阅读说明：本技术 一种hpv33类病毒颗粒的核酸适配体hpv3301及其应用 (Nucleic acid aptamer HPV3301 of HPV33 virus-like particles and application thereof ) 是由 吴冬 于 2021-08-11 设计创作，主要内容包括：本发明涉及一种HPV33类病毒颗粒的核酸适配体HPV3301及其应用,该核酸适配体HPV3301的序列为：5’-TAGGCGTTGCTCCCCTTCGTCCCCCCTT TAGCGCAGCCCCCCCCAATACGTTCTCTTGTCCTCTCGAGGTTGTTACGC-3’；本发明的核酸适配体HPV3301能高亲和力、高特异性地与HPV33类病毒颗粒结合,该核酸适配体HPV3301在HPV33感染的诊断和治疗方面具有广阔的应用前景和重要的科学、社会价值,特别是它具有阻断HPV33感染的作用,可作为潜在的HPV33感染治疗药物。(The invention relates to a nucleic acid aptamer HPV3301 of HPV33 virus particles and application thereof, wherein the sequence of the nucleic acid aptamer HPV3301 is as follows: 5'-TAGGCGTTGCTCCCCTTCGTCCCCCCTT TAGCGCAGCCCCCCCCAATACGTTCTCTTGTCCTCTCGAGGTTGTTACGC-3', respectively; the aptamer HPV3301 of the invention can be combined with HPV33 virus particles with high affinity and high specificity, and the aptamer HPV3301 has wide application prospect and important scientific and social value in diagnosis and treatment of HPV33 infection, especially has the effect of blocking HPV33 infection, and can be used as a potential HPV33 infection treatment drug.)

1. An HPV 33-type virion aptamer HPV3301, characterized in that: its sequence is shown below:

5'-TAGGCGTTGCTCCCCTTCGTCCCCCCTTTAGCGCAGCCCCCCCCAATACGTTCTCTTGTCCTCTCGAGGTTGTTACGC-3', respectively; and 100mM Na at 25 deg.C⁺，1mM Mg²⁺Under the condition of (2), the spatial structure is as follows:

2. the aptamer HPV3301 of HPV 33-like virions according to claim 1, characterized in that: and carrying out chemical modification on the 5 'end or the 3' end of the aptamer HPV3301 by using a fluorescent group, amino, biotin, digoxigenin or polyethylene glycol.

3. Use of the aptamer HPV3301 of HPV 33-like virions according to claim 1 for the manufacture of a medicament for the treatment of HPV33 infection.

4. Use of the aptamer HPV3301 of HPV 33-like virions according to claim 1 for the preparation of a reagent for the isolation and enrichment of HPV 33-like virions in a sample.

5. Use of the aptamer HPV3301 of HPV 33-like virions according to claim 1 for the preparation of an HPV33 detection reagent or kit.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a high-affinity aptamer HPV3301 specifically bound with HPV33 virus particles and application thereof.

Background

Human Papilloma Virus (HPV) is a circular double-stranded DNA Virus without envelope coating. Currently, HPV has been found in over 200 types, most of which do not show obvious symptoms after infecting humans, and a few of which show signs of viral infection, such as causing various papillomas or warts of the skin and proliferative lesions of the genital tract epithelium. According to research results of WHO International cancer research Institute (IARC) and other international organizations, the national drug administration medical instrument technical evaluation center issues "Human Papillomavirus (HPV) nucleic acid detection and genotyping, reagent technical review guide principles" to list HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59 and HPV68 as high risk types, and HPV26, HPV53, HPV66, HPV73 and HPV82 as medium risk types. The high-risk type is closely related to the occurrence of cervical cancer and is one of the main carcinogenic factors of women.

The lack of effective HPV antiviral therapies is currently a worldwide consensus. Although some antiviral drugs, such as "nucleoside" antiviral drugs and interferons, are currently on the market, none of these drugs have any definite or even no therapeutic effect. Antiviral therapies specifically directed against HPV have not been developed yet. Therefore, in all guidelines for prevention and treatment of HPV, there is no mention (or recommendation) of using any drug to kill HPV viruses. Therefore, there is an urgent need for safe and effective drugs for treating HPV infection.

