CN104789569A - DNA (Deoxyribose Nucleic Acid) aptamer for detecting grouper iridovirus infection, as well as screening method and application of DNA aptamer - Google Patents

Abstract

The invention discloses a DNA nucleic acid aptamer for detecting grouper iridescent virus infection, a screening method and application thereof. During each round of screening, two steps of reverse screening were introduced. First, the single-stranded DNA library of the previous round was combined with normal cells to remove non-specific ssDNA combined with normal grouper cells, and then the supernatant was combined with the normal cells. The grouper iridovirus-infected cells were combined for screening, the ssDNA isolated from the grouper iridovirus-infected cells was combined with normal cells, and the supernatant was obtained. PCR amplifies the library to prepare a single-stranded DNA library. Repeat the above screening process, compared with the number of normal cells in the first round of screening, increase the number of normal cells in the screening process by 2-6 times, compared with the binding time of the library and cells in the first round of screening, in the subsequent During the screening process, the binding time between the library and normal cells was increased from 0.5h to 1h, and the binding time between the library and virus-infected cells was shortened from 1h to 0.5h to improve the screening efficiency of each round.

Description

A DNA nucleic acid aptamer for detecting grouper iridescent virus infection and its screening method and application

Technical field:

The invention belongs to the field of molecular biology, and in particular relates to a DNA nucleic acid aptamer which can be used for detection of grouper iridescent virus (SGIV) infection at the cell level and tissue level, and a screening method and application thereof.

Background technique:

Nucleic acid aptamer is a new type of detection and treatment tool screened by exponential enrichment ligand system evolution (SELEX). Ligand system evolution technology with exponential enrichment is a kind of biological library technology for novel detection and treatment that has attracted wide attention. It uses artificially synthesized random oligonucleotide library with a capacity of about 10 ¹⁴ to 10 ¹⁵ to bind to the target substance, and obtains the nucleic acid aptamer of the target substance after multiple rounds of screening. The nucleic acid aptamers screened by this technology have high specificity and high affinity comparable to monoclonal antibodies. Since the screening is a chemical process in vitro, simple systems such as metal ions and organic dyes, to viruses, Complex systems such as cells and tissues can be used as screening objects, and nucleic acid aptamers are not immunogenic, denaturation caused by temperature changes is reversible, screening time is short and the cost is low, and the obtained nucleic acid aptamers are easy to synthesize and modify. Therefore, nucleic acid aptamers not only have broad prospects in the field of new drug research and development, but also show broad application prospects in the fields of biomedical basic research and disease diagnosis of major diseases.

my country is a big country in aquaculture, and grouper is one of the most valuable marine fishes with high economic value. However, the outbreak and prevalence of iridescent virus in grouper in recent years has seriously threatened the mariculture of grouper and caused huge economic losses. At present, the international rapid diagnosis methods for aquatic animal diseases, including grouper iridescent virus, mainly include traditional methods based on bacteriology, virology, parasitology and histopathology, and antibody-based immunological detection methods for specific pathogens. Antibody serological detection technology and molecular biology technology, etc. These methods have their own advantages and serious shortcomings, so efforts must be made to develop new nucleic acid aptamers with stronger specificity, higher sensitivity, and easier operation to detect and control grouper iridescent virus (SGIV) infection.

Invention content:

The first object of the present invention is to provide a DNA nucleic acid aptamer for detecting grouper iridescent virus infection with high specificity, high sensitivity, non-immunogenicity, stability and easy modification, and convenient synthesis and storage.

The DNA nucleic acid aptamer for detecting grouper iridovirus infection of the present invention has a nucleotide sequence as shown in SEQ ID NO: 1, and a marker for detection is bound to the nucleotide sequence.

Any position on the nucleotide sequence of the above-mentioned DNA nucleic acid aptamer can be phosphorylated, methylated, aminated, thinned or isotoped.

