CN102392024A - Nucleic acid aptamer combining hepatitis B virus surface antigen and sequence thereof - Google Patents
Nucleic acid aptamer combining hepatitis B virus surface antigen and sequence thereof Download PDFInfo
- Publication number
- CN102392024A CN102392024A CN2011103958663A CN201110395866A CN102392024A CN 102392024 A CN102392024 A CN 102392024A CN 2011103958663 A CN2011103958663 A CN 2011103958663A CN 201110395866 A CN201110395866 A CN 201110395866A CN 102392024 A CN102392024 A CN 102392024A
- Authority
- CN
- China
- Prior art keywords
- hepatitis
- surface antigen
- rna
- virus
- sequence
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a nucleic acid aptamer of a targeted human infectious hepatitis B virus cell and a sequence thereof and belongs to the fields of gene engineering and biological medicine. According to the invention, a novel combining chemical technology SELEX is utilized to screen out an RNA aptamer capable of specifically binding to a hepatitis B virus surface antigen from single chain RNA random library by using the hepatitis B virus surface antigen as a target protein. The RNA aptamer has a sequence of 5'-GUUGAUUGCGUGUCAAUCAUGGCCGUCUAUAAUGAUCGUAAACGACGGGUCAUGUGUAUGUUGGGGAUUGGGACCUGAUUGAGUUCAGCCCACAUAC-3', can form a special loop structure in a random sequence area thereof and combine with the hepatitis B virus surface antigen singularly and with high affinity or combine to hepatocyte infected by the hepatitis B virus by target bonding. The RNA aptamer provides a specific efficient mark molecule for hepatitis B diagnosis and treatment and also provides a new option for exploiting diagnostic reagent and treatment medicament for chronic hepatitis B.
Description
Technical field
The invention belongs to genetically engineered and biomedicine field.The present invention relates to utilize the hepatocellular nucleic acid aptamer of a kind of target hepatitis b virus infection of SELEX technology screening acquisition, and the nucleotide sequence of this adaptive son.
Background technology
Hepatitis B virus (Hepatitis B Virus; HBV) infecting is one of major reason that causes chronic hepatitis, liver cirrhosis and hepatocellular carcinoma; About 75% is distributed in the Asia among the 3.5 hundred million chronic HBV infection persons of the whole world, and China is the maximum country of global HBV number of the infected.Therefore, the study on prevention to hepatitis B is the key areas of China's health care scientific research always.Though the planned immunization of domestic Hepatitis B virus vaccine greatly reduces the sickness rate of hepatitis B, the HBV chronic infection that has existed is still lacked permanently effective antiviral, and most drug and nonspecific action are in liver.Medicine is combined with liver target property carrier, make its selectivity be targeted to specific liver cell, thereby make medicine be delivered to liver more efficiently, the therapeutic action of bringing into play medicine better is the regimen that has prospect.Can utilize HBsAg as target at the surface antigen HBsAg of cells infected surface expression virus behind the HBV infected liver cell, medicine is transported to play a role in the cells infected be very promising a kind of treatment imagination.
SELEX technology (systematic evolution of ligands by exponential enrichment) be the early 1990s-a kind of new combinatorial chemistry technique that grows up.It utilizes jumbo random oligonucleotide storehouse and target molecule to interact; Therefrom filter out oligonucleotide with the target molecule specific combination; And combine the PCR amplification in vitro technological; Make it obtain exponential enrichment,, finally obtain the oligonucleotide aptamer of high-affinity, high specific through multi-turns screen.With respect to traditional protein antibody molecule, the oligonucleotide aptamer molecule that obtains through SELEX screening is littler, and immunogenicity is low, can infiltrate cell sooner, is the novel agent of the prevention, diagnosis and the treatment disease that have potentiality.
Summary of the invention
The purpose of this invention is to provide the adaptive son of a kind of RNA, combine the liver cell of hepatitis b virus infection in the liver to realize the target property that the treating hepatitis B medicine transports, can be used for the Clinics and Practices of chronic hepatitis B through specificity.
