CN110184336B - Folic acid receptor FOLR2 and application of coding gene thereof - Google Patents
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Abstract
The invention discloses application of a folate receptor FOLR2 and a coding gene thereof as a biomarker in preparation of a product for diagnosing neural lyme disease or a medicament for treating the neural lyme disease. Clinical diagnosis and treatment of the neural lyme disease are not completely clear at present, so that the specific high expression of the FOLR2 in the neural lyme disease has important significance for diagnosis, treatment and pathogenesis research of diseases.
Description
Technical Field
The invention relates to the technical field of biomarkers, in particular to application of a folate receptor FOLR2 and a coding gene thereof.
Background
Lyme Neuroreliosis (LNB) refers to an infectious disease of the nervous system caused by infection with Borrelia burgdorferi, which is one of the most serious clinical manifestations of Lyme disease. Borrelia burgdorferi is highly neurotropic, can remain latent in the central or peripheral nervous system for a long time, and can generate different neuropathy at different stages, and the central nervous system is mostly expressed by lymphocytic meningitis (encephalopathy). Neurolyme disease is characterized by recurrent attacks and progressive exacerbations, and some patients may develop hypomnesis, dementia, personality disorders, and the like. Since LNBs can cause extensive nervous system damage and have complicated serious disability, the harm is serious and in recent years, they have received attention from clinicians and researchers.
The early symptoms of the neural lyme disease are atypical, the onset of the disease is insidious, the latent period can be as long as months to 1 year, and tuberculosis, viral meningitis, bell paralysis, multiple sclerosis and the like are sometimes misdiagnosed. It is also necessary to identify the pathological changes of skin, heart, joints and nervous system caused by other causes, such as rheumatic fever, erythema multiforme, rheumatoid arthritis, etc., and clinical diagnosis and treatment still have certain difficulties.
Therefore, how to provide a biomarker which has practical significance for the diagnosis, treatment and pathogenesis research of the neural lyme disease becomes a technical problem to be solved in the field.
Disclosure of Invention
In view of the above, the folate receptor FOLR2 is used as a biomarker, and it is determined that FOLR2 is significantly increased in occurrence and development of the neural lyme disease and is closely related to occurrence and development of the disease. The specific expression and obvious increase of FOLR2 have important significance for the diagnosis, treatment and mechanism research of the neural lyme disease.
The invention provides the following technical scheme:
the application of folate receptor FOLR2 as a biomarker in the preparation of products for diagnosing the neural lyme disease.
The application of the coding gene of the folate receptor FOLR2 as a biomarker in the preparation of products for diagnosing the neural lyme disease.
The application of folate receptor FOLR2 as a biomarker in the preparation of drugs for treating neural lyme disease.
The application of the coding gene of the folate receptor FOLR2 as a biomarker in the preparation of drugs for treating the neural lyme disease.
According to the technical scheme, the invention provides the application of using the folate receptor FOLR2 and the coding gene thereof as the biomarker, and provides more possibilities for the diagnosis, treatment and mechanism research of the neural lyme disease.
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FIG. 1 shows Borrelia burgdorferi under high power microscope after 7 days of culture.
FIG. 2 is a chart showing Fold Change (FC) heatmap of FOLR2 gene expression level in experimental and control groups.
FIG. 3 shows the relative expression amounts of FOLR2 mRNA in the experimental group and the control group.
FIG. 4 shows the FOLR2 protein expression levels of the experimental group and the control group.
FIG. 5 shows the electrophoretogram of total protein of the staining-free gel.
Detailed Description
The technical solutions in the embodiments of the present invention are 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. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1 Resuscitation and culture of Borrelia burgdorferi
Borrelia burgdorferi 4680 (Borreliaella burgdorferi, B31) was purchased from German Collection of microorganisms and cell cultures (DSMZ).
The recovery and culture of the borrelia burgdorferi specifically comprises the following operation steps:
1. an 18mL centrifuge tube was prepared and 9.5mL BSK II medium was added.
2. 0.5ml of the borrelia burgdorferi frozen at-80 ℃ is added into the centrifuge tube.
3.37℃、5％CO 2 And culturing in 100% wet-heat incubator for 3 days.
4.3 days later, mix the bacteria solution, take 5 μ L bacteria solution from the centrifuge tube and drip on the slide glass, after the cover slip, count under the microscope, and observe its activity, culture until the thalli number is 60-120/HPF.
5. The above-mentioned culture solution was transferred to a 50mL centrifuge tube, and 30mL of BSK II medium was added thereto.
6. Tightening the cap at 37 ℃ and 5% CO 2 And continuously culturing for 4 days in a 100% damp-heat incubator.
After 7.4 days, the cells were mixed, 5. Mu.L of the cells were dropped on a glass slide from the centrifuge tube, and the cells were counted under a microscope after being covered with a glass slide, and the activity was observed and cultured until the number of cells was about 160/HPF (FIG. 1).
