CN1611509B - Nucleic acid adaptor for specific identifying human cerebral acetylcholinesterase and its use - Google Patents
Nucleic acid adaptor for specific identifying human cerebral acetylcholinesterase and its use Download PDFInfo
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Images
Abstract
The invention offers oligonucleotide adapter of specific combining human-brain acetylcholinesterase containing SEQ ID: 1, SEQ ID: 2 or SEQ ID: 3 sequence, method of detecting or discriminating human-brain acetylcholinesterase, method and diagnostic reagent and medicine to diagnose or therapy the disease relating to human-brain acetylcholinesterase (especially neural tube defect and neurotic disorder), and so on.
Description
Technical field
The present invention relates generally to the oligonucleotide aptamers of specific combination human brain acetylcholinesterase, the composition that contains it, detection or differentiates method and the diagnostic reagent and the medicine etc. of the disease (particularly neural tube defect and nervous dysfunction) that method, diagnosis or the treatment of human brain acetylcholinesterase is relevant with human brain acetylcholinesterase.
Background technology
The SELEX technology is a kind of new combinatorial chemistry technique of development in recent years research nucleic acid construct, function and the evolution etc. of getting up, it not only has good application at aspects such as fundamental research, biological screenings, and (the Brody EN﹠amp that also shows wide prospect aspect clinical diagnosis; Gold L, J.Biotechnol., 2000,75:5-13).The oligonucleotide aptamer that filters out (aptamer) but high-affinity with discern different molecules with high specificity.Its avidity and specificity can compare favourably with antibody, are considered to a kind of novel agent of " challenge " antibody status, play an increasingly important role in the diagnosis of disease and treatment.The SELEX technology generally needs number wheel or tens of the wheel just can obtain its corresponding aglucon.Recently, Santa-Coloma TA etc. has reported high specific " polyclonal oligobody (polyclone oligonucleotide aptamers) ", " monoclonal oligobody (mono-clonal oligonucleotide aptamers) " or " synthetic oligobody (synthetic oligonucleotide aptamers) " (the Bianchini M et al with the preparation of target handoff technique, J.Immunol.methods, 2001,252:191-197) can discern corresponding proteins matter specifically, be applied to western blotting, immunohistochemical methods, co-immunoprecipitation etc. and analyze in the experiment.
The protein production of antibodies with use to have following restriction: during with toxicity antigen, immune animal is held and can't stand (1), and immunogenicity is weak is difficult to produce antibody; (2) hybridoma results from mouse, uses limited at human body; Heterologous antibody also produces nonspecific reaction (false positive) in diagnosis; (3) the cost height, waste time and energy, rare antibody needs a large amount of screenings just can obtain; (4) effective preservation of clonal cell line is difficult for, and some hybridoma is difficult to grow in vivo; (5) criticize between quality differ, to optimize again during diagnosis; (6) the interior and external identification specificity of body has difference; (7) the interactional kinetic parameter of antibody-target molecule can't be according to requiring change; (8) life-span limited, to temperature sensitive, irreversible denaturation takes place.And nucleic acid (adaptive son or aptamers) has following superiority: (1) external but not screen under (animal, cell) condition in the body, and characteristic can change according to requiring; (2) kinetic parameter can change according to the requirement of in-vitro diagnosis condition; (3) the weak suffered restriction of antigen of nontoxicity antigen and immunogenicity; (4), can guarantee time, quality and quantity in external chemosynthesis; (5) specificity and affinity do not organized or sample in the interference of non-target protein; (6) can be when synthetic accurately, fixed point, arbitrarily connect other functional group and molecule, as sulfydryl, amino and fluorescein, vitamin H, enzyme etc.; (7) littler than antibody molecule, more be applicable to diagnostic imaging and treatment in the body.As connect thiophosphoric acid, can be used for diagnosis and treatment in the cell; (8) sex change and renaturation are reversible, and speed is fast, and use repeatedly, prolonged preservation and transportation at room temperature (Jayasena SD, Clin.Chem., 1999,45:1628-1650).
