CN113528530B

CN113528530B - Aptamer specifically combined with mycoplasma hyorhinis and application thereof

Info

Publication number: CN113528530B
Application number: CN202110597749.9A
Authority: CN
Inventors: 叶茂; 谭蔚泓; 张毅彬
Original assignee: Hunan University
Current assignee: Hunan University
Priority date: 2021-05-31
Filing date: 2021-05-31
Publication date: 2022-07-12
Anticipated expiration: 2041-05-31
Also published as: CN113528530A

Abstract

The invention discloses a nucleic acid aptamer specifically combined with mycoplasma hyorhinis and application thereof. The aptamer has strong specificity and high sensitivity, and can be combined with mycoplasma hyorhinis in a targeted manner and cells infected by the mycoplasma hyorhinis in a targeted manner; as a reagent for detecting mycoplasma hyorhinis infection, as a reagent for detecting mycoplasma hyorhinis infected cells and as a reagent for detecting in vivo imaging of mycoplasma hyorhinis infected cells. The invention provides a new method and a new way for detecting, diagnosing and treating tumors.

Description

Aptamer specifically combined with mycoplasma hyorhinis and application thereof

Technical Field

The invention belongs to the technical field of tumor microbiology, and relates to a nucleic acid aptamer for targeted recognition of mycoplasma hyorhinis and cells infected by the mycoplasma hyorhinis and the targeted combination of the nucleic acid aptamer and the mycoplasma hyorhinis; the applications of the compound in a reagent for detecting mycoplasma hyorhinis infection, a reagent for detecting mycoplasma hyorhinis infected cells and a reagent for detecting in-vivo living body imaging of the mycoplasma hyorhinis infected cells.

Background

The malignant tumor is a malignant disease which is caused by the phenomena of hyperproliferation, metastasis, infiltration and the like of normal cells in organisms under the action of a plurality of internal causes and external causes, thereby harming the functions of normal organs of human bodies and leading to death. The tumor-causing factors include the genetic factors of the host and the external physical, chemical and microbial causes. According to the existing research reports, the infection of pathogenic microorganisms is an important external factor causing the occurrence and development of tumors, 16 percent of human cancers can be attributed to the infection of pathogenic microorganisms, such as gastric cancer caused by helicobacter pylori, liver cancer promoted by hepatitis B virus, nasopharyngeal carcinoma induced by EB virus, gene mutation, protein expression change, cell morphological change and even malignant transformation, abnormal proliferation, migration and invasion capacity increase of cells caused by the colonization of pathogenic microorganisms.

Mycoplasma hyorhinis, a normal flora originally found in the respiratory tract of swine, can cause pneumonia, serositis, arthritis, tympanitis and otitis media of swine under specific conditions, but in recent years, it has been found to be closely related to the occurrence and development of human tumors, and according to reports, the infection in human tumor tissue specimens was detected by an antibody PD4 specifically recognizing Mycoplasma hyorhinis, and it was found that the infection rate was 39% -55% in patient samples of stomach cancer, colon cancer, esophageal cancer, lung cancer and breast cancer, and it was also found that Mycoplasma hyorhinis infection was found in 59.3% of ovarian cancer and 52% of prostate cancer, and another study showed that 89% of patients with liver cancer CTC circulating tumor cells had Mycoplasma hyorhinis infection and that the prognosis of infected persons was shorter than those who had not been infected. At present, mycoplasma detection is not listed as a diagnosis project for tumor screening in clinical work for a while, more mycoplasma detection is applied to in vitro cell culture, and in the aspect of treatment, antibiotics are mostly used for treatment, but the drug resistance of the antibiotics is very common at present, certain side effects and toxicity can still be generated on eukaryotic cells, specificity is lacked, and the inherent defect that the normal flora in a human body is killed is also the defect. Therefore, the development of a novel rapid detection method for in vivo and in vitro and a treatment method without toxic and side effects has important significance.

The aptamer can be used as a high-sensitivity molecular detection probe and can also be used as a targeting ligand loaded drug for disease treatment, and compared with an antibody, the aptamer has many additional advantages, such as low synthesis cost, good stability, easy modification and no immunogenicity, can be used for disease diagnosis and treatment, and has great academic and application values.

Disclosure of Invention

The invention mainly aims to provide a novel aptamer which is combined with mycoplasma hyorhinis with high affinity and high specificity and application thereof.

The aptamer has strong specificity and high sensitivity, and can be combined with mycoplasma hyorhinis in a targeted manner and cells infected by the mycoplasma hyorhinis in a targeted manner; as a reagent for detecting mycoplasma hyorhinis infection, as a reagent for detecting mycoplasma hyorhinis infected cells and as a reagent for detecting in vivo imaging of mycoplasma hyorhinis infected cells.

A nucleic acid aptamer specifically binding to mycoplasma hyorhinis, which has the following sequence:

SEQ NO.1:

5’-ACCGACCGTGCTGGACTCACGTCGTCCATTTCCTTGAAAAAGGCACGGGTTCCATGAACTCACTATGAGCGAGCCTGGCG-3’；

or base deletion is carried out on the 3 'end segment of SEQ NO.1 to obtain the aptamer with the same function as the 3' end segment;

or the base exchange is carried out in the middle section of SEQ NO.1 to obtain the aptamer with the same function as the base exchange.