Centrifuging at 8.2000 Xg for 10min, pouring out the upper culture medium, washing with PBS buffer solution for 2 times, adding 3mL 1640 complete culture medium containing 10% fetal calf serum, and mixing well.
9. Taking 10 mu L of the bacterial liquid, slowly adding the bacterial liquid from the grooves on the two sides of the middle platform of the counting plate along the lower edge of the cover glass, filling the counting area with the bacterial liquid by utilizing the surface tension of the liquid without generating bubbles, and sucking the excess bacterial liquid flowing out of the grooves by using absorbent paper.
10. Standing for a moment, placing the counting plate on an objective table, stably clamping, finding a counting area under the low-power lens, and then switching the high-power lens to observe and count.
11. Calculating the concentration of the bacterial liquid, wherein the bacterial liquid per mL contains the number of the borrelia burgdorferi = the average number (N) of each borrelia burgdorferi x coefficient (K) x bacterial liquid dilution multiple (d) (K =4 x 10) 6 If the concentration of the bacteria liquid is too high and the counting is influenced, the bacteria liquid can be diluted first and then counted).
12. The bacterial suspension was prepared in 1640 complete medium containing 10% fetal calf serum at a concentration of 1X 10 7 the/mL bacterial suspension is ready for use.
Example 2 Borrelia burgdorferi coculture with frontal cortex of monkey brain
The rhesus monkey used in the experiment is presented by the institute of medical biology of academy of Chinese medical sciences, and the license number of the unit is SCXK (Yunnan) K2015-0004, which is a breeding base of the experimental monkey in Yunnan.
1. 3 healthy rhesus monkeys were randomly selected, sacrificed after anesthesia, and were harvested and the frontal cortex tissues of the monkeys were immediately and completely soaked in PBS buffer.
2. Experimental grouping and preparation
(1) Borrelia burgdorferi and monkey brain frontal cortex tissue sample coculture group (experimental group): 1X 10 of the product prepared in example 1 are taken 7 Perml bacterial suspension, added to 25cm 2 4mL per bottle.
(2) Borrelia burgdorferi-free infection group (control group): adding 1640 culture medium containing 10% fetal bovine serum to 25cm 2 4mL per bottle.
3. The frontal cortex tissue of monkey brain was washed 2 times with PBS solution, cut into 0.5g of sheet tissue samples, and then transferred to the culture flasks of each group, and 3 tissue samples were placed in each flask.
4. Placing the culture flask in which the tissue sample is placed at 37 deg.C, 5% 2 Co-culturing in an incubator.
5. After 6h,12h and 24h of culture, tissue samples were collected at each time point. Tissue samples from each group were collected in 1.8mL cryovials, and each tissue sample was individually stored in 1 vial (3 vials per vial) at-80 ℃.
Example 3 transcriptomic sequencing and bioinformatics analysis
Total RNA was extracted from the frontal cortex tissue of monkey brains collected at each time point in example 2 by Trizol. Wuhan Kingrendering was entrusted with high-throughput sequencing using an Illumina sequencer and a sequencing library matched thereto was constructed.
The detailed steps of RNA library construction are as follows:
1. after the total RNA sample is qualified, taking 5 mu g of total RNA for subsequent library building experiments;
2. enriching mRNA with magnetic beads carrying Oligo (dT), and then adding Fragmentation Buffer to fragment the mRNA into short fragments;
3. taking the mRNA after the fragment as a template, synthesizing single-strand cDNA by using a hexabasic random primer, and then adding a buffer solution, dNTPs and DNA polymerase I to synthesize double-strand cDNA;
4. carrying out end repair on the eluted and purified double-stranded cDNA, adding a base A, and adding a sequencing joint for treatment;
5. recovering target fragments with different sizes by using magnetic beads and carrying out PCR amplification;
6. performing quality inspection on the constructed library by using an agarose electrophoresis method;
7. quantifying the library by utilizing the Qubit 2.0 to determine whether the concentration of the library is suitable for being operated on the computer;
8. and after the quality of the library is qualified, performing on-machine sequencing on different libraries on an Illumina sequencer according to the requirements of effective concentration and target off-machine data volume.
After obtaining sequencing raw data (raw data), firstly filtering the raw data to obtain high-quality sequencing data (clean data), and comparing the sequencing data (clean data) to a reference genome of a project species to obtain comprehensive transcript information.
Comparing clean data with reference genome to obtain comprehensive transcript information. The mRNA of the gene encoding the protein annotated to the genome is analyzed, and the expression level of the gene is counted, and the correlation of the expression characteristics of the sample gene between the groups and the genes with different expression is evaluated. Evaluating the difference multiple and the significance level, and screening the difference expression genes; the gene was expressed as a differentially expressed gene using a log FC with an absolute value of >1 and fdr <0.05 as a standard. And classifying different expression regulation and control modes by using a hierarchical clustering method. And further carrying out gene expression quantification on the differential expression gene and GO and KEGG Pathway analysis.