Acetylcholinesterase (Acetylcholinesterase, AChE, EC 3.1.1.7) is neural important hydrolase, and its major function is the ACh of hydrolysis postsynaptic membrane, thereby keeps the normal delivery of nerve impulse.AChE has molecular diversity (Grisaru D, et al.Eur.J.Biochem., 1999; 264:672-686).Molecular cloning discloses, and causes three kinds of different AChE hypotypes by the alternative splicing of the mRNA precursor of AChE.Cynapse type AChE (AChE-S) mainly is distributed in neural system and muscle, and it has a wetting ability C end that contains 40 amino-acid residues.Erythrocytic form AChE (AChE-E) is distributed in the mature erythrocyte surface, may (Butyrylcholinesterase BChE) participate in to remove some ester compounds in the blood together with pseudocholinesterase.Recently the rare type of reading over AChE (AChE-R) is found, and it is present in embryo and the tumour cell with the soluble and monomeric form, and AChE-R content significantly rises in the brain when acute physiological stress, closure craniocerebral injury or contact AChE inhibitor.The sequence major part of cynapse type and erythrocytic form AchE is identical, only its C-terminal amino acid residue difference.Because different montage and the posttranslational modifications of mRNA precursor, erythrocytic form AChE (557aa) lacks 26 residues than brain type AChE (583aa).
(neural tube defects NTDs) is the modal congenital malformation of China to neural tube defect, and average attack rate is 7/1000ths.NTD is the deformity due to neurocele patent embryonic stage, mainly comprises anencephalus, spina bifida and brain bulging.NTD baby's birth brings lifelong disaster, brings heavy burden for family and society.NTD is a disease of multifactorial inheritance, and the Gestation period lacks folic acid and such environmental effects is close to closing.The antenatal diagnosis of NTD mainly contains following several method: (1) female blood and AFAFP (AFP) are checked; (2) amniotic fluid AChE measures: human brain AChE is secreted by nervous tissue, when spinal cord and cerebral tissue are exposed in the amniotic fluid, can measure AChE in the amniotic fluid, does not detect AChE in the normal amniotic fluid.May cause false positive owing to blood sample pollutes during amniocentesis, so it is very useful in the NTD antenatal diagnosis to distinguish the reagent of human brain AChE and HRBC AChE; (3) method such as ultrasound diagnosis is inapplicable in early days in gestation.(Dong Xuejun, Chinese aristogenesis and Journal of Heredity, 1996,4:114-115).
Purpose of the present invention mainly is to produce the oligonucleotide aptamers that can distinguish cynapse type and erythrocytic form AChE.They can be applicable to the diagnosis and the treatment of neural-tube defect and nervous dysfunction (e.g. presenile dementia).
Summary of the invention
The difference of human brain AChE and HRBC AChE is the different of C-end.We utilize " target switching " (target switching) method to start with from the difference peptide section of the two, obtain special polyclone and mono-clonal oligonucleotide aptamers at human brain AChE.The oligonucleotide aptamers specificity height that filters out can clearly be differentiated HRBC AChE and human brain AChE.In addition with people BChE also no cross reaction.Finish the present invention thus.
Therefore, on the one hand, the invention provides the oligonucleotide aptamers of specific combination human brain acetylcholinesterase.
Particularly, oligonucleotide aptamers of the present invention comprises following nucleotide sequence:
A) comprise sequence A AACAX
nThe long 15-50nt of GYCCC (comprises any integer between the 15-50, preferred 15-40nt, more preferably 20-30, most preferably 23-28nt, particularly 25nt) or more nucleotide sequence, wherein, each X is respectively A, T, C or G independently, n is that 0-20 (comprises any integer between the 0-20, for example 0,1,2,3,4 ..., 10,11,12,18,19,20, preferred 0-15, more preferably 1-10,2-4 most preferably, particularly 2 and 4) integer or more, Y is G or C;
B) sequence of SEQ ID NO:3;
C) under stringent condition with a) or b) the sequence of sequence hybridization; Or
D) a) or b) or complementary sequence c).
