CN113462694B - Nucleic acid aptamer for megaphone iridovirus infected cells and application thereof - Google Patents
Nucleic acid aptamer for megaphone iridovirus infected cells and application thereof Download PDFInfo
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Abstract
The invention discloses a nucleic acid aptamer aiming at a megaphone jewfaciens iridovirus infected cell and application thereof, wherein the nucleotide sequence of the nucleic acid aptamer is 5'-GCCCGAGCACGCAGATCTTGCGCATAAAGCTTACGACCTCTTGTTACGTTGCCTTCA-3' (SEQ ID NO: 1) or 5'-GACGCTTACTCAGGTGTGACTCGGCCCGAGCACGCAGATCTTGCGCATAAAGCTTACGACCTCTTGTTACGTTGCCTTCACGAAGGACGCAGATGAAGTCTC-3' (SEQ ID NO: 2). The aptamer has specificity and high sensitivity to cells infected by the largemouth black bass iridovirus, and has no immunogenicity. The aptamer has stable structure, is easy to modify, is convenient to synthesize and store, and can be used for rapidly and accurately detecting cells infected by the largehead jewfish iridovirus.
Description
Technical Field
The invention relates to a ssDNA aptamer and application thereof, in particular to a nucleic acid aptamer aiming at a megaphone iridovirus infected cell and application thereof.
Background
The common name of the micropterus salmoides (Micropterus salmoides) is micropterus salmoides, and the micropterus salmoides have the characteristics of quick growth, low temperature resistance, delicious meat quality, easy capture and the like, and are popularized to all parts of the world from the 70 th century, and the annual output of the current micropterus salmoides in China is about 10 ten thousand t. However, an ulcer syndrome is prevalent in cultured micropterus salmoides and tends to be aggravated year by year. When the fish infected with viruses breaks the skin and muscle, the spleen and the kidney become large, the death rate of the diseased fish is up to 60 percent, and huge losses are caused to breeders, which forms a serious threat to the industry of culturing the largemouth bass. Through researches, the infection is mainly caused by an iridovirus, and after the largehead jewfish is infected by the iridovirus, plaque hemorrhagic ulcers appear on the body surface, red and swollen tail handles are ulcerated, and the liver of the sick fish is swelled, white or yellow.
At present, no effective treatment method exists for the viral disease, so that early rapid diagnosis of the disease and determination of the pathogen become important prevention and treatment means. At present, scientific researchers in China have developed various detection technologies aiming at the largemouth black bass iridovirus, including a polymerase chain reaction technology (PCR) based on molecular biology, a loop-mediated isothermal amplification technology (LAMP), an immunodetection technology based on antibodies and the like, and the operability and the technical level of partial products reach higher levels. The PCR technology is based on high sensitivity and high accuracy, and is the most commonly used pathogenic detection technology in the laboratory at present, but the PCR technology has various defects of complex operation, expensive instrument, long detection time consumption, harsh reagent preservation conditions and the like, so that the PCR technology is only suitable for detecting a small amount of samples under laboratory conditions by professional technicians, and cannot meet the requirement of carrying out on-site high-throughput rapid detection on LMBV viruses in the cultivation process of micropterus salmoides. The antibody-based immunological detection technique has some disadvantages, such as the need of strict low-temperature preservation condition of the antibody, long antibody preparation time, high cost and difference of batch-to-batch quality, and a certain degree of cross reaction on the structure similar compound is easy to cause false positive result.
Therefore, the rapid detection technology and functional products of the LMBV virus of the micropterus salmoides have the advantages of convenient and fast development and operation, low cost, short time consumption and high accuracy, and can be used for the cultivation site, which is important for early detection and determination of pathogens, and further, the treatment scheme is formulated in a targeted manner to control the pathogen diffusion and reduce the loss.
The exponential enrichment ligand system evolution technique (Systematic Evolution of Ligands by Exponential Enrichment technology, SELEX) is a biological library screening technique based on molecular biology recognition: the specific interaction of nucleic acid molecule and protein molecule in vivo is the ordered progress of gene transcription, nucleic acid replication, protein expression and other basic life activitiesEnsuring. Basic principle of SELEX technology: artificial synthesis of single-stranded nucleic acid library (single-stranded DNA or RNA) containing about 40nt of random sequences, the possible sequences of which can theoretically reach 4 40 A kind of module is assembled in the module and the module is assembled in the module. The diversity of the primary sequence also results in diversity of the secondary and tertiary structures of single stranded DNA or RNA. The nucleic acid with different sequences and different space structures can be screened for a plurality of cycles, and a plurality of target substances, such as proteins, nucleic acids, small peptides, amino acids and other organic molecules, even metal ions, can theoretically find the nucleic acid sequence which is specifically and firmly combined with the nucleic acid. Through multiple rounds of cyclic screening and amplification by PCR/RT-PCR technology, and cloning, sequencing and identification, the nucleic acid chain-ligand of the specific sequence combined with the target substance, also called nucleic acid aptamer or aptamer, can be produced and purified in large quantity.