The present invention adopts following technical scheme:
External synthetic single-stranded DNA banks that contains 25 stochastic sequences that length is 97 Nucleotide constructs the adaptive sub-random library of single stranded RNA through in-vitro transcription; Adopting the method vivoexpression hepatitis B virus surface proteins of yeast expression, is target protein with it, adopts the SELEX technology screening to have the adaptive son of HBsAg specific RNA of high-affinity; Secondary structure through the adaptive son of structure prediction software RNA Structure Program analyses and prediction; Combine determination experiment, the adaptive son of gel retardation assasy evaluation and screening specificity and avidity through film, obtained an adaptive son with HBsAg avidity height, high specificity, called after S-A22 target protein; The nucleotides sequence of the adaptive son of this RNA is classified as: 5 '-GUUGAUUGCGUGUCAAUCAUGGCCGUCUAUAAUGAUCGUAAACGACGGGUCAUGUG UAUG UGGGGAUUGGGACCUGAUUGAGUUCAGCCCACAUAC-3 ' can form loop-stem structure.This adaptive son ability specificity, high-affinity ground combine hepatitis B virus surface proteins or target to be bonded to the liver cell of hepatitis b virus infection.The special competition hepatitis B virus surface antigen of this adaptive son ability of ELISA experiment confirm combines with antibody.
Beneficial effect of the present invention:
(1) obtained a kind of adaptive son that can differential high efficient combines hepatitis B virus surface antigen.This adaptive son is artificial preparation in a large number, and method is simple, and is with low cost.
(2) be that basic chronic hepatitis B diagnosis and medicine laid a good foundation for further screening with the nucleic acid aptamer.
Description of drawings
Fig. 1: the schema that is the adaptive son of SELEX screening hepatitis B virus surface antigen specificity bind nucleic acid.
Fig. 2: the secondary structure analysis figure that is the nucleic acid aptamer of screening acquisition.
Wherein Fig. 2 arrow indication is the stochastic sequence zone of adaptive son, and this zone has formed a special loop-stem structure.
Fig. 3: explained through 12 take turns the screening enrichment the RNA library can the specificity high-affinity combine the figure as a result of hepatitis B virus surface antigen.
Wherein left side figure is that film combines experiment to detect the binding ability of different rounds library and HBsAg, has shown that the 12nd takes turns bonded high-affinity between library and HBsAg; Right figure the 12nd takes turns library and HBsAg bonded gel retardation assasy.
Fig. 4: competitive ELISA experiment shows that adaptive sub-S-A22 can block combining of HBsAg and HBsAb, possibly seal the binding site of HBsAg and HBsAb after S-A22 being described and HBsAg combining.
Embodiment
Below in conjunction with Figure of description and embodiment the present invention is further described, but is not restriction the present invention.
Embodiment 1H1 protein-specific combines the SELEX screening of the adaptive son of RNA
The SELEX screening process is as shown in Figure 1; Initial oligonucleotide random library of chemosynthesis and primer, sequence is following: 5 '-TTAATACGACTCACTATAGTTGATTGCGTGTCAATCATGG-25N-GGTCATGTGTA TGTTGGGGATTAGGACCTGATTGAGTTCAGCCCACATAC-3 ' (25N represents 25 random nucleotides); Use
Primer 1:5 '-TTAATACGACTCACTATAGTTGATTGCGTGTCAATC-3 ',
Primer 2: 5 '-GTATGTGGGCTGAACTCAAT-3 '.The single-stranded DNA banks amplification is double-stranded DNA, and product is through 2% agarose gel electrophoresis and cut glue recovery purifying; Double-stranded DNA to reclaim is a template, and in-vitro transcription goes out the single stranded RNA random library, and transcription product is through the PAGE purifying.Remove and membrane-bound RNA molecule through anti-sieve of nitrocellulose filter in 68 μ g RNA libraries, hatches 40min for 37 ℃ with 2 μ g HBsAg then, and reaction solution filters through nitrocellulose filter, the washing filter membrane; Then filter membrane is shredded, (1mmol/L EDTA boils 5min in 0.2%SDS), and is centrifugal, gets supernatant for 7mol/L urea, 0.5mol/L ammonium acetate, absolute ethyl alcohol precipitated rna, and being dissolved in again in the 20 μ l DEPC water to place elution buffer; With RNA is template RT-PCR amplifying doulbe-chain DNA, and in-vitro transcription goes out the RNA library and is used for the next round screening; Every RT-PCR that takes turns in the screening process obtains size and is 115bp double-stranded DNA library, is the adaptive word bank of RNA that the template in-vitro transcription goes out 97nt with this double-stranded DNA, and screening is carried out 12 altogether and taken turns.