The base deletion is carried out on the 3' end segment of the SEQ NO.1 to obtain the aptamer with the same function as the base deletion; nucleic acid aptamers of the following sequence are preferred:

SEQ NO.7:

5’-ACCGACCGTGCTGGACTCACGTCGTCCATTTCCTTGAAAAAGGCACGGGTTCCATGAACTCACTATGAGC-3’。

carrying out base replacement on the middle section of SEQ NO.1 to obtain the aptamer with the same function as the middle section; nucleic acid aptamers of one or more of the following sequences are preferred:

SEQ NO.2:

5’-ACCGACCGTGCTGGACTCAAGCGGTTTCCGTGAACCTGCGAGTTCCCTGAATACTTGTACTATGAGCGAGCCTGGCG-3’

SEQ NO.3:

5’-ACCGACCGTGCTGGACTCAGATCCATTTCCTTGAAAAAGGCACGGGTTCCCAGAACTCAACTATGAGCGAGCCTGGCG-3’

SEQ NO.4:

5’-ACCGACCGTGCTGGACTCAATCGGGATCCATGTATCCAGGTTCGTTCCTTGAATGGCACACTATGAGCGAGCCTGGCG-3’

SEQ NO.5:

5’-ACCGACCGTGCTGGACTCAACTGCTGAGCCAATTCTTAATAAAGCACGGGTTCCTAGAACTCAACTATGAGCGAGCCTGGCG-3’

SEQ NO.6:

5’-ACCGACCGTGCTGGACTCAATCGGGATCCTAGATCCAGCGAGTTCCCCGAATGCTTTGCACTATGAGCGAGCCTGGCG-3’。

the nucleic acid aptamer as described above, wherein the aptamer,

the functions include one or more of the following:

(1) targeting to bind mycoplasma hyorhinis;

(2) targeting cells that bind to mycoplasma hyorhinis infection;

(3) as a reagent for detecting mycoplasma hyorhinis infection;

(4) as a reagent for detecting mycoplasma hyorhinis infected cells;

(5) as a reagent for detecting in vivo imaging of mycoplasma hyorhinis infected cells.

The aptamer is connected with a fluorescent substance, a radioactive substance, a therapeutic substance, biotin or an enzyme-labeled substance to obtain the aptamer derivative with the same function.

The second purpose of the invention is to provide the application of the nucleic acid aptamer or the nucleic acid aptamer derivative in targeted combination with the mycoplasma hyorhinis or the mycoplasma hyorhinis infected cells. Used for scientific research and excluding diagnosis.

The third purpose of the invention is to provide the application of the nucleic acid aptamer or the nucleic acid aptamer derivative in preparing a reagent for targeting and combining with the mycoplasma hyorhinis.

The fourth purpose of the invention is to provide the application of the nucleic acid aptamer or the nucleic acid aptamer derivative in preparing a cell reagent for targeting and combining with the mycoplasma hyorhinis infection.

The fifth purpose of the invention is to provide the application of the nucleic acid aptamer or the nucleic acid aptamer derivative in preparing a reagent for detecting the mycoplasma hyorhinis infection.

The sixth purpose of the invention is to provide the application of the nucleic acid aptamer or the nucleic acid aptamer derivative in preparing a reagent for detecting mycoplasma hyorhinis infected cells.

The seventh purpose of the invention is to provide the application of the nucleic acid aptamer or the nucleic acid aptamer derivative in preparing a reagent for detecting in vivo living imaging of mycoplasma hyorhinis infected cells.

The invention is characterized in that:

preparing a nucleic acid aptamer containing 10 by using mycoplasma hyorhinis infected cells and a cell-SELEX screening technology¹⁴-10¹⁵A library of different DNA sequences, using cells infected with Mycoplasma hyorhinis as positive sieve cells and cells not infected with Mycoplasma hyorhinis as negative sieve cells, during the screening process, removing DNA bound to the negative sieve cells, retaining DNA bound to the positive sieve cells and performing PCR amplification, repeating the screening process and repeatingThe binding capacity of DNA to cells is detected by fluorophores attached to the DNA molecules until a defined DNA sequence is obtained by sequencing.

The aptamer is a nucleic acid molecular probe and a nucleic acid drug which are similar to antibodies, but have the advantages of small molecular weight, good thermal stability, easy chemical modification and low immunogenicity. The nucleic acid aptamer capable of identifying and targeting the mycoplasma hyorhinis can be combined with the mycoplasma hyorhinis and cells infected by the mycoplasma hyorhinis with high affinity, high specificity and can be used for detecting whether the cells are infected by the mycoplasma hyorhinis, the identification mechanism is that the nucleic acid aptamer is combined with p37 protein of the mycoplasma hyorhinis on the cell surface, the cells infected by the mycoplasma hyorhinis can be blocked and the cells infected by the mycoplasma hyorhinis can be treated by combining the p37 protein of the mycoplasma hyorhinis, the nucleic acid aptamer has the effects of inhibiting cell migration and invasion capacity enhancement brought by the mycoplasma hyorhinis, and the drug sensitivity of the mycoplasma hyorhinis infected cells to gemcitabine can be increased.

In a word, the nucleic acid aptamer for identifying the mycoplasma hyorhinis can be used for preparing a diagnostic reagent for detecting the mycoplasma hyorhinis, can also be used for preparing a reagent for preventing and treating the mycoplasma hyorhinis infection, can be used for preparing a therapeutic reagent for inhibiting cell migration and invasion caused by the mycoplasma hyorhinis, can reverse gemcitabine drug resistance caused by the mycoplasma hyorhinis, and the like, and has a good application prospect.

Drawings

FIG. 1 is a graph showing the flow cytometry detection of the binding capacity of a DNA library to Mycoplasma hyorhinis infected Eca109-mycoplasma infected cells and uninfected Eca109 cells during aptamer screening;

in FIG. 1, the abscissa represents the fluorescence intensity of DNA, and the ordinate represents the number of cells.

FIG. 2 is a graph showing the binding capacity of aptamers of the present invention (SEQ NO.1/SEQ NO.2/SEQ NO.3/SEQ NO.4/SEQ NO.5/SEQ NO.6/SEQ NO.7) to Eca109-mycoplasm infected cells and uninfected Eca109 cells;

in FIG. 2, the abscissa represents the fluorescence intensity of DNA, and the ordinate represents the number of cells.