The aptamer can specifically bind to a target substance firmly, and thus can largely correspond to the action of an antibody, and can also act in a field where some antibodies cannot yet be used. The aptamer can be used as a diagnostic reagent, can be applied to various diagnostic modes alone or in combination with an antibody, and can also be applied to clinical treatment and the like.
The aptamer has the advantages of easy screening, easy modification, strong stability, high specificity for identifying target molecules and the like, and is developed into a novel detection and treatment tool which is widely focused. First, the SELEX technique has a short screening cycle, and typically requires 6-30 SELEX cycles for aptamer screening, about 3-8 weeks. The screening of target molecules, the preparation of specific biological antibodies and the like need 3-8 months, and the antibodies have the problems of high cost, easy inactivation, severe preservation conditions, difference in quality of the antibodies among batches and the like. Secondly, the SELEX technology is a chemical screening process performed in vitro, so that simple systems such as Fan Cong metal ions, organic dyes and the like with a wide range of target molecules can be used as target molecules for SELEX screening, especially target molecules with cytotoxicity, no immunogenicity or weak immunogenicity, from complex systems such as viruses, cells and tissues and the like, and the SELEX technology can be used for screening high-specificity nucleic acid aptamers in vitro. Therefore, research on aptamer is increasingly receiving extensive attention from researchers in multiple disciplines such as biochemistry, biomedicine, nanomaterials, protein science, etc. at home and abroad. At present, the aptamer has been developed into a novel detection and treatment tool which is widely focused, and has a wide application prospect in the fields of biomedical basic research and disease diagnosis of serious diseases.
Disclosure of Invention
The invention aims to provide a nucleic acid aptamer which has high specificity, high sensitivity, no immunogenicity, stability, easy modification and convenience in synthesis and storage and is used for detecting cells infected by the iridovirus of the micropterus salmoides, so as to at least solve the problem that the existing biological detection technology cannot rapidly and accurately detect the cells infected by the iridovirus of the micropterus salmoides on site.
The invention aims at providing a nucleic acid aptamer capable of specifically identifying and detecting cells infected by the largemouth black bass iridovirus, wherein the nucleotide sequence of the nucleic acid aptamer is 5'-GCCCGAGCACGCAGATCTTGCGCATAAAGCTTACGACCTCTTGTTACGTTGCCTTCA-3' (SEQ ID NO: 1).
Further, the nucleotide sequence of the nucleic acid aptamer is 5' -GACGCTTACTCAGGTGTGACTCGGCCCG AGCACGCAGATCTTGCGCATAAAGCTTACGACCTCTTGTTACGTTGCCTTCACGAAGGACGCAGATGAAGTCTC-3’(SEQ ID NO：2)。
Further, the nucleotide sequence of the aptamer has a label bound thereto.
Still further, the label is selected from one or more of biotin, an enzyme, a luminescent group.
Further, the luminescent group is one or more selected from fluorescent substances or luminescent materials such as fluorescein isothiocyanate (fluorescein isothiocyanate, FITC), hydroxyfluorescein (6-carboxy-fluorescein, FAM), CY5 fluorescein (CY 5 fluorescent dye), CY3 fluorescein (CY 3 fluorescent dye), carboxytetramethyl rhodamine (TAMRA), and fluorescence quenching agent (Black Hole Quencher, BHQ).
It is still another object of the present invention to provide a fluorescent molecular probe (Aptamer LBVA3-based Fluorescent Molecular Probe, LBVA 3-AFMP) for rapid detection of pathogenic megaphone iridovirus infected cells based on nucleic acid Aptamer, wherein the molecular probe contains the nucleic acid Aptamer. The cells infected with the largemouth black bass iridovirus were labeled with the above-described SEQ ID no:1 or SEQ ID:2, after the nucleic acid aptamer is incubated, combined and washed, detecting by a flow cytometer, and detecting cells infected by the megaphone iridovirus according to the change of the fluorescence value. The LBVA3-AFMP can be applied to the development of a rapid detection kit for the cells infected by the megaphone iridovirus based on the nucleic acid aptamer, is suitable for the rapid detection of the large batch for diagnosing whether the megaphone is infected by the megaphone iridovirus, and has the advantages of short time consumption, simple and convenient operation, strong stability, high sensitivity and the like.