As shown in FIG. 2, the differentially expressed genes were significantly changed at the time points of 6h,12h and 24h in the experimental group compared with the control group. Wherein, yellow represents that the gene expression level of the experimental group is up-regulated compared with the control group, blue represents that the gene expression level of the experimental group is down-regulated compared with the control group, the brighter yellow represents the up-regulation, and the brighter blue represents the down-regulation. The FOLR2 gene has obviously different expression quantity changes from other genes in 6h,12h and 24 h: FOLR2 is down-regulated in gene expression level at 6h and up-regulated in gene expression level at 12h and 24 h; the FC of FOLR2 at 12h and 24h was 2.3536 and 5.8588, respectively, indicating that FOLR2 was significantly increased in the development of neural lyme disease.
Rhesus folate receptor FOLR2 Gene ID 718405; NCBI Reference Sequence NM _001260645.1.
Example 4 real-time fluorescent quantitative PCR detection of FOLR2 mRNA expression level
Taking the two groups of tissue samples collected at each time point in the example 2, adding Trizol, standing for 5min at room temperature, centrifuging for 5min at 12,000rpm, and removing precipitates; adding chloroform into 200 μ L chloroform/mL Trizol, shaking, mixing, standing at room temperature for 15min, centrifuging at 4 deg.C for 15min at 12,000 Xg, and sucking the upper water phase into another centrifuge tube; adding 0.5mL of isopropanol/mL of Trizol into the isopropanol, uniformly mixing, standing at room temperature for 5-10min, centrifuging at 4 ℃ for 10min at 12,000 Xg, removing supernatant, and depositing RNA at the bottom of the tube; adding 75% ethanol into 1mL of 75% ethanol/mL of Trizol, gently oscillating the centrifuge tube, suspending and precipitating, centrifuging at 4 ℃ for 5min at 8,000 Xg, and discarding the supernatant as much as possible; air-drying or vacuum-drying at room temperature for 5-10min, and adding a proper amount of TE buffer to dissolve the RNA sample.
RNA samples were reverse transcribed into cDNA using a cDNA synthesis kit and stored at-20 ℃.
The concentration of cDNA obtained by reverse transcription of RNA is used as a standard substance, 6 centrifuge tubes are prepared, 20 mu L of Easy Dilution is added into each centrifuge tube, 5 mu L of initial cDNA solution is added into a first tube, the mixture is gently mixed, 5 mu L of initial cDNA solution is absorbed and added into a second tube for mixing, and the steps are repeated until reaching a sixth tube, so that a series of cDNA standard substances are obtained.
The Kunming Shuozhi organism company was assigned to synthesize primers whose sequences are shown in Table 1.
TABLE 1
Detection was performed using SYBR Fluorogenic quantitative QRT-PCR reaction kit, reaction system (25. Mu.L) as in Table 2.QRT-PCR reaction conditions are shown in Table 3 (two-step method).
TABLE 2
TABLE 3
QRT-PCR was performed using cDNA standards as templates, and a standard curve was obtained by Bio-Rad CFX96TM software, which determines the coefficient (R) 2 ) Should be greater than 0.98, the amplification efficiency (E) should be between 95% and 105%.
And (4) result judgment standard: the QRT-PCR detection result is the threshold cycle number (Ct value) obtained by the Bio-Rad CFX96TM software. When the Ct value range of the target gene is 15-35 and the Ct value range of the internal reference gene is 15-25, the QRT-PCR detection result is reliable.
By utilizing a standard curve, the software can automatically calculate quantitative results of target genes and reference genes, wherein the quantitative results of the target genes/the quantitative results of the reference genes are correction values, the correction values of an experimental group/a control group are relative values, the results are shown in figure 4, FOLR2 mRNA in the experimental group is obviously higher than that in the control group in 12h and 24h, and the statistical significance is achieved. This result coincides with the expression level of FOLR2 in the RNA sequencing result of example 3.
Example 5 detection of FOLR2 expression amount by Western blotting
The tissue samples collected at each time point in the two groups of example 2 were transferred to a tissue grinder, 1mL of RIPA lysate containing 1mM PMSF was added, and the lysate and tissue samples were thoroughly contacted with each other by a number of blows using a gun to extract proteins. Transferring the lysate to a freezing tube for centrifugation under 12000 Xg for 5min, taking the supernatant, performing all the steps at 4 ℃, and determining the protein concentration by using a BCA method.