Aptamers are also known as "synthetic antibodies", "chemical antibodies", and their chemical nature is that a single-stranded oligonucleotide molecule (ssDNA or RNA) folds into a specific three-dimensional structure that binds to a target substance with high affinity and specificity. Aptamers were obtained by in vitro screening procedures using Systematic evolution of ligands by exponentiation technology (SELEX). The aptamer has the characteristics of high affinity, high specificity, capability of being synthesized in vitro, capability of changing the function and the pharmacokinetic characteristic thereof through modification, no immunogenicity, economy and the like. Aptamer drugs developed based on the above advantages can specifically block biological functions of targets, for example, as blocking agents for viral infection, neutralizing antagonists for toxins, inhibitors of cytokines, tumor therapeutic drugs for blocking transcription factors, and the like.

Viroid particles are highly compatible with viral structures, and structurally still possess proviral characteristics, but lack self-replicating capacity, for reassembly of the remaining material after removal of viral genetic material. Viroid particles enable a human to generate an immune response against the virus, and thus the production of antibodies enables a human to have a good defense against the attack of the virus. The existing first-generation and second-generation cervical cancer vaccines use "viroid" similar to natural HPV viroid as vaccine antigen. In addition, HPV virus particles are also used as infection models and widely used in the research field of screening anti-HPV infection medicines. At present, the aptamer of the HPV16 virus particle is screened out and found to have potential effect of inhibiting HPV16 infection. However, even one HPV virosome vaccine can only prevent one HPV type due to the differences between HPV types. Therefore, screening the aptamer which is combined with HPV33 virus particles with high specificity and high affinity and has important scientific research and clinical value as a blocking agent for high-risk HPV33 infection.

Disclosure of Invention

One of the objects of the present invention is to provide a nucleic acid aptamer HPV3301 of HPV 33-type viral particles with high specificity and high affinity; the invention also aims to provide the application of the aptamer HPV3301 in the preparation of separation and enrichment reagents of HPV33 virus particles in samples, the preparation of HPV33 detection reagents or kits, the preparation of HPV33 infection blocking drugs and the like.

The purpose of the invention is realized by the following technical scheme: a nucleic acid aptamer HPV3301 of HPV33 virus particle, the sequence of which is shown as follows:

5'-TAGGCGTTGCTCCCCTTCGTCCCCCCTTTAGCGCAGCCCCCCCCAATACGTTCTCTTGTCCTCTCGAGGTTGTTACGC-3'(SEQ ID NO:1)

the aptamer HPV3301 of the HPV33 viroid is obtained by an in vitro SELEX screening technology based on the aptamer, wherein a PVDF membrane is used as a solid phase medium, HPV33 viroid is used as a target, and the aptamer specifically bound with the HPV33 viroid is screened from a ssDNA library and named as the aptamer HPV 3301.

The nucleic acid aptamer HPV3301 of the HPV33 viroid can be chemically modified by fluorescent group, amino group, biotin, digoxin or polyethylene glycol at the 5 'end or 3' end.

The nucleic acid aptamer HPV3301 of the HPV33 viroid has the effect of inhibiting HPV33 infection at a cell model level, and can be used as a potential HPV33 infection blocking agent. The nucleic acid aptamer HPV3301 of the HPV33 virus-like particle is applied to preparation of HPV33 infection treatment medicines.

The nucleic acid aptamer HPV3301 of HPV33 virus particles is applied to preparation of a separation and enrichment reagent of HPV33 virus particles in a sample.

The nucleic acid aptamer HPV3301 of the HPV33 virus particle is applied to preparation of an HPV33 detection reagent or kit.

Compared with the prior art, the invention has the advantages that:

1. the nucleic acid aptamer HPV3301 of the invention is non-toxic, small in molecular weight, good in permeability and easy to synthesize and mark.

2. The synthesis cost of the aptamer HPV3301 is lower than that of antibody preparation, and the method has the advantages of short period and good reproducibility.

