CN106636101A - Nucleic acid aptamer for binding human-derived beta-microglobulin - Google Patents

Abstract

The invention belongs to the field of biotechnology and relates to screening of short-chain nucleotide aptamer with high specificity and high affinity to beta-microglobulin. A preparation method comprises steps as follows: designing and synthesizing a random single-chain nucleotide library and screening a nucleotide aptamer of beta-microglobulin.

Description

For with reference to the aptamer of people source β-microglobulin

Technical field

This patent disclosure relates generally to aptamer, and more particularly relate to the nucleic acid combined with people source Beta2- microglobulins Fit and its screening technique.

Background technology

Beta2- microglobulins are widely present in blood, cerebrospinal fluid, saliva and urine, are cell surface Main Tissues phases The Main Ingredients and Appearance of capacitive antigen MHC--I, MHC-I molecules are almost present in the surface of all cells in addition to red blood cell^[1,2].Determine Beta2-microglobulin content in serum or blood plasma contributes to clinically determining the biologically active and tumour of cell immune system The activity of mark.

Beta- microglobulin molecules amount is 12,000D, the chondritic that it is made up of the domain of multiple beta- lamellas, Itself there is no the domain of film, be one of four subunits of composition MHC-I type molecules, played maintenance MHC-I molecular surfaces With the effect of polypeptide binding site structure, but be not involved in itself combine.

Microglobulin plays an important role during human immunity, and it is synthesized by lymphatic system, while it It is the necessary condition of cell expression MHC-I.The mutant mice of beta- microglobulins disappearance, is nearly no detectable in cell surface MHC-I molecules, without the participation of MHC-I, cd8 cell cannot differentiation and maturation, the T cell without CD8 hypotypes, human body is just The immunity to invading antigen can not possibly be obtained, therefore it relate to immunologic process important in vivo.

In addition to it may make up MHC-1 class surface moleculars, beta2 microglobulins may also participate in the molecule to form other I types, such as CD1 and Qa molecules. additionally, it can also be participated in adjusting the pinocytosis that iron enters small intestine together with HFE albumen, this function The default excess and hemochromatosis for causing iron.

In clinical diagnosis, the patient of chronic hemodialysis, its beta2-microglobulin can assemble amyloidogenic fibre in articular cavity Dimension, can cause Dialysis-related amyloidosis.

Beta2-microglobulin content in urine, by the beta2- in glomerulus and reabsorption, therefore urine Microglobulin content indicates the disorder of kidney filtration.The content of beta2-microglobulin can take off in clinical diagnosis in blood urine Show various diseases^[3].The concentration of beta2 microglobulins is less than 2.5mg/L in normal human serum, and the elderly is then less than 3.5mg/ L；Content in urine in 0-0.3mg/L. in (1) malignant tumour, such as liver cancer, cancer of pancreas, courage cancer, lung cancer, cancer of the stomach, ovary In the serum and urine such as love, palace body gland cancer, knot (straight) intestinal cancer, Huppert's disease, NHL and hundred blood diseases Beta2-microglobulin level is increased significantly, can be used as an index of malignant tumour state of an illness monitoring.Other can cause Beta2-microglobulin level is elevated to also have (2) kidney diaseases, urgent, chronic nephropyeltis high^[4], wolf property ephritis, ephrosis synthesis Beta2-microglobulin level is raised in disease, the blood of renal failure, urine；(3) systemic loupus erythematosus, rheumatoid arthrosis Beta2-microglobulin level is raised in inflammation, leucoderma, nettle rash, herpes zoster, AIDS blood；(4) kidney rejection Beta2-microglobulin level is raised in urine；(5) other diseases, such as diabetic nephropathy, preeclampsia, acute, chronic hepatitis, popularity Beta2-microglobulin level is raised in the blood such as Hemorrhagic fever^[3]；Lymphatic vessel disease, (6) peripheral artery disease^[5].Therefore, blood is determined The content of microglobulin in liquid or urine, can be with the process of these diseases of assisted diagnosis.

