CN103305519B - Aptamer sequence of hepatitis B virus (HBV) core antigen, and applications thereof - Google Patents

Abstract

The invention belongs to the molecular immunity field, and relates to an aptamer sequence of an HBV core antigen, and uses thereof. A signature sequence TATTT having a specific binding HBV core antigen is prepared by adopting a gene recombinant plasmid to express the core antigen and screening an aptamer specially bound with the core antigen. The signature sequence is a base of the specific binding the aptamer and HBc, can be used for designing drugs and preparing drugs or other products, and the aptamer containing the signature sequence can be used for designing and preparing anti-HBV drugs or preparations as an anti-HBV probe or target spot.

Description

A kind of nucleic acid aptamer sequence of hepatitis B virus core antigen and application

Technical field

The invention belongs to molecular immune field, relate to sequence of a kind of aptamer of hepatitis B virus core antigen and uses thereof.

Background technology

Hepatitis B (hepatitis B) is the Infectious Diseases of serious harm our people health, add up according to the World Health Organization (WHO), China's hepatitis B virus (hepatitis B virus, HBV) carrier reaches 1.3 hundred million, liver problem sufferer up to 3,000 ten thousand, every year because all kinds of hepatopathy death toll reaches 300,000.Hepatopathy has become current and has threatened one of principal disease of Chinese population health, the existence of the patients with chronic liver of enormous amount, and the appearance of annual new discovery hepatitis cases, causes great consumption to social medical resource.Research display, hepatitis B virus is that the principal causative of hepatitis B is former, and cause of disease is clear and definite.The result for the treatment of of current hepatitis B is not very desirable, and clinical practice display hepatitis B virus creates resistance to most of medicine, therefore, urgently develops the new treatment means to hepatitis B virus resisting.

Prior art discloses the DNA sequence dna of hepatitis B virus, it mainly containing four coding regions, is respectively S, P, C, X open reading frame; Wherein, C open reading frame coding core antigen (HBcAg), this cAg is one of important symbol of diagnosis hepatitis B virus infection.Have research to point out, to hepatitis B virus self life cycle closely-related albumen or the enzyme intervention be one of the available strategy killing and suppress virus, be also the ideal orientation of following Diagnosis and Treat hepatitis B.

At present, known nucleic acid aptamers (aptamer) is oligonucleotide (DNA or the RNA) fragment of energy specific combination metal ion, polypeptide, protein and even the whole cell gone out through in-vitro screening technology screening, its specificity, as synantibody, has the avidity of strict recognition capability and height to combinative part.The in-vitro screening technology of aptamer is called as Fas lignand system evolution (the Systematic evolution of ligand byexponential enrichment of index concentration, SELEX) technology, SELEX technical modelling natural evolution process, selective pressure (in conjunction with target) is applied to random oligonucleotide library, elutriation and target high special binding fragment (as shown in Figure 1); Compare the aptamers (peptide aptamer) of the polypeptide classes such as antibody, the Dominant Facies of aptamer when obviously, such as: prepare simple and fast, stable chemical nature, be not reported so far there is immunogenicity or toxicity, target molecule scope is wide, avidity is high, high specificity, be easy to carry out transformation modification etc.

The many advantages of aptamer makes it in fundamental research, clinical detection, new drug development etc., have purposes widely.In clinical detection, nearly all can substitute with aptamer by the technology that antigen antibody reaction is carried out detecting at present, as a kind of adaptor molecules sensor---the RiboReporter of Archemix company exploitation ^tMnamely the protein signal detected directly can be changed into optical signal record and analyze, thus achieve the direct rapid detection to the high molecular weight protein in biological mixed solution (as serum, cell extract).In new drug development, most typical example is first aptamer medicine Macugen by U.S. FDA approval listing.It is the aptamer of anti-vascular endothelial cell growth factor (VEGF), is used to treatment wet age-related macular degeneration (wetAMD).The example of another aptamer medicine is the AGR0100 developed by Aptamera company.Preclinical test shows, AGRO100 all has restraining effect to multiple cancer cells.