3. The nucleic acid aptamer HPV3301 of the invention can be combined with HPV33 virus particles with high affinity and high specificity, the dissociation constant is 103.7pM, and the nucleic acid aptamer HPV3301 is not combined with other control HPV virus particles.

4. The nucleic acid aptamer HPV3301 of the invention has wide application prospect and important scientific and social value in diagnosis and treatment of HPV33 infection, especially has the effect of blocking HPV33 infection, and can be used as a potential HPV33 infection treatment drug.

Drawings

FIG. 1 is a bioinformatics mimic of the secondary structure of aptamer HPV 3301.

FIG. 2 is a diagram of the specificity of the aptamer HPV3301 analyzed by fluorescence binding rate experiments. In FIG. 2, the abscissa represents the analyzed protein, and the ordinate represents the fluorescence binding rate.

FIG. 3 is a curve plotting dissociation constants of nucleic acid aptamer HPV3301 binding to HPV33 virus particles according to fluorescence binding rate experiment. Dissociation constant (Kd) was 103.7 pM. In FIG. 3, the abscissa represents the DNA concentration (pM) and the ordinate represents the fluorescence binding rate.

FIG. 4 is a dose-inhibition curve for flow cytometry analysis of the aptamer HPV3301 to inhibit the false infection of HPV 33-type viral particles. DNA concentration (pM) on the abscissa and relative infection rate on the ordinate.

Detailed Description

The invention is described in detail below with reference to the drawings and examples of the specification:

a nucleic acid aptamer HPV3301 of HPV33 virus particle, the sequence is as follows:

5'-TAGGCGTTGCTCCCCTTCGTCCCCCCTTTAGCGCAGCCCCCCCCAATACGTTCTCTTGTCCTCTCGAGGTTGTTACGC-3'(SEQ ID NO:1)

the nucleic acid aptamer HPV3301 of the HPV33 viroid is 100mM Na at 25 DEG C⁺，1mM Mg²⁺Under the conditions of (a), the spatial structure thereof is as follows:

the aptamer HPV3301 of the HPV33 virosome is subjected to chemical modification including but not limited to fluorescent group, amino group, biotin, digoxin, polyethylene glycol and the like on the 5 'end or the 3' end of the aptamer HPV 3301.

The aptamer HPV3301 of the HPV33 virus particle is obtained by carrying out chemical modification including but not limited to fluorescent group, amino group, biotin, digoxin and polyethylene glycol on a product obtained by carrying out truncation or extension or partial base replacement on the aptamer HPV 3301.

The aptamer HPV3301 of the HPV33 viroid is obtained by an in vitro SELEX screening technology based on the aptamer, a PVDF membrane is used as a solid phase medium, HPV33 viroid is used as a target, and the aptamer specifically bound with the HPV33 viroid is screened from an ssDNA library.

The screening method of the aptamer HPV3301 of the HPV33 virus particles comprises the following steps:

(1) preparation of screening library: a random ssDNA library was prepared as shown by the following sequence:

5’-TAGGCGTTGCTCCCCTTCGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN CCTCTCGAGGTTGTTACGC-3’；

(2) transferring HPV33 virus particles to a PVDF membrane to prepare a PVDF transfer membrane;

(3) subjecting the ssDNA library to a thermal activation treatment;

(4) incubating the ssDNA library obtained in the step (3) with the PVDF transfer membrane obtained in the step (2);

(5) separating the PVDF transfer film after the step (4), and washing off ssDNA which is not combined, weakly combined and non-specifically combined on the surface of the PVDF transfer film; heating the PVDF transfer film, and collecting ssDNA (single-stranded deoxyribonucleic acid) specifically bound with HPV33 virus particles, namely a ssDNA enrichment library;

(6) and (3) PCR amplification: and (3) carrying out PCR amplification on the ssDNA enrichment library obtained in the step (5), wherein primers used for the PCR amplification are as follows:

primer HPVup: 5 '-FAM-TAGGCGTTGCTCCCCTTCG-3' (SEQ ID NO:2)

Primer HPVdown: 5 '-Biotin-GCGTAACAACCTCGAGAGG-3' (SEQ ID NO: 3);

(7) purification of PCR products: purifying the PCR product by using a small fragment DNA purification kit; incubating the purified dsDNA with streptavidin magnetic beads, washing the streptavidin magnetic beads combined with the dsDNA, melting the dsDNA, separating by using a magnetic frame, and collecting supernatant; precipitating the supernatant by ethanol to obtain a secondary ssDNA library for the next round of screening;

(8) and (3) circulating screening: and (3) taking the FAM-labeled secondary ssDNA library obtained in the step (7) as a secondary library for the next round of screening, and repeating the screening processes of the steps (3) to (7).