Aptamer is invented in nineteen ninety by Gold laboratories, and they pass through a now referred to as process of SELEX, The RNA molecule that can be combined with T4 archaeal dna polymerases is obtained, this is the earliest aptamer molecule for finding. after 2 years, continue Stark reality Room and Gilead Sciences companies are tested, has obtained what is can combined with organic dye molecule and human thrombin with similar method Single stranded DNA is fit.At present DNA or RNA aptamers are widely used for preparing different bioactivators^[6][7][8], wherein Including lysozyme^[9], fibrin ferment^[10], human Aids virus trans-acting original paper (HIV TAR)^[11], ferroheme^[12], interferon^[13], VEGF (VEGF)^[14], dopamine^[15].With updating for aptamer technology of preparing, Gold leaders' The single fit research and development time is tapered to the time of three days from six weeks for SomaLogic companies.

The preparation process of aptamer is exactly to screen from artificial design, the single stranded DNA/RNA libraries of synthesis briefly A series of oligonucleotides with high-affinity and high specific for obtaining.The oligonucleotide molecules of these synthesis are specifically being delayed Under rushing system, different three-D space structures can be formed by active force in hydrogen bond, Van der Waals force, hydrophobic effect equimolecular, Such as hairpin structure (Hairpin), false knot (pseudoknot), G- tetrads (G-quartet), then recognize simultaneously with targeting molecule With reference to, uncombined sequence is rejected, retain binding sequence, and amplify with reference to aptamer by the way that PCR is exponential Quantity, and uncombined adaptor molecules are removed by anti-sieve competition law, through the screening of several wheels or more than ten wheels, obtain high parent With power and the aptamer part of high specific.This process is referred to as the SELEX (Systemic of aptamer now Evolution of Ligands by Expotential Enrichment) index concentration Fas lignand system evolve screened Journey, its general principle is strong and weak with the binding ability of targeting molecule incubation using artificial synthesized oligonucleotide library, reaches powerhouse The result of enrichment.

Compared with antibody, aptamer has many irreplaceable advantages：Target molecule scope is more extensive, from little metal The target that ion can serve as screening to hypertoxic molecule, even complete cell etc.；High-affinity and high specific；By changing Method synthesizes, easy to maintain, is difficult by biological pollution or degraded；Non-immunogenicity；Tissue permeability is strong；Easily carry out chemistry to repair Decorations etc..The medical value of aptamer is also being found .2004 to be ratified earliest by food and drug administration (FDA) For the nucleic acid aptamer medicament Macugen listing of the treatment AMD (AMD) of Osi Pharm Inc..Additionally, its It is used to treat acute disease and the APTAMER MEDICINAL of chronic disease is also in clinical testing, is such as used to treat acute coronary syndrome (ACS) vWF ELISA (vWF) antagonist (ARC1779) of a kind new medicine will start the second stage of clinical examination the end of the year in 2007 Test.

It is more fit constantly to develop in clinical diagnosis field, such as Ellington laboratory and Colorado It is fit group for plasma protein analyzing and diagnosing that SomaLogic companies are developing, and they are referred to as fit plasma protein Matter group.This technology aptamer chips, once analyzes the changes of contents of the protein component of multiple blood plasma, for distinguishing, diagnosing The disease and health status of patient.

[1]″Entrez Gene：Beta-2-microglobulin″.

[2] G ü ssow D., et al., (1987) The human beta 2-microglobulin gene.Primary structure and definition of the transcriptional unit″.J.Immunol.139(9)：3132- 8.

[3] Ma Aiqun, Lv Yi (2008) clinical laboratory handbook, Science Press PP：505.

[4] Salvaggio E., et al, the microglobulin in the diagnosis of of (1988) Beta 2 reflux nephropathy in childhood.Pediatr Med Chir.198810：83-8.

[5] United States Patent (USP) (2011) Beta-2 mieroglobulin as a biomarker for peripheral artery disease US patent 7,867,719.

[6] Neves, M.A.D.；O.Reinstein, M.Saad, P.E.Johnson (2010). " Defining the secondary structural requirements of a ***e-binding aptamer by a thermodynamic and mutation study″.Biophys Chem 153：9-16.

[7] Baugh, C.；D.Grate, C.Wilson (2000). " 2.8 angstrom crystal structure of the malachite green aptamer.″.J.Mol.Biol.301：117-128..

[8] Dieckmann, T.；E.Fujikawa, X.Xhao, J.Szostak, J.Feigon (1995). " Structural Investigations of RNA and DNA aptamers in Solution″.Journal of Cellular Biochemistry：56-56.