At present both at home and abroad major part is engaged in the scientist of aptamer research and biotech company mainly for cardiovascular disorder, and the Chinese common disease of less concern, as hepatitis, liver cancer, cancer of the stomach etc., therefore the present invention intends considering how aptamer is applied to the major disease of above-mentioned serious threat our people health.With regard to hepatitis B virus, existing research reports the peptide aptamers for hepatitis B virus core antigen, but there is no the report or open for the aptamer of hepatitis B virus so far.Therefore, develop the aptamer for hepatitis B virus, provide powerful support for for the diagnosis of hepatitis B virus and hepatitis B virus resisting treatment provide, there is important clinical value.

Summary of the invention

The object of this invention is to provide a kind of sequence of aptamer of hepatitis B virus core antigen, be specifically related to the sequence (in the present invention called after HBcAp3) in a kind of aptamer of HbcAg with specific combination HbcAg.

In the present invention, the characteristic sequence containing specific combination HbcAg in the aptamer (sequence 1) of described HbcAg, described characteristic sequence is TATTT.

In the present invention, described hepatitis B virus core antigen is selected from B-type hepatitis strain A, B, C, D, E, F, G, F, G or H gene type.

A further object of the present invention is to provide the purposes of described nucleic acid aptamer sequence, and according to this sequential analysis, the medicine of design anti HBV infecting also prepares medicine or the goods of anti HBV infecting further.

The present invention's gene recombination plasmid expresses cAg, and screening and the aptamer of its specific combination, obtain the characteristic sequence TATTT (in the present invention called after HBcAp3) with specific combination HbcAg.

The present invention is achieved through the following technical solutions:

Adopt known molecule clone technology, based on the recombinant plasmid pBS_HBV3.6II (sequence 2) containing two copy HBV gene group sequence, according to HBc gene order design primer:

HBc_L：5’-GCCCATATGGACATTGACCCGTA-3’

HBc_R：5’-GCCCTCGAGTCAAACAACAGTAGTTT-3’

HBc gene is amplified by polymerase chain reaction (PCR), cut with restriction enzyme NdeI and NotI enzyme, cut pET28a plasmid (winning biotech firm purchased from Beijing bit) with identical restriction enzyme simultaneously, then connect in the NdeI/NotI restriction enzyme site of pET28a with T4 ligase enzyme, obtain the recombinant plasmid pET28a-HBc (plasmid construct as shown in Figure 2) containing HBc gene;

By e. coli bl21 (DE3) (purchased from Shanghai Jie Qing biotech firm) the single colony inoculation containing pET28a-HBc to receiving in antibiotic LB substratum containing card, overnight incubation, culture is transferred in 20OmL fresh culture and cultivates, collected by centrifugation thalline after IPTG abduction delivering;

With 5mL lysate (50mM Tris-HCl (pH8.5 ~ 9.0), 2mM EDTA, 100mM NaCl, 0.5%Triton X-100,1mg/ml N,O-Diacetylmuramidase) resuspended bacterium, 100w ultrasonication in ice bath; Get 1ml supernatant to mix with 500ulNi-NTA Agarose pearl, wash twice with 2ml PBS+Mg (1mM MgCl2), then use 2mlPBS+Mg resuspended, sucking-off albumen-bead mixtures; Add Roche cOmplete Mini EDTA-free protein protective agent to preserve.