The label used for detection is preferably: fluorescein isothiocyanate (FITC), carboxytetramethylrhodamine (TAMRA), biotin, digoxin, fluorescent substances, nano-luminescent materials, polyethylene glycol, Peptide, protein, folic acid or enzyme labeling.

The above-mentioned DNA aptamers, whether partially substituted or modified, have basically the same or similar molecular structure, physical and chemical properties and functions as the original aptamers, and can be used for the detection of grouper iridescent virus (SGIV) .

The second object of the present invention provides a kind of screening method of above-mentioned DNA nucleic acid aptamer, it is characterized in that, comprises the following steps:

(1), synthesize the following random single-stranded DNA library and primers:

Random library Library50:

5'-GACGCTTACTCAGGTGTGACTCG(50N)CGAAGGACGCAGATGAAGTCTC

5' primer: 5'-FITC-GACGCTTACTCAGGTGTGACTCG-3';

5' primer: 5'-TAMRA-GACGCTTACTCAGGTGTGACTCG-3';

3' primer: 5'-Biotin-GAGACTTCATCTGCGTCCTTCG-3';

(2), Cell-SELEX screening process: Infect normal grouper splenocyte GS cells with grouper iridescent virus, the cells continue to be cultivated for 48h, wash the infected GS cells with buffer after removing the culture medium, and step (1 After dissolving the random library in ), first incubate with normal GS cells on ice for 0.5h to obtain the supernatant, then incubate the supernatant with infected GS cells for 1h, remove the liquid after incubation and wash the GS cells with buffer, Then heat the washed GS cells at 85-94°C for 2-10 minutes, centrifuge to obtain supernatant 1, incubate supernatant 1 with normal GS cells on ice, and obtain supernatant in the same way as supernatant 1 2. It is the specific nucleic acid aptamer library of GS cells infected by iridovirus;

(3), library amplification: utilize the synthetic primer in step (1) to carry out PCR amplification to the specific nucleic acid aptamer library screened in step (2), obtain double-stranded DNA of amplified product;

(4) Preparation of single-stranded DNA library: Incubate the streptavidin-labeled magnetic beads with the double-stranded DNA in step (3) at room temperature, and use the label on the double-stranded DNA to bind the streptavidin on the magnetic beads. Binding double-stranded DNA to the surface of magnetic beads, adding lye to the magnetic beads to react, recovering the supernatant, and collecting the supernatant through a desalting column to a solution containing a DNA single-stranded library ;

(5), repeat multiple rounds of screening: replace the random library in step (2) with the DNA single-stranded library collected in step (4), repeat the above steps (2)-(4) at least 14 times, and the first round of screening Compared with the number of normal cells, the number of normal GS cells in the screening process is increased by 2-6 times. Compared with the binding time of the library and cells in the first round of screening, in the subsequent screening process, the library is combined with normal GS cells The time was increased from 0.5h to 1h, and the binding time between the library and virus-infected cells was shortened from 1h to 0.5h, so as to improve the screening efficiency of each round, that is, to obtain DNA aptamers for detecting grouper iridovirus infection.

Preferably, the medium described in step (2) is L15 medium.

Preferably, the buffer described in step (2) is PBS buffer.

Preferably, for the PCR amplification described in step (3), the amplification system is 1000 μl: 10×buffer 100 μl, dNTP (2.5mM) 80 μl, template 100 μl, 5’ primer 30 μl, 3’ primer 30 μl, rTaq enzyme 10 μl, dsH ₂ O 650μl; amplification program: 94°C for 2min, 94°C for 1min, 60°C for 30sec, 72°C for 1min, after 12 cycles, 72°C for 5min.

The supernatant obtained in the recovery described in step (4), is preferably recovered by a magnetic separation rack.

A third object of the present invention is to provide a kit for detecting grouper iridescent virus infection, which is characterized in that it contains the DNA nucleic acid aptamer shown in the above SEQ ID NO: 1.

The fourth object of the present invention is to provide the application of the above-mentioned DNA nucleic acid aptamer for detecting grouper iridescent virus infection in the detection of grouper iridescent virus infection.