The secondary structure analysis of the adaptive son of embodiment 2RNA
Take turns the library clone that obtains of screening to the pMD19-T carrier with the 12nd, be converted into bacillus coli DH 5 alpha, 48 clones of random choose, order-checking.Obtain the sequence information of the adaptive son that screens; RNA secondary structure through all order-checking clones of structure prediction software RNA Structure Program prediction; It can form a special loop-stem structure in stochastic sequence zone (23-47nt) to obtain a kind of adaptive son, and is as shown in Figure 2.
The HBsAg of embodiment 3 specificity high-affinities combines the acquisition of adaptive son
The adaptive son of RNA that will have above-mentioned secondary structure is got 1 μ g respectively, with 37 ℃ of digestion of calf intestinal alkaline phosphatase (CIP) 1h, the dephosphorylized RNA of purifying and recovering; Through T4 polynucleotide kinase mark [γ-
32P] ATP is in dephosphorylized RNA molecular end.The adaptive son of the radiolabeled RNA of 10nmol is hatched 40min with HBsAg37 ℃ of different concns (10-200nM) respectively; Each group reaction liquid filters through nitrocellulose filter, washing filter membrane, dry filter membrane; Liquid scintillation counter is measured residual exit dose on the filter membrane, the parallel mensuration of doing twice of same sample.Calculate the dissociation constant of each adaptive son and HBsAg, obtain the highest adaptive son of avidity, called after S-A22.
32The 5th of P mark is taken turns library R5, the 6th and is taken turns library R6, the 10th and take turns library R10 and the 12nd and take turns library R12 and hatch 40min with HBsAg at 37 ℃ respectively; Each group reaction liquid filters through nitrocellulose filter; The washing filter membrane; Dry filter membrane, liquid scintillation counter is measured residual exit dose on the filter membrane, the parallel mensuration of doing twice of same sample.Appearance is to 6% non-sex change PAGE gel on the afterreaction liquid for same hatching of dividing into groups, and 120V electrophoresis 2h takes out gel, the imaging of phosphorus screen, and the result is as shown in Figure 3.
The binding ability of embodiment 4 adaptive sub-S-A22 blocking-up HBsAg and HBsAb
(R12), the system of transcribing is following for R0, R6: 1 μ g dna profiling for adaptive sub-S-A22, S-A21 and different round RNA library that in-vitro transcription 2 ' fluorine is modified; 5 μ l 10 * transcribe damping fluid, 400 μ g/ μ l PEG, 0.5M MgCl2,100U/ml IPP; 50U RNasin, 2.5mM 2F '-CTP, 2.5mM 2F '-UTP, 2.5mM ATP; 2.5mMGTP reach 0.36 μ g/ μ l T7RNA polymerase, moisturizing to final volume 50 μ l transcribe for 37 ℃ and spend the night.Transcription product is through the PAGE purifying.Respectively RNA is mixed with HBsAg with the mol ratio of 10: 1 or 100: 1, hatch after 30 minutes as testing sample, get 50 μ l and add the enzyme plate that is coated with HBsAb for 37 ℃; Hatch after 30 minutes for 37 ℃ and abandon supernatant, PBS washes 3 times, adds the HBsAb of HRP mark; Hatch after 30 minutes for 37 ℃ and abandon supernatant; PBS washes 3 times, adds colour developing liquid, and ELIASA is surveyed the absorbancy of 450nm.The result is as shown in Figure 4, and experiment shows that adaptive sub-S-A22 can block combining of HBsAg and HBsAb, possibly seal the binding site of HBsAg and HBsAb after S-A22 being described and HBsAg combining.