Mixing 4 XProtein SDS PAGE Loading Buffer with Protein samples according to the following ratio of 3The chamber was allowed to equilibrate to room temperature and briefly centrifuged before loading. Use of BIO-RAD Mini-
Protein detection was performed in the Tetra System by adding 200mL of the electrophoresis solution (Tris 3.02g, glycine 18.8g, SDS 1g, dissolved in 1L distilled water) to the internal buffer chamber, adding 800mL of the electrophoresis solution to the external buffer chamber, and rapidly washing the loading well by sucking the electrophoresis solution with a 1mL sample gun. The loading was 20. Mu.g per well, marker was added to the left-most well, and loading buffer and ddH were used for protein-free wells 2 Supplementing O; electrodes were connected, 250V,35min. After electrophoresis is completed, the power supply is turned off, the electric wire is pulled out, the cover is opened, the rubber plate is taken out, the rubber plate and foam of the sample adding hole are washed by clear water, the rubber plate is pried open, the dyeing-free glue is taken out gently and placed on the rubber plate to be cut, the electrophoresis liquid is sprayed, and the rubber is cut. The cut gel was placed in an imager and a Stain free was selected for total protein imaging (FIG. 5).
The non-staining gel was placed in the membrane transfer solution [5 × transfer buffer (Trans-
Turbo TM ) Distilled water, absolute ethyl alcohol =1:3:1]And (5) balancing for 10min. 2 pieces of filter paper with the thickness are put into the membrane transferring liquid to be fully soaked. PVDF membrane ddH after 30s of methanol infiltration 2 And O washing for 2min, and soaking the PVDF membrane in the membrane transferring liquid to balance for 10min. The sandwich is sequentially placed by the filter paper, the dyeing-free glue, the PVDF membrane and the filter paper from top to bottom, after the sandwich is completed, the cover is pressed down to lock, the drawer is placed on a rotary membrane meter, the membrane rotating time is set for 2min, and the corresponding option is selected to start membrane rotating.
Taking out the electrotransformation membrane, placing in a tank box, pouring the confining liquid (5 g skimmed milk powder +100mL TBST liquid, mixing well), placing in a horizontal shaking table, slowly shaking for 2h at a speed of once per second, and removing the confining liquid. Adding the primary antibody LifesPan and LS-C152098-50 diluted by TBST into a tank box, shaking at room temperature for incubation for 2h, taking out the membrane, placing the membrane into a clean tank box, slowly adding a proper amount of TBST from the side of the membrane, then placing the membrane on a room-temperature water flat shaking bed for 10min, discarding TBST waste liquid, adding new TBST liquid, and washing for 4 times in total. The secondary antibody (Abcam, ab 6721) was diluted with TBSTIncubate for 1h and wash 3 times as before. 2 ECL color developing solutions with 1mL are prepared, the membrane is placed in an imager, ECL color developing agents are added on the membrane, corresponding programs are selected to start imaging, and the exposure time is taken into consideration. Use of Trans-
Turbo TM Image Lab carried by universal protein transfer printing system TM The software processes the collected total protein and the immunoblot image.
The test is repeated for 3 times, the result data are statistically analyzed by SPSS18.0 statistical software, and the comparison of the experimental group and the control group is carried out by unpaired t test, which is considered to have statistical significance when P is less than 0.05.
The total protein quantification method is used for calculating the expression level of the protein, as shown in figure 3, the FOLR2 protein expression level of the neurolyme disease group is obviously higher than that of the control group at 6h,12h and 24h, and the statistical significance is achieved. The gene expression level of the 6h control group is higher, the gene expression level is lower in 12h, but the protein expression level is low at three time points, and the transcriptional regulation of gene expression may exist.
In conclusion, FOLR2 is significantly increased in occurrence and development of the neural lyme disease and is closely related to occurrence and development of the disease. Therefore, the specific expression and the obvious increase of FOLR2 have important significance on the diagnosis and treatment of the neural lyme disease and the research on pathogenesis, and the FOLR2 can be used as a biomarker of the neural lyme disease to develop products such as neural lyme disease diagnosis kits or medicines for treating the neural lyme disease.
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Claims (2)
1. The application of folate receptor FOLR2 as a biomarker in the preparation of products for diagnosing the neural lyme disease.
2. The application of the coding gene of the folate receptor FOLR2 as a biomarker in the preparation of products for diagnosing the neural lyme disease.
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| CA2025178A1 (en) * | 1989-10-02 | 1991-04-03 | William G. Weisburg | Nucleic acid probes for the detection of lyme disease spirochetes |
| US6087097A (en) * | 1994-05-12 | 2000-07-11 | Mayo Foundation For Medical Education And Research | PCR detection of Borrelia burgdorferi |
| CN102703587B (en) * | 2012-05-18 | 2013-11-27 | 中国疾病预防控制中心传染病预防控制所 | Loop-mediated isothermal amplification method for detecting lyme disease spirochete |
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