CN111118014B - Anti-iridovirus aptamer and construction method and application thereof - Google Patents
Anti-iridovirus aptamer and construction method and application thereof Download PDFInfo
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Abstract
An anti-iridovirus aptamer comprising the nucleotide sequence: CCCAACTCGCTAAGATTAACCAAATGAAAGGCGCCGTAACCTGGATGCTT. The aptamer provided by the invention has the advantages of small molecular weight, short preparation period, good reproducibility, convenience for in-vitro chemical synthesis and marking, and stable sequence and easiness in transportation and storage. The aptamer has high specificity and affinity to the grouper iridovirus, and has no immunogenicity.
Description
Technical Field
The invention belongs to the technical field of aquatic pathogenic bacteria detection, and particularly relates to an anti-iridovirus aptamer, and a construction method and application thereof.
Background
The development of the ocean economy is promoted by the force of marching towards the ocean, and the important strategy of China is developed. Guangxi is an international transportation hub connected with the east Union, is adjacent to the main consumer market of marine products such as southeast Asia and the like, and the mariculture industry is rapidly developed in recent years. The grouper has fine and smooth meat quality and rich nutrition, is used as a large and rare marine culture fish, has the annual output of over 13 million tons in domestic grouper culture at present, has direct industrial output value of more than billions yuan, and has extremely high economic value. However, with the increase of the culture density and the expansion of the culture scale of the groupers, the offshore culture environment tends to be poor, and the increasingly poor culture water area environment causes frequent outbreaks of various diseases, thereby causing huge economic loss. Among them, grouper iridovirus is the main viral pathogen causing the disease of groupers, and the fish disease fatality rate caused by the virus is extremely high, which is called as 'marine foot-and-mouth disease'. At present, systematic research on the grouper iridovirus is carried out internationally, mainly comprises epidemiological investigation, pathogen separation and identification, sensitive cell line construction, virus ultrastructure observation, virus diagnosis technology development and the like, and the infection and pathogenic mechanism of the grouper iridovirus are disclosed to a certain extent. Suspected iridovirus disease of grouper occurs in pond-cultured grouper in northern sea area of Guangxi in 2018, and the iridovirus disease of grouper (SGIV) is proved to be a main pathogenic pathogen by analyzing and identifying pathogenic microorganisms in diseased grouper through Polymerase Chain Reaction (PCR) detection technology. Therefore, the research and development of the detection technology with convenient operation, low cost, short time consumption and high accuracy is extremely important for controlling the damage of the SGIV virus of the grouper. However, the current diagnosis method aiming at the grouper SGIV virus mainly comprises a common PCR technology, a nested PCR technology, a fluorescent quantitative PCR technology and the like. The detection result of the PCR technology is accurate and reliable, but the PCR technology has the defects of complex operation, long time consumption, expensive instrument and reagent and the like, and can not meet the requirement of rapid and accurate detection and diagnosis on site. Therefore, the technology and the functional product for quickly detecting the SGIV virus of the grouper on the culture site are developed, are convenient to operate, low in cost, short in time consumption and high in accuracy, and are important for finding and determining the pathogen as soon as possible and further purposefully making a treatment scheme to control pathogen diffusion and reduce loss.
The aptamer is a single-stranded oligonucleotide capable of specifically recognizing a target substance, which is obtained by in vitro multiple rounds of screening from an artificially synthesized random sequence library by using an Exponential Enrichment ligand phylogenetic technology (SELEX). The aptamer has the advantages of high specificity, high affinity, strong stability, easy chemical synthesis and chemical modification and the like. Based on the characteristic that the aptamer can recognize pathogenic microorganisms or pathological cells with high specificity, the aptamer is widely used for construction of biosensors, and accurate detection and diagnosis of pathogens or diseases are realized through signal amplification and the like. At present, the aptamer serving as a novel and widely-focused novel detection and treatment tool has wide application prospects in the fields of human medical research, disease diagnosis, virus infection mechanism research and the like.
Disclosure of Invention
The invention aims to provide an anti-iridovirus aptamer, a construction method and application thereof, so as to realize high-sensitivity and high-specificity detection of the grouper iridovirus.
According to one aspect of the present application, there is provided an anti-iridovirus aptamer comprising the nucleotide sequence of SEQ ID No. 1.
Preferably, it comprises the nucleotide sequence of SEQ ID NO.2.