The first embodiment is as follows: screening of aptamer HPV3301

The screening method of the aptamer HPV3301 of the HPV33 virus particles comprises the following steps:

(1) preparation of screening library: designing a random ssDNA library, the sequence of which is: 5 '-TAGGCGTTGCTCCCCTTCGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCCTCTCGAGGTTGTTACGC-3', which includes a fixed sequence region at both ends (19 nucleotides at the 5 'end and 3' end, respectively) and a random sequence region in the middle (40 random sequence nucleotides), and was synthesized by Competition institute of Industrial engineering, bioengineering, Inc.

(2) Preparation of PVDF transfer films for HPV 33-type virus particles: the HPV 33-like virions were derived from the E.coli expression system and were purchased from Creative Diagnostics, USA with a purity of > 95% (SDS-PAGE), and the PVDF membrane was purchased from Biotechnology engineering, Inc. HPV33 type virus particles are mixed in 5 xSDS-PAGE loading buffer and boiled for 10min by boiling water. HPV 33-type virus particles were separated by SDS-PAGE electrophoresis. Soaking a PVDF membrane in methanol for 3-5 seconds in advance until the PVDF membrane is saturated, and then placing the PVDF membrane in a membrane transfer buffer solution for balancing; the HPV33 virus particles in the SDS-PAGE gel are transferred to a PVDF membrane by a BILE BIO-RAD full-function protein rapid membrane transfer instrument under the condition of a power supply of 10V, wherein the time is 60 min. And (3) washing the membrane for 5min by using PBS buffer solution, putting the PVDF transfer membrane into PBS sealing solution containing 5% skim milk, sealing in a shaking table at 37 ℃ for 2h, and rinsing the PVDF transfer membrane for 5min by using PBS.

(3) 1nmol of the random ssDNA library was dissolved in 500. mu.L of selection buffer (50mM Tris-HCl, 100mM NaCl, 1mM MgCl)₂5mM KCl, pH 7.4) and then heat activated. The thermal activation treatment method comprises the following steps: after denaturation at 95 ℃ for 5min, the mixture was immediately placed in an ice-water bath for 10min, followed by room temperature for 10 min.

(4) And (3) incubating the ssDNA library obtained in the step (3) with the PVDF transfer membrane (with the load of HPV33 viroid particles being 20ng) of the HPV33 viroid particles obtained in the step (2) and yeast tRNA (with the molar quantity being 5 times of that of the ssDNA library) at room temperature for 1 h.

(5) Taking out the PVDF transfer film of the HPV33 virus particles after the step (4), and washing off ssDNA (single stranded deoxyribonucleic acid) which is not combined, weakly combined and non-specifically combined on the surface of the PVDF transfer film of the HPV33 virus particles by using a selection buffer solution containing 0.2% BSA (bovine serum albumin); then placing the PVDF transfer membrane of the HPV33 viroid particles in 200 mu L ddH₂And O, after a hot water bath at 100 ℃ for 5min, centrifuging at a high speed to collect supernatant, and obtaining ssDNA (single stranded deoxyribonucleic acid) specifically bound with HPV33 type virus particles, namely a ssDNA enrichment library.

(6) And (3) PCR amplification: adding the ssDNA enrichment library obtained in the step (5) into 1mL of PCRmix; after vortex oscillation and uniform mixing, subpackaging according to 50 mu L of each tube for PCR amplification, wherein the amplification conditions are as follows: pre-denaturation at 94 deg.C for 5 min; denaturation at 94 ℃ for 30S, annealing at 63 ℃ for 30S, and extension at 72 ℃ for 30S, 15-25 cycles.