Utilize in-vitro screening technology and the SELEX technology of aptamer subsequently, with HBc albumen-bead mixtures for just to sieve target, to be the anti-target that sieves containing the e. coli expression product-bead mixtures of pET28a empty plasmid, from the random oligo DNA library (5 '-acgctcggatgccactacagNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNctcatggacgtgctggtgac-3 ') of external synthesis, filter out the aptamer with HBc specific combination.By the sequence primer Aptamer_L filtered out (5 '-FAM-acgctcggatgccactacag-3 ') and Aptamer_R (5 '-biotin-gtcaccagcacgtccatgag-3 '), being undertaken increasing and carrying out TA is cloned into pMD19-T carrier (winning photo bio company purchased from Shanghai), transforms DH5a bacterium (purchased from Beijing Tian Gen biotech firm).Choosing white colony carries out after PCR determines positive colony with primer RV-M (5 '-GAGCGGATAACAATTTCACACAGG-3 ') and M13 (-47) (5 '-CGCCAGGGTTTTCCCAGTCACGAC-3 '), extracting plasmid and with M13 (-47) (5 '-CGCCAGGGTTTTCCCAGTCACGAC-3 ') for sequencing primer carries out sequencing reaction, upper sequencer; According to sequencing result analytical characteristic sequence.

In the present invention, described nucleic acid aptamer sequence is selected from the sequence of natural existence or synthetic, or the same sequence in any other source;

Described nucleic acid aptamer sequence contains the whole identical sequence with the Nucleotide of described characteristic sequence, and namely all aptamers are all containing described characteristic sequence.

Characteristic sequence of the present invention is the basis of aptamer and HBc specific binding, can be used for medicinal design, prepare medicine or other goods, the described aptamer containing this characteristic sequence can be used as probe or the target spot of Anti-HBV activity, for designing, preparing medicine or the preparation of hepatitis B virus resisting.

Accompanying drawing explanation

Fig. 1 is the in-vitro screening technology of aptamer and the general flow figure of SELEX technology.

Fig. 2 is pET28a-HBc plasmid construct figure.

Embodiment

Embodiment 1:HBc gene clone

With known molecule clone technology, based on the recombinant plasmid pBS_HBV3.6II containing two copy HBV gene group sequence, according to HBc gene order design primer:

HBc_L：5’-GCCCATATGGACATTGACCCGTA-3’

HBc_R：5’-GCCCTCGAGTCAAACAACAGTAGTTT-3’

Configuration PCR system (cumulative volume 50ul): H ₂o 38ul, 10 × PCR damping fluid 5ul, 25mM MgCl ₂3ul, dNTP (10mM) lul, primer mixture 1.5ul (H ₂, DNA profiling 1ul, PrimeStar enzyme 0.5ul O: 100uM HBc_L: 100uM HBc_R=4: 0.5: 0.5).By following condition, PCR instrument increases: after 94 DEG C of heating denaturation 2min, 94 DEG C of sex change 30s, 55 DEG C of annealing 30s, 72 DEG C extend 20s, 30 circulations, and 72 DEG C extend 8min, 4 DEG C of insulation < 1h.

After reaction terminates, get 5ul product 100V constant voltage electrophoresis in the sepharose of 1%, qualification amplifies and expects after the DNA fragmentation that size conforms to, and purifies reclaim test kit and carry out PCR primer and purify by the PCR primer of Axygen.

With restriction enzyme NdeI and NotI double digestion, cut pET28a plasmid with identical restriction enzyme simultaneously, in the sepharose of 1%, reclaim test kit with Axygen glue after 100V constant voltage electrophoresis reclaim endonuclease bamhi, under the effect of T4 ligase enzyme, connect above-mentioned two sections of fragments, obtain the recombinant plasmid pET28a-HBc containing HBc gene.PET28a-HBc and pET28a empty plasmid is transformed in e. coli bl21 (DE3) cell with the Efficient Conversion test kit of sky root respectively.

Embodiment 2:HBc expression and purification

Single for intestinal bacteria containing recombinant expression plasmid pET28a-HBc colony inoculation is contained in the antibiotic LB substratum of Kan to 5mL, 37 DEG C of overnight incubation.By 1: 100 volume ratio, overnight culture is transferred in 20OmL fresh culture, cultivates 2 ~ 4h, treat OD ₆₀₀when value reaches 0.6, add IPTG (O.1mM final concentration is), 30 DEG C of inducing culture 8h.The centrifugal 30min of 4000rpm collects thalline.