FIG. 3 shows the detection of binding of aptamers of the present invention (SEQ ID NO. 1/2/3/4/5/6/7) to Eca109-mycoplasm infected cells using confocal laser microscopy.

FIG. 4 is a graph showing the affinity dissociation constant K of the aptamer (SEQ NO.1/SEQ NO.2/SEQ NO.3/SEQ NO.4/SEQ NO.5/SEQ NO.6/SEQ NO.7) with Eca109-mycoplasma infested cells in the example of the present invention_DThe value is obtained.

FIG. 5 shows the results of competitive binding experiments between the aptamer of SEQ NO.1 and SEQ NO.1/SEQ NO.2/SEQ NO.3/SEQ NO.4/SEQ NO.5/SEQ NO.6/SEQ NO.7 according to the present invention.

FIG. 6 shows the detection of co-localization of Mycoplasma hyorhinis and the aptamer of SEQ NO.7 on cells using confocal laser microscopy.

FIG. 7 is a graph showing the ability of the aptamer of SEQ NO.7 of the present invention to bind directly to Mycoplasma hyorhinis using flow cytometry.

FIG. 8 shows the detection of the targeting enrichment effect of the aptamer of SEQ NO.7 on the mouse transplantable tumor model of cells infected with Mycoplasma hyorhinis using a small animal in vivo imager.

FIG. 9 is a schematic diagram of the analysis of the specific protein targets of the aptamer of SEQ NO.7 using aptamer pull-down assay and polyacrylamide gel electrophoresis.

FIG. 10 is a table of mass spectrometric analyses for identifying specific proteins in a protein band of interest of polyacrylamide gel electrophoresis.

FIG. 11 shows that the aptamer-based ELONA assay detects the affinity dissociation constant of p37 pure protein and SEQ NO. 7.

FIG. 12 is a graph showing the ability of a nucleic acid aptamer of SEQ NO.7 to inhibit the migration of Mycoplasma hyorhinis-infected Eca109 cells.

FIG. 13 is a graph showing the ability of the nucleic acid aptamer of SEQ NO.7 to inhibit the invasion of swine mycoplasma rhinotracheale infected Eca109 cells.

FIG. 14 shows the blocking of Mycoplasma hyorhinis infection of Eca109 cells using the nucleic acid aptamer of SEQ NO. 7.

FIG. 15 is a graph showing the reduction of Mycoplasma hyorhinis content in Eca109 cells infected with Mycoplasma hyorhinis using the nucleic acid aptamer of SEQ NO. 7.

FIG. 16 is a graph showing the use of the nucleic acid aptamer of SEQ NO.7 to enhance the sensitivity of Mycoplasma hyorhinis infected Eca109 cells to gemcitabine drugs.

Detailed Description

The following examples are intended to further illustrate the invention without limiting it.

Example 1 construction of a cell model infected with Mycoplasma hyorhinis

Cell source: eca109 cells (esophageal cancer cells) used in this experiment were from the cell bank of the department of Chinese Wuhan Hospital. The selection process was carried out using Eca109 cells infected with Mycoplasma (Eca109-mycoplasma infested) as the forward selection and Eca109 cells not infected as the reverse selection.

Mycoplasma hyorhinis (ATCC 17981) was purchased from ATCC of America, cultured in Hayflick medium for 5 days until the color of the medium changed from orange red to yellow, and the concentration of Mycoplasma was measured to be 10 by gradient dilution⁶CCU/ml, culturing Eca109 cells to 80% density, diluting 10 times mycoplasma hyorhinis bacterial liquid with a cell culture medium, culturing together with the cells, culturing for 2 days, subculturing, adding the mycoplasma bacterial liquid again, repeating the process, subculturing the cells for 2 times, stopping adding the mycoplasma, continuously culturing the cells normally, subculturing, and detecting the degree of infection of the cells by the mycoplasma by using a mycoplasma PCR detection kit to confirm that the cells are completely infected by the mycoplasma.

Example 2 cell-SELEX technology for screening aptamers.

Designing a nucleic acid library:

5 '-ACCGACCGTGCTGGACTCANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNACTATGAGCGAGCCTGGCG-3' (N represents A, T, C, G four bases); SEQ NO.8

A forward primer: 5 '-FITC-ACCGACCGTGCTGGACTCA-3' SEQ NO.9

Reverse primer: 5 '-biotin-CGCCAGGCTCGCTCATAGT-3' SEQ NO.10

Screening process

The invention takes Eca109-mycoplasma infected cell infected by mycoplasma hyorhinis as a positive screening cell and takes an uninfected Eca109 cell as a counter screening cell.

1. Positive screening:

a. dissolving the library by using a binding buffer solution, carrying out denaturation at 95 ℃ for 5min, immediately placing on ice for renaturation for 10min, then removing a culture medium of Eca109-mycoplasma infested cells, adding a library solution, placing at 4 ℃ for incubation for 1 h, removing a supernatant solution after the incubation is finished, washing the cells for multiple times by using a washing buffer solution, discarding the cells, then adding sterile water, hanging the cells, adding the cells into a centrifuge tube, heating and cracking at 95 ℃ for 5min, cooling on ice for 10min, centrifuging at 6000rpm for 5min, and sucking the supernatant to obtain a product after the first round of positive screening.

b. And (b) performing PCR amplification by taking the product obtained in the step a as a PCR template and the primer as a PCR primer, wherein the amplification conditions are as follows: amplifying for 10 cycles at 95 ℃ for 30s, 58 ℃ for 30s, 72 ℃ for 30s and 72 ℃ for 5min to obtain a primary amplification product, and then amplifying again by taking the amplification product as a template to obtain a larger amount of product.

c. And (b) incubating the agarose strain modified by streptavidin and the PCR product obtained in the step b so as to connect the antisense strand of the double strands of the DNA product to agarose beads, adding 0.2M NaOH to denature the double strands of the DNA, collecting the FITC labeled DNA sense strand, and dissolving the DNA sense strand in enzyme-free water after removing salt ions in the DNA sense strand.