Wherein 1mL of PCRmix contains: 10 XPCR buffer 100. mu.L; pfu enzyme 3. mu.L; dNTP 20 u L; primer HPVup: 5 '-FAM-TAGGCGTTGCTCCCCTTCG-3' and primer HPVbrown: primer HPVdown: 3. mu.L of each of 5 '-Biotin-GCGTAACAACCTCGAGAGG-3'; the primer HPVup and the primer HPVbrown are both synthesized by the agency of bioengineering, Inc.

(7) Purification of PCR products: PCR products labeled with biotin and a fluorophore FAM at both ends, respectively, were purified using a small fragment purification kit (said small fragment purification kit is available from Biotechnology, Ltd.), the purified dsDNA was incubated with streptavidin magnetic beads (available from Invitrogen-Dynal) at 37 ℃ for 20min, the dsDNA-bound streptavidin magnetic beads were washed three times with a washing buffer (5mM Tris-HCl, pH 7.5, 1M NaCl, 500. mu.MEHPVA), and then incubated with 50. mu.L NaOH solution (0.1M) at 37 ℃ for 30min to melt the dsDNA; the supernatant was collected by magnetic frame separation and ethanol precipitated to obtain the FAM-labeled secondary ssDNA library and dissolved in selection buffer as the secondary library for the next round of screening.

(8) The screening process was performed for 12 rounds. From the second round, the secondary libraries were used in an amount of 30pmol each.

Example two: acquisition and analysis of the aptamer HPV3301 sequence:

(1) after 12 rounds of screening, the enriched ssDNA library was collected and submitted to the Beijing Xinnuo Bai Shi medical laboratory Ltd to analyze the sequence of the library by high throughput sequencing technology, the analysis process was: amplifying the enrichment library by PCR, and adding a sequencing joint and an Index part; selecting a purified library by gel electrophoresis; using Nanodrop one to measure the concentration and purity of DNA for quality control analysis; using Illuminate NovaSeq^TM6000 platform, using single chain library as template to do bridge PCR amplification, sequence primer annealing, synthesizing and sequencing; and comparing and enriching the sequencing result.

(2) According to the enrichment degree of the aptamer in the library, selecting ssDNA with high enrichment degree as a candidate aptamer, wherein the aptamer HPV3301 accounts for 16.9% of the enrichment library, and the sequence of the aptamer is shown as SEQ ID NO: 1.

(3) Analysis at 25 ℃ with UNAFold network platform 100mM Na⁺，1mM Mg²⁺Under the conditions of (1), the secondary structure of the nucleic acid aptamer HPV3301 sequence. The schematic diagram of the secondary structure of the analyzed nucleic acid aptamer HPV3301 sequence is shown in FIG. 1.

Example three: specific analysis of aptamer HPV 3301:

(1) FAM-labeled aptamer HPV3301 was chemically synthesized in vitro and dissolved in selection buffer.

(2) Referring to step (2) of example one, BSA (available from Sigma), HPV 16-type virus particles, HPV 18-type virus particles, HPV 31-type virus particles, HPV 33-type virus particles, HPV 35-type virus particles, HPV 39-type virus particles, HPV 45-type virus particles, HPV 51-type virus particles, HPV 52-type virus particles, HPV 56-type virus particles, HPV 58-type virus particles, HPV 59-type virus particles, and HPV 68-type virus particles (available from Creative Diagnostics, usa) were transferred onto PVDF membranes, respectively, to prepare PVDF transfer films containing various HPV-type virus particles.

(3) And (3) mixing 200 mu L of the aptamer HPV3301 solution obtained in the step (1) with the PVDF transfer films prepared in the step (2), and incubating for 1h at room temperature in a cassette.

(4) Washing the PVDF transfer membrane obtained in step (3) with 0.1% PBST for 3 times, and eluting the aptamer bound to the PVDF transfer membrane by boiling 200. mu.L of a selection buffer at 100 ℃ for 5 min.