With 5mL lysate (50mM Tris-HCl (pH8.5 ~ 9.0), 2mM EDTA, 100mM NaCl, 0.5%Triton X-100,1mg/ml N,O-Diacetylmuramidase) resuspended bacterium, be divided in the pipe of 3 2ml ,-80 degree 30min.100w ultrasonication in ice bath (2s ~ 5s, 60 ~ 80 times).Get 1ml supernatant and 500ul Ni-NTA Agarose pearl mixes, add purification column, with 2ml PBS+Mg (1mM MgCl ₂) wash twice after, by 2ml PBS+Mg resuspended sucking-off albumen bead mixtures.Often pipe adds 7 × Roche cOmplete Mini EDTA-free protein protective agent, preserves as just sieving target for 4 degree.

Equally the intestinal bacteria containing pET28a empty plasmid are carried out that same operation is counter is sieved target.

Embodiment 3: aptamer screening (takes turns)

First run oligo DNA library sequence:

5’-acgctcggatgccactacagNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNctcatggacgtgctggtgac-3’

Enrichment the primer:

Aptamer_L：5’-FAM-acgctcggatgccactacag-3’

Aptamer_R：5’-biotin-gtcaccagcacgtccatgag-3’

Oligo DNA library is measured OD ₂₆₀after, centrifugal drying.Dissolve library with 300ul PBS+Mg, get 200pmol (first round 2.5nmol), 95 DEG C of 8min, put rapidly on ice, centrifugal fast after a while.

Anti-sieve: the suction of the library of 200pmol is equivalent to 40pmol and is just sieving in the anti-sieve target of target, supply 800ul with PBS+Mg.37 DEG C of shaking table vibration 30min.Suck Milipore super filter tube fast centrifugal (< 1000rpm), get filtrate.

Just sieve: get 40pmol and just sieve target, 800rpm is centrifugal, sops up supernatant, then wash twice with 1ml PBS+Mg.Filtrate is added 40pmol just sieving in target, 37 DEG C of shaking table vibration 30min.Suck Milipore super filter tube fast centrifugal (< 1000rpm), abandon filtrate.Three times are washed with PBS+Mg ultrafiltration.Being drawn onto just sieving target in another pipe from film with 600uLPBS+Mg subsequently, adding 60uL pancreatin, 37 DEG C of 10min.Add water and supply 1mL, 95 DEG C of 10min, put rapidly on ice.After turning cold, the centrifugal 5min of 5000rpm.Supernatant is transferred in a clean EP pipe.

Enrichment: configuration PCR system (cumulative volume 1000ul): H ₂o 740ul, 10 × PCR damping fluid 100ul, dNTP 80ul, primer mixture 25ul (H ₂, DNA profiling (just sieving supernatant 50ul, Taq HS enzyme 5ul O: 100uM Aptamer_L: 100uM Aptamer_R=4: 0.5: 0.5).

By following condition, PCR instrument increases: after 94 DEG C of heating denaturation 2min, 94 DEG C of sex change 30s, 58 DEG C of annealing 20s, 72 DEG C extend 20s, 20 circulations, and 72 DEG C extend 4min, 4 DEG C of insulation < 1h.

Purifying: purification column MilliQ washes one time, and PBS washes twice, after adding the StreptavidinSepharose of 150ul GE, PBS washes twice, add PCR primer, shaking table vibration 20min, releases liquid, after PBS washes twice, add after 0.5mL NaOH leaves standstill 2-3min and release liquid, desalting column on relief liquor, adds 1mL MilliQ water elution when liquid almost flows to end from desalting column, starts to meet elutriant 1mL simultaneously, this elutriant is the oligo DNA library be enriched, and can carry out next round screening.