2. And (3) reverse screening:

vacuum freeze-drying the collected DNA sense strand into powder, dissolving the powder in a binding buffer, incubating the DNA sense strand with the anti-sieve Eca109 cells for 1 hour according to the method in the step a, collecting supernatant to eliminate DNA sequences bound with the anti-sieve cells, and performing the next round of positive screening on the collected supernatant and the positive sieve cells.

3. And (3) circulation of a screening process: the process of forward and reverse screening was repeated for about 20 rounds until a DNA library was screened that bound strongly to Eca109-mycoplasma infested cells.

4. Monitoring of the screening process: the DNA sense strands obtained by screening in different rounds are lyophilized into powder, dissolved in a binding buffer solution, Eca109-mycoplasma infested cells and uninfected Eca109 cells are cultured to 80% density, the cells are digested with 0.2% EDTA, cell counting is carried out, every 20 ten thousand cells and DNA sense strands with the final concentration of 250nM are incubated for 0.5 hour at 4 ℃, then the nucleic acid sequences in the supernatant are removed by centrifugation, the cells are washed 3 times with a washing buffer solution, and the fluorescence detection of the cells is carried out by a flow cytometer, and the result is shown in figure 1.

5. High-throughput sequencing: DNA products after 21 rounds of screening were subjected to high throughput sequencing.

Example 3 flow cytometry detection of the binding Capacity of nucleic acid sequences sequenced by high throughput to cells

The nucleic acid sequence with the highest enrichment rate obtained by high-throughput sequencing is named as SEQ NO.1, and the sequence is as follows:

base exchange is performed in the middle segment of SEQ NO.1 to obtain an aptamer with the same function as the aptamer:

SEQ NO.2:

SEQ NO.3:

SEQ NO.4:

SEQ NO.5:

SEQ NO.6:

and (3) carrying out base deletion on the 3' end segment of the SEQ NO.1 to obtain the aptamer with the same function as the aptamer SEQ NO.7:

the sequence was synthesized by Shanghai Biotech, labeled with FITC, and the synthesized DNA powder was dissolved in a binding buffer.

Eca109-mycoplasma infested cells and uninfected Eca-109 cells were cultured to 80% density, the cells were digested with 0.2% EDTA solution, every 20 ten thousand cells were incubated with the nucleic acid sequence at a final concentration of 250nM for 0.5 hour at 4 ℃, then the nucleic acid sequence in the supernatant was removed by centrifugation, the cells were washed 3 times with wash buffer, fluorescence detection of the cells was performed by flow cytometry, and the fluorescence intensity of the binding of the initial DNA screening library to the cells was used as a negative control, and the results are shown in FIG. 2.

Example 4 confocal laser microscopy was performed to detect binding of the aptamers of the present invention of SEQ NO.1/SEQ NO.2/SEQ NO.3/SEQ NO.4/SEQ NO.5/SEQ NO.6/SEQ NO.7 to Eca109-mycoplasm infected cells.

Eca109-mycoplasma infected cells are inoculated into an optical culture dish to reach the density of 80%, a supernatant culture medium is discarded, DPBS is added for rinsing once, then a binding buffer solution is added, a aptamer with the final concentration of 250nM and labeled with FITC fluorescein is added, the mixture is incubated for 1 hour at room temperature, a supernatant is discarded, rinsing is carried out for 3 times by using the washing buffer solution for 5 minutes each time, and then the condition that the aptamer is bound with the cells is observed by using a laser confocal microscope through 488nM exciting light, and the result is shown in figure 3. The results in the figure show that the binding of SEQ NO.1/SEQ NO.2/SEQ NO.3/SEQ NO.4/SEQ NO.5/SEQ NO.6/SEQ NO.7 on the cell surface all fluoresce green, while Lib does not show fluorescence.

Example 5 flow cytometry detection of aptamers of SEQ NO.1/SEQ NO.2/SEQ NO.3/SEQ NO.4/SEQ NO.5/SEQ NO.6/SEQ NO.7 affinity dissociation constants to Eca109-mycoplasma infested cells

Culturing Eca109-mycoplasma infested cells to 80% density, digesting the cells with 0.2% EDTA, counting the cells, dividing into 2 groups, one group incubating 20 ten thousand cells with 25nM, 50nM, 75nM, 100nM, 200nM, 300nM, 400nM, 600nM aptamer SEQ NO.1/SEQ NO.2/SEQ NO.3/SEQ NO.4/SEQ NO.5/SEQ NO.6/SEQ NO.7 for 0.5 hour, the other group incubating 20 ten thousand cells with 25nM, 50nM, 75nM, 100nM, 200nM, 300nM, 400nM, 600nM initial screening library Lib for 0.5 hour, centrifuging to remove the nucleic acid sequence in the supernatant, washing the cells 3 times with washing buffer, performing flow cytometry to detect the fluorescence intensity of the cells, calculating the average fluorescence intensity at each concentrationIntensity and corresponding concentration values, the fluorescence value of the initial screening library Lib at the same concentration was subtracted from the fluorescence value of the aptamer at each concentration of SEQ No.1/SEQ No.2/SEQ No.3/SEQ No.4/SEQ No.5/SEQ No.6/SEQ No.7 to subtract the fluorescence value resulting from non-specific binding, by the formula Y ═ B_maxX/(K_D+ X) calculation of K for each aptamer_DValues wherein Y is the fluorescence value, B_maxIs the maximum fluorescence value, X is the nucleic acid concentration, K_DThe results are shown in FIG. 4 for the affinity dissociation constant.