(5) The fluorescence intensities of the initial solution and the eluate were measured by a fluorescence quantitative analyzer, and the fluorescence binding ratio ═ (initial fluorescence intensity-elution fluorescence intensity)/initial fluorescence intensity × 100% was calculated, and the binding ratio of the aptamer HPV3301 to the target molecule was preliminarily represented by the calculated value.

As shown in FIG. 2, the binding rate of the aptamer HPV3301 and HPV33 virosome is significantly higher than that of other HPV virosome, indicating that the binding of the aptamer HPV3301 and HPV33 virosome has better specificity.

Example four: affinity assay for aptamer HPV3301

(1) And respectively mixing FAM labeled aptamer HPV3301 solutions with different concentrations with an HPV33 virus particle PVDF transfer film, and incubating for 1h at room temperature in a cassette.

(2) Referring to the step (4) and the step (5) in the third example, the fluorescence binding rates of the aptamer HPV3301 solutions with different concentrations and the PVDF transfer films of the HPV33 viroid particles are obtained and calculated through experiments.

(3) And (3) utilizing the calculated value of the fluorescence binding rate to draw a saturation binding curve of the aptamer HPV3301 bound to HPV33 virus particles, and calculating the dissociation constant of the aptamer HPV3301 bound to HPV33 virus particles through nonlinear regression analysis.

As shown in FIG. 3, the present invention obtains a saturation binding curve of aptamer HPV3301, and the dissociation constant of aptamer HPV3301 is calculated to be 103.7pM, which shows that the aptamer HPV3301 has strong binding ability to HPV33 type virus particles, and the dissociation constant is in picomolar scale.

Example five: study of aptamer HPV3301 to inhibit HPV33 infection

(1) Cell culture: 293TT cells in DMEM medium at 37 ℃ and 5% CO₂Cultured under conditions supplemented with 5% Gibco fetal bovine serum, 100IU/mL penicillin and 100mg/mL streptococcal mycin.

(2) The research method for inhibiting HPV33 infection by aptamer HPV 3301:

pseudoinfection with HPV33 type viroid serves as a cellular model for HPV33 infection. 293TT cells were seeded in 24-well plates (1X 10)⁵Perwell), incubated overnight at 37 ℃. Infection mixtures were prepared with 5000InU of HPV33 virus-like particles and varying concentrations of the aptamer HPV3301 in a final volume of 80. mu.L of PBS buffer. Random sequence ssDNA was used as a blank at the same concentration. The infection mixture was gently stirred at room temperature for 20min and then added to 500. mu.L of 293TT cell DMEM solution. After 20min, the supernatant was discarded to remove the infection mixture, and then 1mL of pre-warmed DMEM supplemented with 5% fetal bovine serum was added. After 72h, the cells were harvested by centrifugation at 1000rpm, the cell pellet washed with PBS buffer and then resuspended in a volume of 0.5mL of PBS buffer for analysis in a flow cytometer using a bandpass filter at 530/30nm (FL 1). With 488nmExcitation by argon laser (1X 10)⁴Cell/time). The relative infection rates at different concentrations were calculated as an indicator of inhibition of HPV33 infection by the aptamer HPV 3301. Relative infection rate ═ rate of infection of aptamer HPV 3301/rate of infection of control.

(3) Results of the study that the aptamer HPV3301 inhibits HPV33 type infection:

as shown in fig. 4, the nucleic acid aptamer HPV3301 was able to inhibit infection by HPV type 33 pseudovirus in a dose-dependent manner. When the concentration of the aptamer HPV3301 reaches 30 mu M, the inhibition effect is saturated, and the inhibition rate (1-relative infection rate) can reach 45%.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that a person skilled in the art may make several changes, improvements and modifications without departing from the spirit of the present invention, and these changes, improvements and modifications should be construed as the protection scope of the present invention.

Sequence listing

<110> Wudong

<120> nucleic acid aptamer HPV3301 of HPV33 viroid and application thereof

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 78

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<213> Artificial Sequence (Artificial Sequence)

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taggcgttgc tccccttcgt ccccccttta gcgcagcccc ccccaatacg ttctcttgtc 60

ctctcgaggt tgttacgc 78

<210> 2

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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taggcgttgc tccccttcg 19

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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gcgtaacaac ctcgagagg 19