In the present invention, the random DNA single-stranded library is first incubated with normal cells on ice, and then the recovered supernatant 1 is incubated with virus-infected GS cells on ice, and the infected cells are collected after high-temperature denaturation and recovered to supernatant 2, and then mixed with Normal cells were incubated on ice, so as to remove non-specific single-stranded DNA combined with normal cells to the greatest extent, and achieve the purpose of improving screening efficiency.

Compared with the existing technology, the advantage of the present invention is that the nucleic acid aptamer screened by the optimized cell SELEX of the present invention has higher affinity and specificity than the existing protein antibody, and also has protein The characteristics that antibodies do not have include non-immunogenicity; small molecular weight, which is convenient for chemical synthesis in vitro; easy to modify and replace different parts of nucleic acid aptamers; stable sequence, easy to transport and store; short preparation period and good reproducibility ; Easy to mark, etc. When the nucleic acid aptamer of the present invention is used to detect the infection of grouper iridescent virus, the operation is simple and fast, and the infection of iridescent virus can be detected at the cell level and tissue level, and the dissociation constants are all within the nanomolar range, and the application related detection kits. This is of great significance for the rapid detection of iridescent virus infection, and has a good application prospect in the field of detection of iridescent virus infection.

Description of drawings:

Fig. 1 is the 9th round, 10th round, 11th round, 12th round, 13th round, 14th round of screening library and GS infected by iridescent virus in flow cytometer detection fluorescein isothiocyanate (FITC) label in the embodiment of the invention The combination of cells, wherein, A is the GS target cell infected with SGIV, and B is the normal GS control cell;

Fig. 2 is the detection result of the nucleic acid aptamer of the fluorescein isothiocyanate (FITC)-labeled sequence 1 obtained by flow cytometry detection and screening in the embodiment of the present invention and the GS cell infected by iridovirus;

Fig. 3 is the nucleic acid aptamer of carboxytetramethylrhodamine (TAMRA) labeled sequence 1 in the embodiment of the present invention and the GS cell that is infected by iridescent virus, normal GS cell dyeing fluorescent intensity difference comparison (two groups of pictures in the figure , the left is the image of bright field, and the right is the image of fluorescence channel, which is the information of different channels in the same photo);

Fig. 4 is the nucleic acid aptamer of carboxytetramethylrhodamine (TAMRA)-labeled sequence 1 in the embodiment of the present invention and the grouper liver tissue that is infected by iridescent virus, the normal grouper liver tissue staining fluorescent intensity difference comparison ( In the two groups of pictures in the figure, the left side is the imaging of bright field, and the right side is the imaging of fluorescence channel, which is the information of different channels in the same picture).

Detailed ways:

The following examples are to further illustrate the present invention, rather than limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. Unless otherwise specified, the experimental materials in the following examples were purchased from conventional biochemical reagent companies.

Example 1:

1. Synthesize the random single-stranded DNA library and primers shown in the following sequence

Random library Library50:

5'-GACGCTTACTCAGGTGTGACTCG(50N)CGAAGGACGCAGATGAAGTCTC

5' primer: 5'-FITC-GACGCTTACTCAGGTGTGACTCG-3';

5' primer: 5'-TAMRA-GACGCTTACTCAGGTGTGACTCG-3';

3' primer: 5'-Biotin-GAGACTTCATCTGCGTCCTTCG-3';

2. Cell-SELEX screening to obtain nucleic acid aptamers that specifically recognize SGIV-infected GS cells

2.1 Add 10% fetal calf serum to the 1L15 medium to cultivate GS cells until 95% of the bottom of the culture bottle is covered, add grouper iridescent virus (SGIV) to the culture bottle, and grouper iridescent virus infects normal grouper splenocytes GS cells, after continuing to cultivate for 48h, remove the medium in the culture flask of the cells infected by the virus, and wash the infected GS cells with 15ml of PBS;