Example 6 flow cytometry detection of aptamers to the competitive assay of binding epitopes of SEQ NO.1/SEQ NO.2/SEQ NO.3/SEQ NO.4/SEQ NO.5/SEQ NO.6/SEQ NO. 7.

Eca109-mycoplasma infected cells are cultured to 80% density, the cells are digested by 0.2% EDTA, the cells are counted, SEQ NO.1 with FITC fluorescent modification is mixed with Lib/SEQ NO.1/SEQ NO.2/SEQ NO.3/SEQ NO.4/SEQ NO.5/SEQ NO.6/SEQ NO.7 without fluorescent modification, the final concentration of the SEQ NO.1 with fluorescent modification is 250nM, the final concentration of the nucleic acid sequence without fluorescent modification is 2.5 MuM, each group is incubated with 20 ten thousand Eca109-mycoplasma infected cells for 0.5 hour, then the nucleic acid sequence in the supernatant is removed by centrifugation, the cells are washed for 3 times by using washing buffer, and then the fluorescence intensity of the cells is detected by flow cytometry, and the result is shown in figure 5.

It can be seen in fig. 5 that FITC-labeled SEQ No.1 can detect stronger fluorescence in conjunction with cells, and has no significant effect on its association after 2.5 μ M unlabeled Lib is added, and that after 2.5 μ M unlabeled aptamer SEQ No.1/SEQ No.2/SEQ No.3/SEQ No.4/SEQ No.5/SEQ No.6/SEQ No.7 is added, the association ability of SEQ No.1 can be significantly weakened, indicating a competitive association phenomenon, so that it can be known that the epitopes to which all aptamers are associated are the same, and have the same target and function.

Example 7 confocal laser experiments to detect co-localization of Mycoplasma hyorhinis and the aptamer of SEQ NO.7 on Eca109-mycoplasma infested cells.

Eca109-mycoplasma infested cells are inoculated in an optical culture dish to reach the density of 80%, a supernatant culture medium is discarded, DPBS (double DPBS) is added for rinsing, then a binding buffer solution is added, 1 mu l of an anti-mycoplasma hyorhinis p37 protein antibody is added, the supernatant is washed off after 1 hour of room temperature incubation, the binding buffer solution is added again, 2 mu l of a secondary antibody coupled with FITC fluorescein is added, a nucleic acid aptamer SEQ NO.7 with the final concentration of 250nM labeled with cy5 fluorescein is added, 10 mu l of hoechst 33342 staining solution with the concentration of 2 mu g/ml is added, the supernatant is discarded after 1 hour of room temperature incubation, rinsing is carried out for 3 times for 5 minutes each time by using the washing buffer solution, and then the localization conditions of mycoplasma and the nucleic acid aptamer are observed by using a laser confocal microscope through excitation light of 405nM, 488nM and 640nM, and the result is shown in figure 6.

In FIG. 6, hoechst 33342 dye stains a part of cells with DNA, when mycoplasma infects cells, the part adheres to cell membranes, and since mycoplasma also has a DNA structure, hoechst 33342 stains and displays mycoplasma on the membranes, p37 protein is a membrane protein which is specific to mycoplasma hyorhinis and can be used as a protein marker of the mycoplasma hyorhinis, therefore, hoechst 33342 dye is used for characterizing that cells are infected by mycoplasma, p37 antibody is used for characterizing that cells are infected by mycoplasma hyorhinis, and nucleic acid aptamer SEQ NO.7 stains the cells in accordance with the staining distribution of the former 2.

Example 8 flow cytometry detection of the aptamer of SEQ NO.7 for the ability to bind directly to Mycoplasma hyorhinis

At 10⁵Adding 5 mu L of antibody of a targeted mycoplasma hyorhinis specific antigen p37 protein into CCU/ml mycoplasma hyorhinis bacterial liquid, adding magnetic beads coupled with goat anti-mouse secondary antibodies for incubation for 2 hours, connecting the mycoplasma hyorhinis to the magnetic beads through the antibody, after excessive liquid is eluted, suspending the magnetic beads in a binding buffer solution, adding aptamer SEQ NO.7 coupled with FITC fluorescence or an initial screening library Lib for incubation for 30 minutes, eluting for 3 times, and analyzing the fluorescence value of the magnetic beads through a flow cytometer, wherein the result is shown in FIG. 7.

In the graph 7, the abscissa is fluorescence intensity, the ordinate is the number of magnetic beads, the mycoplasma hyorhinis cultured in a culture medium is coupled on the magnetic beads through antibodies in the experiment, the FITC-labeled aptamer SEQ NO.7 is combined with the mycoplasma hyorhinis, and a fluorescence value on the magnetic beads is detected by a flow cytometer.

Example 9 application of the aptamer SEQ NO.7 detected by the animal Living body imager in vivo imaging in tumor-bearing mouse model

4 week old BALB/C nude mice were purchased and injected subcutaneously 7X 10 per mouse⁶Eca109 mycoplasma infested cells, when the tumor body grows to 1cm³On the left and right, the mice were randomly divided into 2 groups, one group of mice was injected with 5nmol of aptamer SEQ No.7 labeled with cy5 fluorescent molecule through tail vein, the other group was injected with 5nmol of initial screening nucleic acid library Lib labeled with cy5 fluorescent molecule, 10 minutes later, the enrichment of cy5 fluorescence in the mice was photographed by a living body imager, then, the images were photographed every 30 minutes, 3 hours later, the mice were euthanized, the organs of the mice were depalletized, and the distribution of the fluorescent aptamer of each organ was detected, and the results are shown in fig. 8.