[9] Potty, A.；K.Kourentzi, H.Fang, G Jackson, X.Zhang, G.Legge, R.Willson (2009).″Biophysical Characterization of DNA Aptamer Interactions with Vascular Endothelial Growth Factor.″.Biopolymers 91：145-156.

[10] Long, S.；M.Long, R.White, B.Sullenger (2008). " Crystal structure of an RNA aptamer bound to thrombin″.RNA 14：2504-2512.

[11] Darfeuille, F.；S.Reigadas, J.Hansen, H.Orum, C.Di Primo, J.Toulme (2006).″Aptamers targeted to an RNA hairpin show improved specificity compared to that of complementary oligonucleotides.″.Biochemistry 45：12076- 12082.

[12] Liu, M.；T.Kagahara, H.Abe, Y.Ito (2009). " Direct In Vitro Selection of Hemin-Binding DNA Aptamer with Peroxidase Activity″.Bulletin of the Chemical Society of Japan 82：99-104.

[13] Min, K.；M.Cho, S.Han, Y.Shim, J.Ku, C.Ban (2008). " A simple and direct electrochemical detection of interferon-gamma using its RNA and DNA aptamers.″.Biosensors& Bioelectronics 23：1819-1824.PMID 18406597.

[14] Ng, E.W.M；D.T.Shima, P.Calias, E.T.Cunningham, D.R.Guyer, A.P.Adamis (2006). " Pegaptanib, a targeted anti-VEGF aptamer for ocular vascular disease.″.Nature Reviews Drug Discovery 5：123-132.

[15] Walsh, R.；M.DeRosa(2009).″Retention of function in the DNA homolog of the RNA dopamine aptamer.″.Biochemical and Biophysical Research Communications 388：732-735.

The content of the invention

1. the first object of the present invention is the aptamer for providing the beta2-microglobulin with high-affinity.

2. the core sequence of the beta2-microglobulin aptamer described in is as follows：

YZMG004：GGGCGACGGACCGTGTGTGTGGGGT

3. also above-mentioned aptamer can be modified and transformed, the nucleic acid aptamer derivative of gained is had identical function Nucleic acid aptamer derivative can be：

A) aptamer is deleted the nucleotides of part or increase and decrease partial complementarity, it is obtaining with the aptamer phase The nucleic acid aptamer derivative of congenerous, such as G- ＞ C, A- ＞ T etc..

B) aptamer is carried out into nucleotides replacement or part is modified, having with the aptamer for obtaining is identical The derivative of the aptamer of function.

C) described aptamer skeleton is transformed, such as phosphorothioate backbone, what is obtained has with the aptamer There is the nucleic acid aptamer derivative of identical function.

4. the method and step for being screened is specific as follows：

1. random DNA library is set up, the length of ss DNA is 25 bases, and the two ends in library can include arbitrary nucleic acid Sequence.

2. the Sepharose activated expoxy 6B that described start library is coupled with beta2-microglobulin enter Row incubation, removes uncombined sequence, obtains the nucleotide sequence combined with beta2-microglobulin.

3. the Sepharose activated being coupled aptamer sequence obtained above and bovine serum albumin BSA Expoxy 6B are incubated, and remove the nucleotide sequence for combining, and obtain what is do not combined with Sepharose activated expoxy 6B Nucleotide sequence.

4. " after circulation repeatedly, the aptamer with beta2-microglobulin specific binding is obtained.

Description of the drawings

Fig. 1 is：The Capillary Electrophoresis collection of illustrative plates that the aptamer for obtaining is combined with β-microglobulin

Specific embodiment

1.beta2- microglobulins are coupled with Expoxy-activated Sepharose 6B：

A () 200ug beta2-microglobulins are dissolved in 200ul water.

B () takes 0.08g Expoxy-activated Sepharose, add 1ml water, overturns repeatedly so as to fully molten Swollen, after about 5min, after it is evenly distributed, low-speed centrifugal removes supernatant.Clean repeatedly several times, to remove additives.

C () takes the beta2-microglobulin of step (a), the Expoxy-activated after adding last time to be centrifuged In Sepharose, the NaHCO of 1M is added₃.(20 DEG C, 150rpm, 16h)

2. the active group of unconjugated beta2-microglobulin is closed：

(1) low-speed centrifugal, repeatedly with the NaHCO of 0.1M₃Cleaning three times.