Claims (2)
1. adaptive son of single stranded RNA; It is characterized in that: the nucleotides sequence of the adaptive son of this RNA is classified as: 5 '-GUUGAUUGCGUGUCAAUCAUGGCCGUCUAUAAUGAUCGUAAACGACGGGUCAUGUG UAUGUUGGGGAUUGGGACCUGAUUGAGUUCAGCCCACAUAC-3 ' can form loop-stem structure.
2. the adaptive son of a kind of single stranded RNA as claimed in claim 1 is characterized in that: this adaptive son ability specificity, high-affinity ground combine hepatitis B virus surface antigen or target to be bonded to the liver cell of hepatitis b virus infection.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011103958663A CN102392024A (en) | 2011-12-02 | 2011-12-02 | Nucleic acid aptamer combining hepatitis B virus surface antigen and sequence thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011103958663A CN102392024A (en) | 2011-12-02 | 2011-12-02 | Nucleic acid aptamer combining hepatitis B virus surface antigen and sequence thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102392024A true CN102392024A (en) | 2012-03-28 |
Family
ID=45859405
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2011103958663A Pending CN102392024A (en) | 2011-12-02 | 2011-12-02 | Nucleic acid aptamer combining hepatitis B virus surface antigen and sequence thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102392024A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102808022A (en) * | 2012-07-25 | 2012-12-05 | 安徽出入境检验检疫局检验检疫技术中心 | Application of oligonucleotide aptamer capable of identifying salmonella specifically |
| CN104694544A (en) * | 2015-03-24 | 2015-06-10 | 刘红卫 | Nucleic acid aptamer combined with hepatitis delta virus and application thereof |
| CN104962560A (en) * | 2015-06-16 | 2015-10-07 | 湖南大学 | Nucleic acid aptamer for detecting schistosoma japonicum eggs and application of nucleic acid aptamer to manufacturing detection preparations |
| CN106032534A (en) * | 2015-02-02 | 2016-10-19 | 苏州方舟基因药业有限公司 | Ribonucleic acid aptamer capable of specifically being combined with human non-small cell lung cancer cells and screening method thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101144814A (en) * | 2007-10-22 | 2008-03-19 | 中国人民解放军第三军医大学第一附属医院 | Method for detecting, identifying and/ or quantifying compound using adapter type reagent |
| CN102226185A (en) * | 2011-05-09 | 2011-10-26 | 华中科技大学同济医学院附属同济医院 | RNA aptamer of targeting hepatocyte and nucleotide sequence thereof |
-
2011
- 2011-12-02 CN CN2011103958663A patent/CN102392024A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101144814A (en) * | 2007-10-22 | 2008-03-19 | 中国人民解放军第三军医大学第一附属医院 | Method for detecting, identifying and/ or quantifying compound using adapter type reagent |
| CN102226185A (en) * | 2011-05-09 | 2011-10-26 | 华中科技大学同济医学院附属同济医院 | RNA aptamer of targeting hepatocyte and nucleotide sequence thereof |
Non-Patent Citations (2)
| Title |
|---|
| JIA LIU等: "Development of HBsAg-Binding Aptamers that bind HepG2.2.15 cells via HBV surface antigen", 《VIROLOGICA SINICA》, vol. 25, no. 1, 12 February 2010 (2010-02-12), pages 27 - 35 * |
| 王薇薇,胡康洪: "RNA药物抗乙型肝炎病毒的研究进展", 《武汉大学学报（理学版）》, vol. 56, no. 1, 28 February 2010 (2010-02-28), pages 63 - 68 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102808022A (en) * | 2012-07-25 | 2012-12-05 | 安徽出入境检验检疫局检验检疫技术中心 | Application of oligonucleotide aptamer capable of identifying salmonella specifically |
