordinate's embodiment if no special instructions, is to buy from routine biochemistry reagent shop and obtains.
Worm's ovum is originated:
This experiment screening two kinds of worm's ovums (schistosome ovum and clonorchis egg) used provide by country of Prevention & Control Station of Parasitic Disease, China Diseases Prevention & C parasitosis Reference Lab.
The present invention utilizes the in-vitro screening technology SELEX technology of aptamer, with schistosome ovum for just to sieve target, is the anti-target that sieves with clonorchis egg, from the random oligomerization ssDNA library of external synthesis, filter out the aptamer with described schistosome ovum specific combination.
Embodiment 1: the screening of schistosome ovum specific nucleic acid aptamers
(1) design of nucleic acid library used and primer
Random nucleic acid library:
5’-ACGCTCGGATGCCACTACAG(45N)CTCATGGACGTGCTGGTGAC-3’
Upstream primer: 5 '-fluorescein isothiocyanate-ACG CTC GGA TGC CAC TAC AG-3 ';
Downstream primer: 5 '-vitamin H-GTC ACCAGC ACG TCC ATG AG-3 ';
(2) just screen
2.1 hatch: dissolve above-mentioned random nucleic acid library with binding buffer liquid (D-PBS, 5mM magnesium chloride), 95 DEG C of constant temperature sex change 5min, put into rapidly ice 10min; Then good with pre-treatment schistosome ovum hatches 1.5h at 4 DEG C of shaking tables.
2.2 dissociate: hatched rear centrifugal, and removed solution in centrifuge tube, hatch the schistosome ovum in rear centrifuge tube with lavation buffer solution (PBS, containing the glucose of 0.45%, 5mM magnesium chloride) centrifuge washing; Get supernatant after 95 DEG C of heat denatured 10min, 5500rpm/s are centrifugal, the nucleotide sequence of separation and combination schistosome ovum, be the nucleic acid library of the first round screening of schistosome ovum.
(3) pcr amplification library is carried out: get in step 2 nucleic acid library of screening gained and carry out standard PCR amplification, upstream primer: 5 '-fluorescein isothiocyanate-ACG CTC GGA TGC CAC TAC AG--3 '; Downstream primer: 5 '-vitamin H-GTCACCAGC ACG TCC ATG AG-3 '; Amplification condition is: 95 DEG C, 3min; 95 DEG C, 30s; 59.4 DEG C, 30s, 72 DEG C, 30s, through suitable circulation wheel number, 72 DEG C, 5min.After first round screening, the nucleic acid library that the first round of the schistosome ovum of full income screens being carried out increasing in advance 10 circulations, is then that template increases in a large number again with amplified production.
(4) DNA single chain library is prepared: with the pcr amplification product in Streptavidin dextran microspheres separating bio element markers step (3); Then utilize 0.2M sodium hydroxide that double-stranded DNA sex change is unwind, collect the DNA single chain library of marked by fluorescein isothiocyanate;
(5) instead sieve: the DNA single chain library that step (4) is obtained with instead sieve worm's ovum clonorchis egg and hatches, then collection worm's ovum hatch after supernatant, namely exclude the nucleotide sequence with schistosome ovum non-specific binding;
(6) just sieve: made supernatant liquor and schistosome ovum in step (5) are hatched, wash-out retains the nucleotide sequence in conjunction with schistosome ovum, is the nucleic acid library of programmed screening;
(7) aptamer Cycle Screening: the amplified production obtained with the nucleic acid library step of replacing (2) of step (6) gained, and the screening process of repeating step (4) ~ (6), until screening obtains the nucleic acid library strong with target worm's ovum schistosome ovum binding ability;
(8) the final nucleic acid library that screening obtains is carried out high-flux sequence.
Embodiment 2: the screening of schistosome ovum specific nucleic acid aptamers
Real-time PCR is utilized to detect screening process, get primary libraries, the 1st respectively to take turns screening product, the 3rd and take turns screening product, the 7th and take turns screening product, the 9th and take turns screening product, the 13rd and take turns screening product and carry out PCR, judge to screen the specificity of product by the change of melting curve.