(2) the Expoxy-activated Sepharose after coupling are placed in the monoethanolamine of 1M (40 DEG C, 4h)

(3) with the Na AC (PH=4.0,0.5M Na Cl) and 0.1M Tris-HCL (Cl of Na containing 0.5M) of 0.1M, hand over For cleaning three times.

(4) finally washed twice with 10% ethanol, in being stored in 10% ethanol.

3.SELEX processes

(1) the Expoxy-activated Sepharose in the ethanol of 80ul 10% are taken, with 1X binding buffer Twice, supernatant is removed in centrifugation for cleaning.

(2) Expoxy-activated Sepharose beads and 10x binding buffer (PH=7.5,50mM Hepes, 120mM Na Cl, 5mM KCL, 2mM CaCL₂), 2mM MgCL₂), then mix with nucleic acid.(20 DEG C, 160rpm, 1h is incubated Change)

(3) it is centrifuged, abandons supernatant.(being uncombined DNA in supernatant)

(4) Expoxy-activated Sepharose are cleaned with 1X binding buffer, supernatant is removed in centrifugation. (on It is uncombined DNA in clear liquid)

(5) Expoxy-activated Sepharose are cleaned with 50ul (8M) urea, 20 DEG C, 160rpm, 10 minutes.From The heart, collects supernatant (being the DNA for combining in supernatant).It is repeated twice

(6) Expoxy-activated Sepharose are cleaned with water, it (is to combine in supernatant that supernatant is collected by centrifugation DNA)。

(7) by the supernatant collected (containing DNA is combined), it is centrifuged in the ultra-filtration centrifuge tube of 3KD.

(8) enter performing PCR as template with the nucleic acid collected, prepare single stranded DNA.

(9) repeat the above steps, carry out ten screenings.

(10) after carrying out 4 circulations, counter to be sieved, be mutually coupled by nucleotide sequence and with bovine serum albumin BSA Expoxy-activated Sepharose are incubated, to remove the nucleic acid of non-specific binding.

4. the preparation of double-stranded DNA.

5. the PCR primer of cloning is connected into carrier T. (pMD 18-T simple vector, it is biological public purchased from TAKARA Department)

6. transformed competence colibacillus cell.(Ecoli DH5 a compliment Cells, purchased from TAKARA biotech firms)

7. after bacterium colony PCR, spot is chosen at random and is sequenced.

8. the aptamer sequence combined with β-microglobulin is obtained

9. using capillary electrophoresis detection beta2-microglobulin and the combination situation of resulting aptamer.(see attached Figure)

Claims (9)

1. aptamer, is the sequence 1 of sequence table, and shown DNA fragmentation, sequence is as follows respectively：

YZMG004：GGGCGACGGACCGTGTGTGTGGGGT。

2. it is DNA molecular and other DNA moleculars comprising its intact DNA fragments described in claim 1.

3. as claimed in claim 1, people source β-microglobulin can be used for for object, but while may act on can be with people source β-microballoon Albumen plays the microglobulin in other sources of cross reaction.

4. as claimed in claim 1, including the aptamer of β-microglobulin is in the application of association area.

5. the method for screening β-microglobulin aptamer, with reference to the claims the 5th of number of patent application 2012103692363 Bar.

6. the aptamer described in claim 1, also including connect or mark that its its functional group produces with identical The derivative of function, these groups include the enzyme such as methyl blue, amino, sulfydryl, alkaline phosphatase or horseradish peroxidase, glimmering Nano-metal particle, the sensitising agent such as light element, radio isotope and therapeutic substance, nm of gold and nanometer ruthenium etc..

7. the aptamer described in claim 1, is also included using the nucleotide monomer synthesis of part modification with identical The modification type aptamer of function.These modifications include：The modification of sugared ring side chain, the change of sugared ring configuration, the modification of base, The modification of phosphate.

8. the aptamer described in claim 1, also includes for aptamer being transformed into the peptide nucleic acid with identical function It is fit.

9. the aptamer described in claim 1, after also including being transformed oligonucleotide backbone and has and microballoon Protein bound compound.The transformation of these skeletons include lock nucleic acid, cyclohexene nucleic acids, morphine woods nucleic acid, Asia acid amides nucleic acid, N3 '- The modified compounds such as P5 ' phosphoramidites.