Preferably, at least one nucleotide in its nucleotide sequence is phosphorylated, thiolated, methylated, aminated, or isotopically esterified.
Preferably, the kit further comprises a sequence for coding a marker, wherein the marker is selected from one or more of luminescent substances, biotin or enzymes.
Preferably, the marker is a luminescent substance, and the luminescent substance is one or more selected from hydroxyl fluorescein, fluorescein isothiocyanate or carboxytetramethyl rhodamine.
According to another aspect of the present invention, there is provided a method for constructing the above-mentioned anti-iridovirus aptamer using the SELEX technique, comprising the steps of: (1) Establishing a single-stranded DNA random Library50, wherein the nucleotide sequence of the single-stranded DNA random Library50 is as follows: 5' -GACGCTTACTCAGGTGTGACTCG (50N) CGAAGGACGCAGATGAAGTCTC;
(2) Screening a single-stranded DNA random Library50 by using a grouper somatic cell infected with the grouper iridovirus to obtain a specific DNA Library of the grouper iridovirus; (3) Taking the specific DNA library as a template to carry out PCR amplification, wherein a 5 'end primer adopted by the PCR amplification comprises a nucleotide sequence of SEQ ID NO.3, and a 3' end primer adopted by the PCR amplification comprises a nucleotide sequence of SEQ ID NO. 4.
Preferably, the amplification procedure for PCR amplification is: 5 minutes at 94 ℃,1 minute at 94 ℃, 30 seconds at 56 ℃,1 minute at 72 ℃ and 25 cycles; 5 minutes at 72 ℃.
According to another aspect of the present invention there is provided the use of an anti-iridovirus aptamer as described above for the detection of grouper iridovirus.
According to another aspect of the invention, a fluorescent molecular detection probe for grouper iridovirus is provided: the marker comprises the anti-iridovirus aptamer and the hydroxyfluorescein.
Compared with the existing protein antibody, the anti-iridovirus aptamer provided by the invention has the advantages of small molecular weight, no immunogenicity, short preparation period, good reproducibility and the like, and is convenient for in vitro chemical synthesis; moreover, the aptamer has high stability and is easy to transport and store; in addition, the aptamer is convenient to mark, and different parts of the aptamer can be easily modified and replaced. Compared with the existing protein antibody, the invention adopts SELEX technology in the process of constructing the aptamer, so that the obtained aptamer has higher affinity and specificity to the grouper iridovirus; in particular, the aptamer is partially substituted or modified to obtain a aptamer derivative, and the aptamer derivative can be used for detecting the SGIV virus of the grouper as long as the aptamer derivative has a molecular structure, physical and chemical properties and functions which are basically the same as or similar to those of the original aptamer. The nucleotide sequence of the anti-iridovirus aptamer and the derivative thereof provided by the invention is provided with a marker sequence, and the efficient detection of the iridovirus of the grouper can be realized by simple and rapid operation in combination with a corresponding detection means.
Drawings
FIG. 1 is a diagram showing the prediction of the secondary structure of an aptamer having the nucleotide sequence of SEQ ID NO. 2;
FIG. 2 shows the FAM fluorescence value test results of the samples measured by the flow cytometer in example 2;
FIG. 3 is the observation results of the laser scanning confocal microscope in example 2: a light mirror image of a test group sample, (b) a fluorescence image of the test group sample, (c) a superimposed effect image of the (a) image and the (b) image, (d) a light mirror image of a control group sample, (e) a fluorescence image of the control group sample, and (f) a superimposed effect image of the (d) image and the (e) image.
Detailed Description
In order to make the technical solutions of the present invention better understood by those skilled in the art, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments.
Example 1 screening and preparation of aptamers for detection of Epinephelus iridovirus
S1, construction of random single-stranded DNA (ssDNA) library and synthesis of primers
Constructing a random ssDNA Library50, wherein two ends are fixed sequences, the middle 50 nucleotides are random sequences, and the nucleotide sequences are as follows: 5' -GACGCTTACTCAGGTGTGACTCG (50N) CGAAGGACGCAGATGAAGTCTC. The sequences of the 5' end primers used were as follows: 5' -FAM-GACGCTTACTCAGGTGTGACTCG-3', the 3' end primer sequence used is as follows: 5'-Biotin-GAGACTTCATCTGCGTCCTTCG-3'. Both random ssDNA libraries and primers were synthesized by Shanghai Biotechnology, inc.