The invention also aims to provide an application of the aptamer in detecting cells infected by the largemouth black bass iridovirus.
Compared with the prior art, the invention has the advantages that: the nucleic acid aptamer obtained by screening through the SELEX technology can identify cells infected by the largehead jewfish iridovirus with high affinity and specificity, and has no immunogenicity; the molecular weight is small, and the in vitro chemical synthesis is convenient; the preparation period is short; the repeatability is good; chemical group modification and substitution are easy to be carried out on different parts of the nucleic acid aptamer; stable sequence, easy transportation and preservation, etc. When the rapid detection method (LBVA 3-AFMP) based on the aptamer is used for detecting cells infected by the megaphone jewfaciens iridovirus, the operation is simple and rapid, and the rapid detection method can be used for developing related rapid detection kits. The method has important significance for rapid detection of cells infected by the largemouth black bass iridovirus, and has good application prospect in the field of detection of the largemouth black bass iridovirus.
Drawings
FIG. 1 is a sequence of SEQ ID NO:1 a secondary structure prediction map of a nucleic acid aptamer;
FIG. 2 shows the detection of hydroxyfluorescein (FAM) labeled SEQ ID NO by a confocal laser microscope in an embodiment of the invention: 1 binding to largemouth black bass iridovirus LMBV infected cells, control group Library: hydroxy Fluorescein (FAM) labeled SEQ ID NO:1 binding to normal cells;
FIG. 3 is a flow cytometer showing the detection of a hydroxyfluorescein (FAM) labeled SEQ ID NO:1 with the infection of cells by the largemouth black bass iridovirus LMBV, control group 1: hydroxy Fluorescein (FAM) labeled SEQ ID NO:1 binding to normal cells; control group 2: hydroxy Fluorescein (FAM) labeled SEQ ID NO:1 binding condition of the grouper iridovirus SGIV infected cells; control group 3: hydroxy Fluorescein (FAM) labeled SEQ ID NO:1 binding to the neural necrosis virus NNV infected cells; control group 4: hydroxy Fluorescein (FAM) labeled SEQ ID NO:1 and the infected cells of the Chinese soft-shelled turtle iridovirus STIV.
Detailed Description
In order that those skilled in the art will better understand the present invention, a technical solution of the embodiments of the present invention will be clearly and completely described below, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments.
Example 1 screening of nucleic acid aptamers
Step 1: synthesis of random single stranded DNA library and primers shown in the following sequences
Randomly starting a single-stranded ssDNA Library:
5’-GACGCTTACTCAGGTGTGACTCG-N50-CGAAGGACGCAGAGAAGTCTC-3’
5' primer: 5'-FAM-GACGCTTACTCAGGTGTGACTCG-3';
3' primer: 5' -Biotin-GAGACTTCATCTGCGTCCTTCG-3;
step 2: SELEX screening to obtain nucleic acid aptamer for specifically recognizing aquatic pathogenic bacteria megalopsis iridovirus
2.1 dissolving 10nmol of the random library in 500 μl PBS, carrying out constant-temperature water bath at 92 ℃ for 5min, then rapidly inserting the random library into ice, carrying out ice bath for 10min, and incubating the treated random library and the largehead jewfish iridovirus LMBV infected cells on ice for 40min;
2.2 after incubation and combination are completed, the supernatant is removed by centrifugation, 3mL PBS is used for washing the cells infected by the megaphone jewfaciens iridovirus LMBV, a constant temperature water bath is carried out for 10min at 92 ℃, and 3000g centrifugation is carried out for collecting the supernatant, thus obtaining the single-stranded nucleic acid library for specifically identifying the cells infected by the megaphone jetfaciens iridovirus LMBV.
Step 3: PCR amplification screening library
Taking 100 μl of single-stranded nucleic acid library which is obtained by screening and is used for identifying the cells infected by the largehead jewfish iridovirus LMBV for PCR amplification, wherein the specific amplification strip procedure is as follows: 94℃for 5min,94℃for 1min,56℃for 30sec,72℃for 1min, 25 cycles and 72℃for 5min. The supernatant obtained after the first round of screening was used for all PCR amplification to obtain amplified double-stranded nucleic acid library.