| CN102808022B (en) * | 2012-07-25 | 2014-04-30 | 安徽出入境检验检疫局检验检疫技术中心 | Application of oligonucleotide aptamer capable of identifying salmonella specifically |
| CN106032534A (en) * | 2015-02-02 | 2016-10-19 | 苏州方舟基因药业有限公司 | Ribonucleic acid aptamer capable of specifically being combined with human non-small cell lung cancer cells and screening method thereof |
| CN106032534B (en) * | 2015-02-02 | 2019-08-23 | 苏州方舟生物医药有限公司 | A kind of rna aptamer and its screening technique in conjunction with Non-small cell lung carcinoma cell-specific |
| CN104694544A (en) * | 2015-03-24 | 2015-06-10 | 刘红卫 | Nucleic acid aptamer combined with hepatitis delta virus and application thereof |
| CN104962560A (en) * | 2015-06-16 | 2015-10-07 | 湖南大学 | Nucleic acid aptamer for detecting schistosoma japonicum eggs and application of nucleic acid aptamer to manufacturing detection preparations |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Zhuo et al. | Recent advances in SELEX technology and aptamer applications in biomedicine | |
| CN111849994B (en) | Aptamer of SARS-CoV-2S protein or RBD protein and application thereof | |
| CN102703454B (en) | Nucleic acid aptamer capable of detecting myohemoglobin, microfluidic chip for screening and screening method and application | |
| CN102392024A (en) | Nucleic acid aptamer combining hepatitis B virus surface antigen and sequence thereof | |
| CN102839181B (en) | Nucleic acid aptamer sequence of gastric cancer cells, and uses thereof | |
| Prakash et al. | Aptamers and their significant role in cancer therapy and diagnosis | |
| CN101605908A (en) | The nuclease-resistant rna that inhibition hepatitis c viral replication duplicates is fit | |
| CN105296491A (en) | Aptamer AS1411 subjected to chemical modification and application thereof | |
| CN1958809A (en) | Method for detecting mycobacterium tuberculosis by using adaptor technique | |
| CN102226185A (en) | RNA aptamer of targeting hepatocyte and nucleotide sequence thereof | |
| Martínez-Roque et al. | DNA aptamer selection for SARS-CoV-2 spike glycoprotein detection | |
| CN102703455B (en) | Human cardiac troponin I aptamer, screening method and application | |
| CN105541990B (en) | Aptamers K15 of skate angiogenesis inhibitor 1 and screening method and application thereof | |
| CN102952801A (en) | Group of oligonucleotide aptamers for specifically identifying aflatoxin B2 | |
| Chang et al. | Aptamer-based strategies to address challenges in COVID-19 diagnosis and treatments | |
| Lee et al. | In vitro selection of cancer-specific RNA aptamers | |
| CN105483133A (en) | Staphylococcus aureus toxic shock syndrome toxin-1(TSST-1) aptamer T-7 and preparation method and application thereof | |
| CN110759994A (en) | Nano antibody for resisting hepatitis B virus core antigen and application thereof | |
| Dausse et al. | In vitro selection procedures for identifying DNA and RNA aptamers targeted to nucleic acids and proteins | |
| CN104694544A (en) | Nucleic acid aptamer combined with hepatitis delta virus and application thereof | |
| CN103305521A (en) | Sequence of aptamer of gastric cancer cell, and application thereof | |
| CN105319376B (en) | Kit for detecting nasopharynx cancer and detecting method | |
| CN102367476A (en) | Detection method for slow virus infected cell | |
| CN105301262B (en) | A kind of test kit for human brain disease detection | |
| He et al. | Effects of two novel nucleoside analogues on different hepatitis B virus promoters |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20120328 |