S2.SELEX screening (corresponding to positive screening)
10nmol of the random ssDNA library is dissolved in 500 muL PBS, and is subjected to thermostatic water bath at 92 ℃ for 5min, then is quickly inserted into ice, and is subjected to ice bath for 10min, and the treated random ssDNA library and SGIV virus-infected splenocytes of grouper are incubated on ice for 1h.
After the incubation combination is completed, the supernatant is removed by centrifugation, 10mL of PBS is used for washing the spleen cells of the grouper infected by the SGIV virus, 10min is carried out in a thermostatic water bath at 92 ℃,12000g is used for centrifuging and collecting the supernatant, and the ssDNA nucleic acid library for specifically identifying the cells of the grouper infected by the SGIV virus is obtained.
S3.PCR amplification
Taking 100 mu L of ssDNA library obtained by screening and identifying SGIV virus infected cells, and carrying out PCR amplification on the ssDNA library, a 5 'end primer and a 3' end primer. The PCR reaction system was as follows (1000. Mu.L): 10 XBuffer 100. Mu.L, dNTP Mix (2.5 mM) 80. Mu.L, 5 'primer 40. Mu.L, 3' primer 40. Mu.L, ssDNA library 100. Mu.L, rTaq enzyme 12.5. Mu.L, ddH 2 O627.5. Mu.L. The PCR amplification procedure was: 5min at 94 ℃, 1min at 94 ℃, 30sec at 56 ℃ and 1min at 72 ℃ and are circulated for 25 cycles; 5min at 72 ℃. The supernatants obtained after the first round of SELEX screening were all used for PCR amplification to obtain a double stranded nucleic acid (dsDNA) library.
S4. Preparation of ssDNA library
Incubating 100 mu L of streptavidin-labeled magnetic beads and a dsDNA library prepared from S3 for 20min at normal temperature, utilizing the affinity action of biotin on the dsDNA and streptavidin on the magnetic beads to bond the dsDNA to the surfaces of the magnetic beads, removing supernatant on a magnetic separator, washing the magnetic beads by using 2mL of PBS, then adding 200 mu L of NaOH solution (200 mM) into an EP tube, reacting for 10min at normal temperature to denature the dsDNA, leaving one chain with the biotin bonded with the streptavidin on the magnetic beads, and recovering the supernatant by using a magnetic separation frame after the reaction is finished; adding the supernatant into a desalting column washed by sterile water, and naturally dripping under the action of gravity. To the filtrate was added 500. Mu.L PBS and the solution containing the ssDNA library was collected for the next round of screening.
S5. Iterative screening of ssDNA libraries
The random ssDNA library in S2 was replaced with the ssDNA library obtained in S4, and the SELEX screening, PCR amplification and ssDNA library preparation process shown in S2-S4 were repeated 10 times.
S6. Negative screening
And (3) dissolving the ssDNA library obtained by the second round of S5 and the subsequent round of screening, performing constant-temperature water bath at 92 ℃, incubating the ssDNA library and normal cells of the grouper on ice for 1h, and centrifugally collecting supernatant solution after incubation is completed, wherein the supernatant solution is the ssDNA library subjected to negative screening.
In the step, normal cells of the grouper are used as a control, and the ssDNA library obtained by screening after S5 is subjected to negative screening so as to improve the screening efficiency of the ssDNA library.
S7.11 round of screening
And (3) performing PCR amplification on the supernatant collected in the step (S6) and preparation of the ssDNA library of the step (S4), sequentially repeating the steps of the step (S6), the step (S2), the step (S3) and the step (S4), detecting the change condition of the identification capacity of the obtained ssDNA library on SGIV virus infected cells by using a flow cytometer, and repeating the step (11) of screening, wherein the identification capacity of the obtained ssDNA library on the SGIV virus infected cells is strongest. And (3) after the obtained amplification product is subjected to clone sequencing analysis, finally obtaining the ssDNA aptamer for detecting SGIV virus infected cells, wherein the nucleotide sequence of the ssDNA aptamer is as follows:
CCCAACTCGCTAAGATTAACCAAATGAAAGGCGCCGTAACCTGGATGCTT (SEQ ID NO.1)，
or the like, or, alternatively,
GACGCTTACTCAGGTGTGACTCGCCCAACTCGCTAAGATTAACCAAATGAA AGGCGCCGTAACCTGGATGCTTCGAAGGACGCAGATGAAGTCTC(SEQ ID NO.2)。
MFOLD software (http:// MFOLD. Rna. Albany. Edu/. Similarly, ssDNA aptamers with the nucleotide sequence of SEQ ID No.1 also form specific stem-loop and hairpin structures.