Step 4: preparation of DNA Single-stranded library
Incubating 100 mu l of streptavidin-labeled magnetic beads and the double-stranded nucleic acid library obtained by PCR amplification in the step 3 for 20min at normal temperature, combining the double-stranded DNA on the surfaces of the magnetic beads by utilizing the affinity of biotin on the double-stranded DNA and streptavidin on the magnetic beads, removing the supernatant by utilizing a magnetic separator, washing the magnetic beads by using 2mL PBS, adding 200ul of NaOH solution (200 mM) into an EP tube, reacting for 10min at normal temperature, and recovering the supernatant by utilizing a magnetic separation frame; the forward ssDNA single-stranded nucleic acid in the supernatant is purified and recovered by using a PCR purification and recovery kit, and a solution containing the ssDNA single-stranded library is collected. :
step 5: repeating the screening as above
And (3) replacing the random library in the step (2) with the DNA single-stranded library obtained in the step (4), and repeating the positive screening process, the PCR amplification and the single-stranded DNA library preparation process shown in the step (2-4) for 9 times.
Step 6: negative selection
In the second and subsequent rounds of screening, the ssDNA single-stranded library obtained by the screening in the step 5 is subjected to negative screening by using normal FHM cells as a control so as to improve the screening efficiency. The specific negative screening process is as follows: dissolving the ssDNA library obtained by screening, incubating the ssDNA library with normal FHM cells on ice for 1h after the ssDNA library is dissolved and subjected to constant-temperature water bath and ice bath at 92 ℃, centrifuging after incubation is completed, and collecting supernatant solution, wherein the collected supernatant is the nucleic acid library subjected to negative screening.
Step 7:10 rounds of screening
And (3) sequentially repeating the processes of the step (6), the step (2), the step (3) and the step (4) after the supernatant solution collected in the step (6) is subjected to PCR amplification in the step (3) and single-stranded DNA library preparation in the step (4), and detecting the change condition of the identification capacity of the obtained single-stranded DNA library on the red-mouth black bass iridovirus LMBV infected cells by using a flow cytometer until the identification capacity of the nucleic acid library on the red-mouth black bass iridovirus LMBV infected cells reaches the strongest after 9 rounds of screening. After the obtained amplification product is subjected to clone sequencing analysis, the ssDNA nucleic acid aptamer which can be used for detecting the cells infected by the megaphone jewfaciens iridovirus LMBV in the embodiment is finally obtained, and the sequence of the ssDNA nucleic acid aptamer is shown as SEQ ID NO: 1.
Step 8: on-line prediction of SEQ ID NO using MFOLD software (http:// MFOLD. Rna. Albany. Edu/: 1 secondary structure of the nucleic acid aptamer.
SEQ ID NO: the predicted secondary structure of the aptamer of 1 is shown in figure 1, and the aptamer of SEQ ID NO:1 form a special stem-loop structure.
Step 9: the hydroxy Fluorescein (FAM) labeled SEQ ID NO:1 or SEQ ID NO:2 binding effect of aptamer on megalopsis iridovirus LMBV infected cells and specificity thereof
Nucleic acid aptamer SEQ ID NO: incubation and binding procedure of 1 and largemouth black bass iridovirus LMBV infected cells as described above, the results of the detection by laser confocal microscopy are shown in fig. 2, which demonstrates that the above-described SEQ ID NO:1 or SEQ ID NO:2 has high specific recognition capability to the megaphone jewfish iridovirus LMBV infected cells.
Step 10: the hydroxy Fluorescein (FAM) labeled SEQ ID NO:1 or SEQ ID NO:2 binding effect of aptamer on megalopsis iridovirus LMBV infected cells and specificity thereof
The incubation and binding process of the aptamer and the largemouth black bass iridovirus LMBV infected cells are shown in the above, the detection result of the flow cytometry is shown in fig. 3, and the result proves that compared with the four groups of control group cells, the hydroxyl Fluorescein (FAM) labeled SEQ ID NO:1 or SEQ ID NO:2 has high specific recognition capability to the megaphone jewfish iridovirus LMBV infected cells.
Example 2
In the present example, the SEQ ID NO obtained in example 1 was used: 1 or SEQ ID NO:2, assembling a fluorescent molecular probe (Aptamer LBVA3-based Fluorescent Molecular Probe, LBVA 3-AFMP) kit to form the LBVA3-AFMP detection kit for the cells infected by the megaphone jewfaciens iridovirus. The molecular probe contains the SEQ ID NO:1 or SEQ ID NO:2 nucleic acid aptamer.