2.2 Dissolve 10nmol of the above random library in 300μl binding buffer, bathe in constant temperature water at 95°C for 5min, then quickly insert it into ice, bath in ice for 10min, supplement to 1mL with binding buffer, mix the processed random library with the GS infected by SGIV in 2.1 Cells were incubated on ice for 1 h;

2.3 After incubation and binding, remove the liquid in the culture flask, wash the cells in the culture flask with 10 mL of PBS; scrape off the cells with a cell scraper and mix them in 1 mL of PBS, transfer the cell mixture to an EP tube and store at 94°C The supernatant was collected by centrifugation at 12000 g for 5 minutes in a constant temperature water bath, which was the specific nucleic acid aptamer library of GS cells infected with SGIV.

3. PCR amplification screening library

Take 200 μl of the screened specific nucleic acid aptamer library of GS cells infected with SGIV for PCR amplification. The specific amplification program is: 94°C for 2min, 94°C for 1min, 60°C for 30sec, 72°C for 1min, after 12 cycles , 72°C for 5min. The supernatant obtained after the first round of screening should all be used for PCR amplification to obtain amplification products.

4. Preparation of single-stranded DNA library

Incubate 100 μl of streptavidin-labeled magnetic beads with the double-stranded DNA amplified by PCR in step 3 for 30 min at room temperature, using the affinity between the biotin on the double-stranded DNA and the streptavidin on the magnetic beads , bind the double-stranded DNA to the surface of the magnetic beads, put the EP tube on the magnetic separator to remove the supernatant, wash the magnetic beads with 1mL of PBS, then add 400ul 200mM NaOH solution to the EP tube, react at room temperature for 15min, and recover with the magnetic separation rack Obtain the supernatant; wash the desalting column with 10 mL of sterile water, add the supernatant to the desalting column, and drop it naturally by gravity. Add 1 mL of sterile water to collect a solution containing a DNA single-stranded library.

5. Repeated screening as above

Replace the random library in step (2) with the DNA single-stranded library obtained in step 4, and repeat the positive screening process, PCR amplification and preparation of the single-stranded DNA library shown in steps 2-4 14 times.

6. Negative screening

In the second round and after the second round of screening, normal GS cells were used as a control, and the DNA single-stranded library obtained after step 5 was subjected to negative screening to improve the screening efficiency. The specific negative screening process is as follows: culture GS cells until the bottom of the culture flask is covered, and wash the cells with 15ml PBS; dissolve the DNA library obtained by screening, put it in a constant temperature water bath at 95°C, and put it in an ice bath, and mix it with the normal GS cells treated above Incubate on ice for 0.5h, collect the cell incubation solution after the incubation is completed; then combine this solution with SGIV-infected GS cells in an ice bath for 1 hour; heat the SGIV-infected GS cells in a constant temperature water bath at 85-94°C for 2-10 minutes , 12000g centrifugation to collect supernatant 1, then incubate this supernatant 1 with another bottle of normal GS cells after the above-mentioned treatment on ice for 1 hour, and the collected supernatant 2 is the high-specificity cell that has been reversed twice. Aptamers that recognize SGIV-infected GS cells.

7. 15 rounds of screening

With the supernatant solution collected in step 6, after the PCR amplification in step 3 and the preparation of the single-stranded DNA library in step 4, repeat the process of step 6, step 2, step 3 and step 4 in sequence, and use flow cytometry Compared with the number of normal cells in the first round of screening, the number of normal GS cells in the screening process was increased by 2-6 times, compared with the library of the first round of screening. Compared with the binding time of cells, in the subsequent screening process, the binding time of the library and normal GS cells was increased from 0.5h to 1h, and the binding time of the library and virus-infected cells was shortened from 1h to 0.5h to improve the screening efficiency of each round , until after 15 rounds of screening, the ability of the library to recognize GS cells infected with SGIV reached the strongest. After the obtained amplified product was analyzed by cloning and sequencing, a nucleic acid aptamer of this embodiment that can be used to detect SGIV infection was finally obtained, the sequence of which is shown in SEQ ID NO: 1.