Embodiment 4: aptamer TA clones

In Eppendorf tube, prepare following solutions, full dose is 5ul:pMD19-T Vector 1ul, aptamers PCR primer 1ul, dH2O 3ul.Add the Solution I of 5ul.16 DEG C are reacted 30 minutes.Full dose (10ul) is added in 100ul DH5a competent cell, places 30 minutes in ice.42 DEG C heating 45 seconds after, then in ice place 1 minute.Add 890ul LB substratum, 37 DEG C of shaking culture 60 minutes.LB Agar Plating containing X-Gal, IPTG, Amp is cultivated, forms single bacterium colony.

Embodiment 5: the bacterium colony PCR of aptamer clone and order-checking

PMD19-T bacterium colony PCR and sequencing primer:

RV-M：5′-GAGCGGATAACAATTTCACACAGG-3′

M13(-47)：5′-CGCCAGGGTTTTCCCAGTCACGAC-3′

Join in 4ml LB (Amp 50ng/L) liquid nutrient medium with sterilizing toothpick picking white mono-clonal bacterium colony, 180rpm, 37 DEG C are shaken bacterium and spend the night (< 16h). and get 1ul bacterium liquid and make pcr template, carry out pcr amplification.Using water as negative control.

Configuration PCR system: bacterium liquid 1ul, primer RV-M (10uM) 0.5ul, primer M13 (-47) 0.5ul, 2 × TaqMix 7.5ul, water 5.5ul (total system 15ul).By following condition, PCR instrument increases: 95 DEG C of denaturation 5min; 94 DEG C of sex change 30s, 55 DEG C of annealing 30s, 72 DEG C extend 45s, totally 33 circulations; 72 DEG C of total elongation 10min.

Get 3-6ul bacterium liquid PCR primer after reaction terminates and carry out agarose gel electrophoresis, 140V electrophoresis 20min.If there is bright object size strip and negative control does not have band, illustrate that this bacterium liquid is positive colony.

Row plasmid extraction subsequently: the fresh bacterium liquid getting 4ml incubated overnight, collects thalline, carries out plasmid extraction according to the plasmid extraction kit operation instructions of Omega.Finally be dissolved in water.Nanodrop is adopted to carry out concentration and purity testing to the plasmid extracted.

Sequencing reaction: BDT v3.10.5ul, plasmid 100ng, primer M13 (-47) 0.5ul, adds water to 5ul.After 96 DEG C of heating denaturation 1min, 96 DEG C of sex change 10s, 50 DEG C of annealing 10s, 60 DEG C extend 4min, 33 circulations, 4 DEG C of insulations.Product+1.25ul EDTA (125mM, the pH 8.0)+15ul dehydrated alcohol that checked order by 5ul after reaction terminates is sealed, and shakes 4 times, room temperature places 15min, immediately 3860rpm, tips upside down on immediately on paper handkerchief after room temperature (25 DEG C) centrifugal 40min, centrifugal to 900rpm, stop at once.Add 60ul 70% ethanol, (not needing concussion) seals.3860rpm under room temperature subsequently, centrifugal 15min.Tip upside down on immediately on paper handkerchief, centrifugal to 900rpm, stop at once.Lucifuge drying at room temperature 15min, adds 10ul Hi-Di, seals lid.95 DEG C of sex change 4min, leave standstill 4min on ice at once, of short duration centrifugal, upper 3730xl sequenator.Sequencing result shows, and all there is same section of sequence TATTT in 12 clones.Result confirms, present invention obtains the characteristic sequence with specific combination HbcAg, this section of characteristic sequence is TATTT.

Claims (3)

1. an aptamer for hepatitis B virus core antigen, is characterized in that, the sequence of described aptamer is as shown in sequence 1.

2. the purposes of the aptamer of the hepatitis B virus core antigen of claim 1 in the medicine preparing hepatitis B virus resisting.

3. the aptamer of the hepatitis B virus core antigen of claim 1 detects the purposes in the probe of hepatitis B virus in preparation.