The nucleic acid aptamer obtained by screening through the SELEX technology has good affinity and specificity, has stable structure, still has good affinity and specificity after group marking and modification, can be applied to fluorescent molecular probe detection kits, has the characteristics of short preparation period, good reproducibility and small molecular weight compared with protein antibodies, is convenient for in vitro synthesis, and has good application prospect in the detection field of aquatic pathogenic bacteria megaphone iridovirus.
Finally, it should be noted that the above-mentioned embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the above-mentioned embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made to the specific embodiments of the present invention after reading the present specification, and these modifications and variations do not depart from the scope of the invention as claimed in the pending claims.
Sequence listing
<110> academy of sciences in Guangxi province
<120> a nucleic acid aptamer for a megaphone iridovirus infected cell and application thereof
<130> 2021
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 50
<212> DNA
<213> artificial sequence
<400> 1
gcccgagcacgcagatcttgcgcataaagcttacgacctcttgttacgttgccttca 57
<210> 2
<211> 95
<212> DNA
<213> artificial sequence
<400> 2
gacgcttactcaggtgtgactcggcccgagcacgcagatcttgcgcataaagcttacgac 60
ctcttgttacgttgccttcacgaaggacgcagatgaagtctc 102
Claims (8)
1. A nucleic acid aptamer for a cell infected with a black bass iridovirus, wherein the nucleic acid aptamer has a nucleotide sequence of 5'-GCCCGAGCACGCAGATCTTGCGCATAAAGCTTACGACCTCTTGTTACGTTG CCTTCA-3'.
2. A nucleic acid aptamer for identifying and detecting cells infected by largemouth black bass iridovirus is characterized in that the nucleotide sequence of the nucleic acid aptamer is 5' -GACGCTTACTCAGGTGTGACTCGGCCCGAGCACGCAGATCT
TGCGCATAAAGCTTACGACCTCTTGTTACGTTGCCTTCACGAAGGACG CAGATGAAGTCTC-3’。
3. The nucleic acid aptamer of claim 1 or 2, wherein the nucleotide sequence of the nucleic acid aptamer is conjugated to a label.
4. The aptamer of claim 3, wherein the label is selected from one or more of biotin, an enzyme, and a luminescent moiety.
5. The aptamer of claim 4, wherein the luminescent moiety is selected from one or more of fluorescein isothiocyanate, hydroxyfluorescein, CY5 fluorescein, CY3 fluorescein, carboxytetramethyl rhodamine.
6. A kit for identifying and detecting cells infected with the megalopsis iridovirus, said kit comprising the nucleic acid aptamer according to any one of claims 1 to 5.
7. A method for detecting a fluorescent molecular probe using the aptamer of claim 1 or 2, comprising the steps of:
step 1: labeling the aptamer with hydroxy fluorescein;
step 2: mixing 1-100mg of sample to be detected with the aptamer with the concentration of 100-200nM obtained in the step 1, and incubating and combining on ice for 20-60min; centrifuging to remove supernatant, cleaning a sample to be detected, and detecting by using a flow cytometer;
the use does not include use in disease diagnosis.
8. Use of the nucleic acid aptamer according to claim 1 or 2 for detecting cells infected with the largemouth black bass iridovirus, said use not comprising the use in the diagnosis of a disease.
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| WO2018029301A1 (en) * | 2016-08-11 | 2018-02-15 | Intervet International B.V. | Novel fish pathogenic virus |
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| CN112522274A (en) * | 2020-12-25 | 2021-03-19 | 华南农业大学 | ssDNA nucleic acid aptamer for identifying iridovirus of micropterus salmoides and application thereof |
| CN112522273A (en) * | 2020-12-11 | 2021-03-19 | 浙江工商大学 | Oligonucleotide aptamer for specifically recognizing largemouth bass virus as well as screening method and application thereof |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018029301A1 (en) * | 2016-08-11 | 2018-02-15 | Intervet International B.V. | Novel fish pathogenic virus |
| CN111073892A (en) * | 2019-10-30 | 2020-04-28 | 广西科学院 | Aptamer for identifying grouper iridovirus infected cells and construction method and application thereof |
| CN112522273A (en) * | 2020-12-11 | 2021-03-19 | 浙江工商大学 | Oligonucleotide aptamer for specifically recognizing largemouth bass virus as well as screening method and application thereof |
| CN112522274A (en) * | 2020-12-25 | 2021-03-19 | 华南农业大学 | ssDNA nucleic acid aptamer for identifying iridovirus of micropterus salmoides and application thereof |
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