In FIG. 8 Eca109 mycoplasma infected cell was implanted subcutaneously in immunodeficient mice, and after the tumors grew to a suitable size, the initial screening library was injected intravenously with cy5 fluorescein-labeled aptamer SEQ NO.7 or cy5 fluorescein-labeled from the mouse tail, and the results showed that the aptamer SEQ NO.7 was able to bind to the site of the tumor tissue infected with Mycoplasma hyorhinis in mice, indicating good binding of the aptamer in vivo.

Example 10 aptamer Pull-down experiments and Mass Spectrometry identification of protein targets

The Eca109-mycoplasma infested cell is amplified and cultured in a large quantity, because the previous confocal experiment proves that the aptamer is combined on the surface of a cell membrane, the membrane protein of the cell is extracted by a membrane protein extraction kit, then the membrane protein is incubated with a protein confining liquid for 1 hour, then the membrane protein is incubated with a biotin-modified aptamer SEQ NO.7 or a biotin-modified random DNA library sequence for 1 hour, then 100 mul of streptavidin-modified agarose beads are added for incubation for 1 hour, the supernatant is removed by centrifugation, the agarose beads are washed 5 times by DPBS (deep plasma-coupled plasma) to remove unbound nucleic acid sequences, then 50 mul of protein loading buffer solution is added into the agarose beads, the incubation is carried out for 10min at 95 ℃, the supernatant is centrifuged and is subjected to polyacrylamide gel electrophoresis, after the electrophoresis is finished, the electrophoresis gel is subjected to Coomassie brilliant blue staining, and then nonspecific staining is eluted, the image of the gel was scanned by a scanner and the differential protein bands were excised for mass spectrometry, and the results are shown in fig. 9 and 10.

In FIG. 9, it can be seen that there is a protein in lane SEQ No.7, which is clearly different from the other lanes and indicated by a red box, and this protein is likely to be a protein to which the aptamer specifically binds, i.e., a target protein, and it is cut off with a blade for mass spectrometry.

The score, coverage, unique peptide fragment and relative abundance of the protein in the band obtained by mass spectrometry are shown in fig. 10, and the position listed in the figure is mycoplasma hyorhinis high-affinity transporter p37 which is the target protein of the aptamer SEQ NO. 7.

Example 11 Enzyme-linked Oligonucleotide adsorption assay (ELONA) assay for detecting affinity dissociation constants of aptamer SEQ NO.7 and GST-p37 purified protein

Expressing high-purity GST-p37 and GST protein by using an escherichia coli system prokaryotic system, diluting GST-p37 and GST protein to 5 mu g/mL, adding 100ul to a 96-well ELISA plate, incubating overnight at 4 ℃, removing supernatant, washing wells with PBST solution for 3 times, adding 1% BSA solution to seal for 2 hours, removing sealing solution, washing wells with PBST for 3 times, adding the biotin-modified SEQ NO.7 aptamer of 600nM, 400nM, 200nM, 100nM, 50nM, 25nM, 12.5nM, 6.25nM, 3.125nM, 1.5nM, 0.75nM, 0.375nM and 0.153nM to the GST and GST-p37 wells respectively, incubating an initial screening library Lib modified by biotin with the same concentration to the control wells at the same time, incubating at room temperature for 2 hours, removing the solution, washing the wells with PBST for 3 times, adding horseradish peroxide diluted by 1:2000, and streptavidin solution labeled with 1 hour at room temperature, after washing the wells with PBST for 3 times, adding 200. mu.L of TMB color developing solution for developing for 5 minutes, then adding 50. mu.L of TMB color developing termination solution, detecting the absorbance of OD450 by using an microplate reader, subtracting the absorbance of the initial screening library Lib concentration of the corresponding concentration from the absorbance of the SEQ NO.7 aptamer of each concentration, removing the nonspecific binding value of Lib, and obtaining the final product by the formula Y-B_maxX/(K_D+ X) calculation of affinity dissociationNumber K_DThe values, results are shown in FIG. 11.

The enzyme-linked oligonucleotide adsorption test is similar to ELISA test principle, in the test, an enzyme-labeled 96-well plate is used for adsorbing p37 protein with GST label and GST protein at the bottom of the well plate, the GST protein is negative control protein, aptamer SEQ NO.7 with different concentrations is added into a well adsorbed with GST-p37 protein and GST protein for incubation, initial screening library Lib with different concentrations is also added into a well adsorbed with GST-p37 protein and GST protein for incubation, biotin labeled by nucleic acid sequence is combined with HRP-avidin, then the mixture is developed with substrate TMB, the binding capacity of the SEQ NO.7 or Lib to the protein is reflected, the reading value of the Lib group is subtracted from the reading value of an enzyme-labeled instrument of the SEQ NO.7 group, the differential binding is removed, a standard curve is prepared, and a formula is used for calculating an affinity dissociation constant K_DK of SEQ NO.7 is shown_DThe value was 4.75. + -. 1.16nM and demonstrated high affinity binding to GST-p37 protein and no binding to GST protein, indicating specific binding to p 37.

Example 12 experiment of the ability of the nucleic acid aptamer SEQ NO.7 to inhibit the migration of Eca109-mycoplasma infested cells.

Eca109-mycoplasma infected cells are inoculated into a 96-well plate, when the cell density reaches 95%, cell scratching treatment is carried out by using a scratching instrument, a supernatant culture medium is discarded, the culture medium is replaced by a culture medium with 1% of serum concentration, a nucleic acid aptamer SEQ NO.7 and an initial screening library Lib sequence are added until the final concentration is 2 mu M, and images of cell scratching healing are shot by using a microscope at a time point after 24 hours, wherein the results are shown in figure 12.