8. Using flow cytometry to detect the enrichment of the nucleic acid aptamer library during the screening process

The GS cells infected with SGIV were treated with enzyme-free digestion solution, the supernatant was removed by centrifugation at 1000g, and washed three times with 5mL PBS. Dissolve 300 nM fluorescein isothiocyanate (FITC)-labeled round 9, round 10, round 11, round 12, round 13, and round 14 of the screening library in 1 mL of binding buffer, and then mix with the above-mentioned treated cells in Incubate on ice for 30min, centrifuge at 1000g to remove the supernatant, wash with 5mL PBS three times, and finally mix the cells in 500μl PBS for flow cytometry detection. The cells combined with Library were used as control 1, and the combination of each round of library with normal GS cells was used as control 2. The detection results are shown in Figure 1. The results confirmed that after 14 rounds of screening, the screening library obtained had strong specific recognition ability for GS target cells infected with SGIV ( FIG. 1A ), but had no recognition for normal GS control cells ( FIG. 1B ).

9. Using flow cytometry to detect the binding effect and specificity of the four fluorescein isothiocyanate (FITC)-labeled aptamers in this example to GS cells infected with SGIV

The treatment of cells, the incubation and binding process of cells and nucleic acid aptamers are as described above, and the detection results of flow cytometry are shown in Figure 2. The results confirm that the nucleic acid aptamers of sequence 1 are all effective against the target cells infected with the GS of SGIV. Cells have strong specific recognition ability.

10. Detection of the binding effect and specificity of the four carboxytetramethylrhodamine (TAMRA)-labeled nucleic acid aptamers in this example and GS cells infected with SGIV by fluorescence microscope

Culture GS cells on a cover glass in a six-well plate, continue to culture for 48 hours after being inserted into SGIV, and obtain GS cells infected with SGIV, and culture normal GS cells on another cover glass as a control, remove the medium, and wash with 10 mL PBS cell. Add 1mL binding buffer containing 300nM nucleic acid aptamer in this example above, and bind in ice bath for 30min. After the reaction, remove the supernatant, wash three times with 10mL PBS, dry slightly, put the coverslip on a glass slide and seal with anti-fluorescence quenching agent for observation. As shown in Figure 3, the nucleic acid aptamers of sequence 1 all have specific binding ability to GS cells infected with SGIV, but have almost no binding ability to normal GS cells, which is consistent with the detection results of flow cytometry.

11. Specific binding test of nucleic acid aptamer to grouper liver tissue infected by iridovirus

Carboxytetramethylrhodamine (TAMRA)-labeled Sequence 1 nucleic acid aptamer was used to detect specific binding on frozen sections of grouper liver tissue infected with SGIV, and the nucleic acid aptamer was combined with normal grouper liver tissue frozen sections The combined results were used as a control. First wash the frozen section with 100mL PBS for 3 times, then incubate the section with 200μl binding buffer containing 300nM TAMRA-labeled nucleic acid aptamer at room temperature for 60min, then wash the tissue on a shaker with 100mL PBS, and compare the staining and washing of normal tissue The method is the same. After the tissue was slightly dry, the slide was sealed with anti-fluorescence quenching agent and observed under a fluorescence microscope. As shown in Figure 4, the nucleic acid aptamers shown in Sequence 1 all have strong specific binding ability to the liver tissue of the grouper infected with SGIV, but have almost no binding ability to the liver tissue of the normal grouper. This is consistent with the results of fluorescence microscopy and flow cytometry of grouper cells infected with SGIV.

Claims (9)

1., for detecting the DNA aptamer that grouper irido virus infects, its nucleotide sequence, as shown in SEQ ID NO:1, described nucleotide sequence is combined with the mark for detecting.

2. according to claim 1 for detecting the DNA aptamer that grouper irido virus infects, it is characterized in that, any position on its nucleotide sequence can be phosphorylated, methylate, amination, thinization or isotopologue.