Example 2
2.1 Main Instrument
Attune NxT flow cytometer (seemer feishell technology), FV3000 laser scanning confocal microscope (olympus).
2.2 Experimental procedures
The aptamers of SEQ ID NO.2 constructed in example 1 were labeled with hydroxyfluorescein (FAM), respectively.
Test groups: 10nmol of FAM-labeled SEQ ID NO.2 aptamer is put in 500 mu L PBS and is put in thermostatic water bath at 92 ℃ for 5min, then the mixture is quickly inserted into ice and is subjected to ice bath for 10min, the treated FAM-labeled SEQ ID NO.2 aptamer and SGIV virus-infected grouper splenocytes are incubated on the ice for 1h, and after the incubation and combination are completed, the supernatant is removed by centrifugation.
Control group: 10nmol of FAM-labeled SEQ ID NO.2 aptamer is put in 500 mu L PBS, water bath is carried out at constant temperature of 92 ℃ for 5min, then the mixture is quickly inserted into ice and is subjected to ice bath for 10min, the treated FAM-labeled SEQ ID NO.2 aptamer and normal grouper spleen cells are incubated on the ice for 1h, and after incubation and combination are completed, the supernatant is removed by centrifugation.
Cell precipitates obtained by incubation of the test group and the control group are detected by using a flow cytometer and a laser scanning confocal microscope respectively.
2.3 results of the experiment
The detection result of the flow cytometer is shown in fig. 2, compared with the fluorescence value of the surface of the spleen cell of the normal grouper detected by the flow cytometer in the control group, the fluorescence value of the surface of the spleen cell of the grouper infected by the SGIV virus detected by the flow cytometer in the test group is obviously increased, namely, the FAM-labeled SEQ ID No.2 aptamer has high specific recognition capability on the SGIV virus infected cell.
The detection result of the confocal laser scanning microscope is shown in fig. 3, and the result proves that compared with the normal cells in the control group, the fluorescent effect of the SGIV virus infected cells in the test group is obvious, and the cell samples in the test group emit strong green light. The FAM marked SEQ ID NO.2 aptamer is obviously bound on the surface of spleen cells of SGIV virus infected grouper, namely the FAM marked SEQ ID NO.2 aptamer has high specific recognition capability on SGIV virus infected cells.
Although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the true spirit and scope of the present invention.
SEQUENCE LISTING
<110> Guangxi academy of sciences, guangxi Zhuang autonomous region Marine research institute
<120> an anti-iridovirus aptamer, and construction method and application thereof
<130>
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 50
<212> DNA
<213> Artificial sequence
<400> 1
cccaactcgc taagattaac caaatgaaag gcgccgtaac ctggatgctt 50
<210> 2
<211> 95
<212> DNA
<213> Artificial sequence
<400> 2
gacgcttact caggtgtgac tcgcccaact cgctaagatt aaccaaatga aaggcgccgt 60
aacctggatg cttcgaagga cgcagatgaa gtctc 95
<210> 3
<211> 23
<212> DNA
<213> Artificial sequence
<400> 3
gacgcttact caggtgtgac tcg 23
<210> 4
<211> 22
<212> DNA
<213> Artificial sequence
<400> 4
gagacttcat ctgcgtcctt cg 22
Claims (4)
1. An aptamer against iridovirus, characterized in that:
the nucleotide sequence is SEQ ID NO.2.
2. An aptamer against iridovirus, characterized in that:
a marker is bound on the basis of the nucleotide sequence as defined in claim 1, wherein the marker is one or more selected from the group consisting of luminescent substances, biotin and enzymes.
3. The anti-iridovirus aptamer of claim 2, wherein:
the luminescent material is one or more than one of hydroxyl fluorescein, fluorescein isothiocyanate or carboxyl tetramethyl rhodamine.
4. A fluorescent molecular detection probe for grouper iridovirus is characterized in that:
an aptamer comprising the anti-iridovirus antibody of claim 3, wherein said marker is hydroxyfluorescein.
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