In FIG. 12, the cells are scribed with equal distances by an instrument, the cells in the groove are removed, then the serum content of the culture medium is reduced to 1%, the proliferation capacity of the cells is inhibited, after a certain time, the cells move into the groove by utilizing the migration capacity, and the migration capacity is measured by the width of the groove, and the figure shows that the groove width is wider compared with that of Lib and the group of SEQ NO.7, which indicates that the SEQ NO.7 can inhibit the migration capacity of the cells.

Example 13 experiment of the ability of the aptamer SEQ NO.7 to inhibit the invasion of Eca109-mycoplasma infested cells.

Corning Matrigel was mixed with DMEM medium at a ratio of 1:10, added to the upper chamber of a 8 μ M transwell chamber, incubated at 37 ℃ for 2 hours to coagulate Matrigel at the bottom of the chamber, the unclotted supernatant was discarded, Eca 109-myeloplasma fed cells were seeded in the upper chamber of the chamber with 1% serum concentration DMEM and the aptamer SEQ NO.7 and the initial screening library Lib were added to a final concentration of 2 μ M, 10% serum DMEM medium was added to the lower chamber, the medium was discarded after 48 hours, the uninfected cells in the upper chamber were wiped off with a cotton swab, and then polyformaldehyde fixation and crystal violet staining were performed to photograph the number of stained cells, as a result shown in FIG. 13.

The invasion of cells refers to the capacity of decomposing extracellular matrix by extracellular enzyme protein, Matrigel matrix gel is used for simulating the extracellular matrix in an experiment, the Matrigel matrix gel can penetrate through a transwell chamber after being subjected to enzymolysis, reaches the bottom of the chamber and is further stained by crystal violet, the number of the stained cells can reflect the invasion capacity of the cells, and the figure shows that the number of the stained cells is less than that of the stained cells of Lib and SEQ NO.7 groups, which indicates that the SEQ NO.7 can inhibit the invasion capacity of the cells.

Example 14 experiment of nucleic acid aptamer SEQ NO.7 blocking Mycoplasma hyorhinis infected cells.

Primers for amplifying p37 gene:

p37-qPCR-F:5'-TATCTCATTGACCTTGACTAAC-3'SEQ NO.11

p37-qPCR-R:5'-ATTTTCGCCAATAGCATTTG-3'SEQ NO.12

primers for amplification of GAPDH:

GAPDH-F：5'-TGAAGGTCGGAGTCAACGG-3'SEQ NO.13

GAPDH-R:5'-CCTGGAAGATGGTGATGGG-3SEQ NO.14

eca109 cells not infected with mycoplasma hyorhinis are inoculated in a 24-well plate to reach the density of 80%, an initial screening library Lib processing hole and a nucleic acid aptamer SEQ NO.7 processing hole are arranged, and then mycoplasma hyorhinis is added to 10% of the hole plates⁵CCU/ml, adding aptamer SEQ NO.7 to a final concentration of 0.1. mu.M, 0.2. mu.M, 0.4. mu.M, 0.6. mu.M, 0.8. mu.M, 1. mu.M, 2. mu.M, 4. mu.M or adding initial screening library Lib to the same concentration, placing in an incubator to incubate for 24 hoursDiscarding the supernatant culture medium, rinsing the cells for 3 times with DPBS, digesting the cells with 0.2% EDTA solution and placing the cells in a centrifuge tube, centrifuging to remove the supernatant, adding DPBS for one time, centrifuging to remove DPBS, adding 100 mul deionized ultrapure water, incubating at 95 ℃ for 10min, centrifuging at 12000rpm for 2min, taking the supernatant to perform real-time fluorescence quantitative PCR detection on the CT value, calculating the content of p37 gene, and calculating 2^ with GAPDH gene as reference^-ΔΔCTThe ratio of mycoplasma hyorhinis infection on cells was determined by the ratio and the results are shown in fig. 14.

In FIG. 14, the mycoplasma hyorhinis is added into uninfected cells, then the nucleic acid aptamer SEQ NO.7 or Lib is added, the content of the mycoplasma hyorhinis on the cell surface is gradually reduced through qPCR detection along with the increasing concentration of the SEQ NO.7, and the Lib does not have the effect, so that the nucleic acid aptamer has the effect of blocking the mycoplasma hyorhinis infection.

Example 15 experiment of nucleic acid aptamer SEQ NO.7 for treatment of Mycoplasma hyorhinis infected cells.

Inoculating Eca109-mycoplasma infested cells infected with mycoplasma hyorhinis into a 24-well plate, setting an initial screening library Lib processing well (Lib) and a nucleic acid aptamer SEQ NO.7 processing well (SEQ NO.7), adding a DNA library or a nucleic acid aptamer into the corresponding well to 2 μ M, placing the well in an incubator for 24 hours, discarding the supernatant medium, rinsing the cells 3 times with DPBS, digesting the cells with 0.2% EDTA solution and placing the cells in a centrifuge tube, centrifuging to remove the supernatant, adding DPBS for one time, centrifuging to remove DPBS, adding 100 μ l deionized ultrapure water, incubating at 95 ℃ for 10min, centrifuging at 12000rpm for 2min, taking the supernatant to perform real-time fluorescence quantitative PCR (same primer as in example 14) of p37 and GAPDH, and calculating the factor 2^ with the GAPDH gene as a reference^-ΔΔCTAnd then the infection rate was calculated, and the results are shown in fig. 15.

FIG. 15 shows the decrease in the extent of infection of cells treated with aptamer, when the cells were treated with SEQ NO. 7.

Example 16 aptamer SEQ No.7 enhances the drug sensitivity of mycoplasma hyorhinis infected cells to gemcitabine guitarabine.