The object of this embodiment is for next step high-flux sequence (i.e. the step (8) of embodiment 1) is as selection gist.Have found by the experiment of embodiment 2 library can carrying out high-flux sequence is the 9th library of taking turns.
Embodiment 3: utilize confocal laser scanning microscope Aptamer with just to sieve worm's ovum and instead sieve worm's ovum in conjunction with situation
Get schistosome ovum and the clonorchis egg of about about 8000 respectively, by with marked by fluorescein isothiocyanate aptamer (LC15, LC6) and schistosome ovum and instead sieving clonorchis egg hatches 45min.Hatch complete after, centrifuge washing twice, each 2000r/s 3min, removes supernatant liquor, and adds 500 μ L washingss and conveniently sample observation.The sample taken a morsel carries out observation of taking pictures under being placed in laser confocal microscope, the results are shown in Figure 2.
Application testing in the rabbit hepatic tissue section of the aptamer that embodiment 4:5 '-indoles cyanine type dye (Cy5) marks after schistosomicide
(1) schistosomicide rabbit liver sample tissue is cut into slices after 60 DEG C of oven for baking 1h, be placed in the first cylinder dimethylbenzene 15min at once, then insert 15min in the second cylinder dimethylbenzene and dewax; Again the tissue slice dewaxed is positioned over 10min in dehydrated alcohol successively, 95% ethanol 5min, 70% ethanol 5min, 50% ethanol 5min; To being placed on TE buffer (pH 8.0) after distilled water profit tissue slice, the container filling repair liquid is put in section.After distilled water flushing, then PBS (0.01M, pH7.4) is used to soak 3 times, each 5min (that ensure first time immersion is the PBS newly joined).
By the tissue slice of antigen retrieval and the binding buffer solution incubated at room 60min containing 20%FBS and 1mg/ml herring sperm dna; The aptamer LC15 that washing Cy5 that is rear and 300pmol/L marks, LC6 or start library are in incubated at room 60min.After washing twice with lavation buffer solution, tissue slice is dried, and with 20% glycerine mounting, observe.The results are shown in Figure 3.This figure is a Binding experiment of the rabbit liver tissue slices of LC15, LC6 or start library and schistosomicide; Send red fluorescence in figure, and the hollow circle of ovalize is exactly colonize in the schistosome ovum in rabbit liver; Can see that in laser co-focusing picture fluorescence concentrates on the position of worm's ovum parasitism clearly, result illustrates that LC15, LC6 have the ability better identifying worm's ovum in tissue.
Claims (4)
1. detect an aptamer for SCHISTOSOMA JAPONICUM, it is characterized in that, arbitrary in following sequence:
Sequence 1: aptamer LC6:
5’-ACGCTCGGATGCCACTACAG
TGATAGAATGGTAGTTGAGTAGTTTGTGTATATGTGGGGCCTCATGGACGTGCTGGTGAC-3’
Sequence 2: aptamer LC15:
5’-ACGCTCGGATGCCACTACAG
TGAGATATAAAGGGCAGAAATAAGTAGGGGCTCATGGACGTGCTGGTGAC-3’。
2. the aptamer of detection SCHISTOSOMA JAPONICUM according to claim 1, is characterized in that, is carried out modifying and transforming by aptamer when keeping the conserved nucleotide sequence of LC6, LC15 underscore part constant.
3. the aptamer of detection SCHISTOSOMA JAPONICUM according to claim 2, is characterized in that, aptamer comprises any one in following three kinds:
A) conserved nucleotide sequence of aptamer LC6, LC15 underscore part is constant, and the Nucleotide at constant sequence two ends is deleted;
B) conserved nucleotide sequence of aptamer LC6, LC15 underscore part is constant, and the base of constant sequence two terminal nucleotide carries out artificial bases's replacement;
C) fluorescent substance, radioactive substance, therapeutic substance, vitamin H or enzyme labelling in aptamer LC6, LC15 connection.
4. the application method of the aptamer of the detection SCHISTOSOMA JAPONICUM described in any one of claim 1-3, is characterized in that, by the aptamer of described detection SCHISTOSOMA JAPONICUM for the preparation of the preparation detecting SCHISTOSOMA JAPONICUM.
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