3. according to claim 1 for detecting the DNA aptamer that grouper irido virus infects, it is characterized in that, described is labeled as fluorescein isothiocyanate, carboxyl tetramethylrhodamine, vitamin H, digoxin, fluorescent substance, nano luminescent material, polyoxyethylene glycol, peptide section, albumen, folic acid or enzyme labelling for what detect.

4. the screening method of DNA aptamer according to claim 1, is characterized in that, comprise the following steps:

(1), following random single-stranded DNA banks and primer is synthesized:

Random library Library50:

5’-GACGCTTACTCAGGTGTGACTCG(50N)CGAAGGACGCAGATGAAGTCTC

5 ' primer: 5 '-FITC-GACGCTTACTCAGGTGTGACTCG-3 ';

5 ' primer: 5 '-TAMRA-GACGCTTACTCAGGTGTGACTCG-3 ';

3 ' primer: 5 '-Biotin-GAGACTTCATCTGCGTCCTTCG-3 ';

(2), Cell-SELEX screening process: infect normal cabrilla splenocyte GS cell with grouper irido virus, cell continues to cultivate 48h, the infected GS cell of buffer solution is used after removing substratum, first hatch on ice with normal GS cell after random library in step (1) is dissolved and obtain supernatant in conjunction with 0.5h, then supernatant and infected GS cell are carried out hatching in conjunction with 1h, hatch rear removal liquid and used buffer solution GS cell, then the GS cell after washing is heated 2 ~ 10min in 85 ~ 94 DEG C, centrifugally obtain supernatant 1, supernatant 1 and normal GS cell are hatched combination on ice, supernatant 2 is obtained by the method identical with obtaining supernatant 1, be the specific nucleic acid aptamers library of the GS cell infected by irido virus,

(3), amplified library: utilize the specific nucleic acid aptamers library that in the primer pair step (2) of the synthesis in step (1), screening obtains to carry out pcr amplification, obtain amplified production double-stranded DNA;

(4), the preparation in DNA single chain library: the double-stranded DNA in the magnetic bead of streptavidin mark and step (3) is hatched at normal temperatures, utilize the affinity interaction of streptavidin on mark on double-stranded DNA and magnetic bead, double-stranded DNA is attached to magnetic bead surfaces, add alkaline reaction in magnetic bead after, recovery obtains supernatant liquor, by supernatant liquor by collecting the solution containing DNA single chain library except salt plug;

(5), repeat multi-turns screen: the DNA single chain library of collecting in step (4) is replaced the random library in step (2), repeat above step (2)-(4) at least 14 times, compared with the normal cell number screened with the first round, the number of GS cell normal in screening process is improved 2-6 doubly, the library of screening with the first round is compared with the binding time of cell, in screening process subsequently, library and normal GS Cell binding time are increased to 1h from 0.5h, library and virus infected cell binding time foreshorten to 0.5h from 1h, to improve the screening efficiency of often taking turns, namely the DNA aptamer infected for detecting grouper irido virus is obtained.

5. the screening method of DNA aptamer according to claim 4, is characterized in that, the substratum described in step (2) is L15 substratum.

6. the screening method of DNA aptamer according to claim 4, is characterized in that, the damping fluid described in step (2) is PBS damping fluid.

7. the screening method of DNA aptamer according to claim 4, it is characterized in that, pcr amplification described in step (3), its amplification system is: 1000 μ l:10 × buffer 100 μ l, dNTP (2.5mM) 80 μ l, template 100 μ l, 5 ' primer 30 μ l, 3 ' primer 30 μ l, rTaq enzyme 10 μ l, dsH ₂o 650 μ l, amplification program is: 94 DEG C of 2min, 94 DEG C of 1min, 60 DEG C of 30sec, and 72 DEG C of 1min, take turns circulation through 12,72 DEG C of 5min.

8., for detecting the test kit that grouper irido virus infects, it is characterized in that, containing DNA aptamer according to claim 1.

9. the application of DNA aptamer according to claim 1 in the infection carrying out grouper irido virus detects.