Eca109-mycoplasma infested cells infected with Mycoplasma hyorhinis were seeded in 96-well plates at 5000 cells per well, and the following experimental groups were set up: a non-treated group (NC, nothing except for cells), a GEM group (i.e., only the guitar cuffin group), a GEM + initial screening library Lib-treated group (guitar cuffin + Lib), a GEM + aptamer SEQ NO. 7-treated group (guitar cuffin + SEQ NO.7), the aptamer SEQ NO. 7-treated group wells were filled with the aptamer SEQ NO.7 after inoculation, the initial screening library Lib was filled with the initial screening library Lib-treated group wells to a final concentration of 2. mu.M, the cells were left in an incubator for 24 hours until the cells were attached, the supernatant was discarded, 100. mu.L of fresh medium was added, and then a gemcitabine solution was added to all wells except the NC group to a final concentration of 3. mu.M, 10. mu.L of a CCK8 solution was added to the wells after the plates were left in the incubator for 72 hours, the cell viability was calculated based on the readings of the microplate reader 450 OD, the results are shown in FIG. 16.

In FIG. 16, since it has been reported that Mycoplasma hyorhinis can cause resistance of cells to chemotherapeutic drug gemcitabine, the content of Mycoplasma hyorhinis on cells is reduced after the aptamer SEQ NO.7 is added, and the resistance is reversed, so that the effect of gemcitabine can be enhanced, and the cell survival rate is lower.

Sequence listing

<110> university of Hunan

<120> nucleic acid aptamer specifically bound with mycoplasma hyorhinis and application thereof

<160> 14

<170> SIPOSequenceListing 1.0

<210> 1

<211> 80

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

accgaccgtg ctggactcac gtcgtccatt tccttgaaaa aggcacgggt tccatgaact 60

cactatgagc gagcctggcg 80

<210> 2

<211> 77

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

accgaccgtg ctggactcaa gcggtttccg tgaacctgcg agttccctga atacttgtac 60

tatgagcgag cctggcg 77

<210> 3

<211> 78

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

accgaccgtg ctggactcag atccatttcc ttgaaaaagg cacgggttcc cagaactcaa 60

ctatgagcga gcctggcg 78

<210> 4

<211> 78

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

accgaccgtg ctggactcaa tcgggatcca tgtatccagg ttcgttcctt gaatggcaca 60

ctatgagcga gcctggcg 78

<210> 5

<211> 82

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

accgaccgtg ctggactcaa ctgctgagcc aattcttaat aaagcacggg ttcctagaac 60

tcaactatga gcgagcctgg cg 82

<210> 6

<211> 78

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

accgaccgtg ctggactcaa tcgggatcct agatccagcg agttccccga atgctttgca 60

ctatgagcga gcctggcg 78

<210> 7

<211> 70

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

accgaccgtg ctggactcac gtcgtccatt tccttgaaaa aggcacgggt tccatgaact 60

cactatgagc 70

<210> 8

<211> 78

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

accgaccgtg ctggactcan nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnna 60

ctatgagcga gcctggcg 78

<210> 9

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

accgaccgtg ctggactca 19

<210> 10

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

cgccaggctc gctcatagt 19

<210> 11

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

tatctcattg accttgacta ac 22

<210> 12

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

attttcgcca atagcatttg 20

<210> 13

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

tgaaggtcgg agtcaacgg 19

<210> 14

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

cctggaagat ggtgatggg 19

Claims

1. A nucleic acid aptamer specifically binding to mycoplasma hyorhinis, which is characterized by comprising the following sequence:

SEQ NO.1:

SEQ NO.2:

5’-ACCGACCGTGCTGGACTCAAGCGGTTTCCGTGAACCTGCGAGTTCCCTGAATACTTGTACTATGAGCGAGCCTGGCG-3’；

SEQ NO.3:

5’-ACCGACCGTGCTGGACTCAGATCCATTTCCTTGAAAAAGGCACGGGTTCCCAGAACTCAACTATGAGCGAGCCTGGCG-3’；

SEQ NO.4:

5’-ACCGACCGTGCTGGACTCAATCGGGATCCATGTATCCAGGTTCGTTCCTTGAATGGCACACTATGAGCGAGCCTGGCG-3’；

SEQ NO.5:

5’-ACCGACCGTGCTGGACTCAACTGCTGAGCCAATTCTTAATAAAGCACGGGTTCCTAGAACTCAACTATGAGCGAGCCTGGCG-3’；

SEQ NO.6:

5’-ACCGACCGTGCTGGACTCAATCGGGATCCTAGATCCAGCGAGTTCCCCGAATGCTTTGCACTATGAGCGAGCCTGGCG-3’；

or SEQ NO.7:

2. the nucleic acid aptamer according to claim 1,

the functions of the aptamer comprise one or more of the following:

(1) targeting to bind mycoplasma hyorhinis;

(2) targeting cells that bind mycoplasma hyorhinis infection;

(3) as a reagent for detecting mycoplasma hyorhinis infection.

3. The nucleic acid aptamer according to claim 1,

by connecting a fluorescent substance, a radioactive substance, a therapeutic substance, biotin, or an enzyme-labeled substance to the aptamer, the aptamer derivative having the same function is obtained.

4. Use of the nucleic acid aptamer or nucleic acid aptamer derivative according to any one of claims 1 to 3 in the preparation of a reagent for detecting mycoplasma hyorhinis infection.

5. Use of the nucleic acid aptamer or nucleic acid aptamer derivative according to any one of claims 1 to 3 in the preparation of a reagent for detecting mycoplasma hyorhinis infected cells.

6. Use of the nucleic acid aptamer or nucleic acid aptamer derivative of any one of claims 1 to 3 in the preparation of a reagent for in vivo imaging of cells infected with mycoplasma hyorhinis.