The stringent condition of oligonucleotide hybridization is well known in the art, " the fine works molecular biology experiment guide " that " molecular cloning experiment guide " second edition that can write referring to for example J.Sambrook etc. or Frederick M.Ausubel etc. write.For example, under the temperature that is lower than 15-25 ℃ of Tm value, hybridize, even under the temperature that is lower than 5-10 ℃ of Tm value, hybridize.
In one embodiment, oligonucleotide aptamers of the present invention is DNA.Oligonucleotide aptamers of the present invention also can be RNA, can also be nucleic acid analog, for example peptide nucleic acid(PNA) (PNA).
In a specific embodiments, described oligonucleotide aptamers has the sequence of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.
In another embodiment, oligonucleotide aptamers of the present invention connect or mark other functional group or molecule, for example sulfydryl, amino, radio isotope, fluorescein, vitamin H, toxin or enzyme etc.These functional groups or molecule can be used for the mark purpose so that detect or diagnosis, also can be used for the treatment of purpose or other purpose.
The present invention also provides carrier that comprises described oligonucleotide aptamers and the cell that comprises carrier of the present invention.Carrier can be to be used to clone or express or any carrier of other purpose, includes but not limited to plasmid, virus vector.The oligonucleotide aptamers can adopt suitable ordinary method to be cloned in the carrier.The cell that comprises carrier of the present invention is unrestricted, can be eukaryotic cell or prokaryotic cell prokaryocyte, for example Bacillus coli cells, fungal cell such as yeast cell, insect cell or mammalian cell or clone.The method of conversion or transfectional cell depends on the factors well known to those skilled in the art such as kind of carrier and cell, can adopt any appropriate means to transform or transfectional cell.
On the other hand, the method that detects or differentiate human brain acetylcholinesterase also is provided, comprise the sample that may contain human brain acetylcholinesterase is contacted with oligonucleotide aptamers of the present invention, and detect combining of human brain acetylcholinesterase and described oligonucleotide aptamers.
On the other hand, also provide the method that suppresses the human brain acetylcholinesterase activity or reduce human brain acetylcholinesterase, comprise the oligonucleotide aptamers of the present invention that gives significant quantity.This method can be in vivo or external use.When using in the body, using object can be that this any animal that needs, preferred mammal, for example people are arranged.
The present invention also provides diagnostic reagent or the medicine that comprises oligonucleotide aptamers of the present invention.Described diagnostic reagent or medicine are preferred for diagnosing or treatment is relevant with human brain acetylcholinesterase disease, particularly neural tube defect and nervous dysfunction.Described diagnostic reagent or medicine can also comprise other suitable auxiliary reagents, for example appropriate solvent, vehicle.Described medicine also can be united other activeconstituentss and be used together.The formulation of medicine, dosage, administering mode, frequency can be determined by ordinary method by those skilled in the art in conjunction with embodiments of the invention.
Again on the one hand, also provide the purposes of described oligonucleotide aptamers in preparation diagnostic reagent of the present invention or medicine.
The present invention will be described in more detail in conjunction with the accompanying drawings by the following examples.Described embodiment only is used to illustrate, and scope of the present invention is not constituted any restriction.
Description of drawings
Fig. 1 is the result of radiolabeled secondary library and human brain decapeptide dot blot.
Fig. 2 shows the polyclone oligonucleotide aptamers that the present invention obtains and the result of people's brain homogenate co-immunoprecipitation.A) people's brain homogenate and the effect of biotinylation polyclone aptamers; B) positive control: people's brain homogenate and the effect of anti-human brain AChE antibody.
Fig. 3 shows the mono-clonal oligonucleotide aptamers that the present invention obtains and the result of following composition western blotting: A) purifying HRBC AChE; B) people's brain homogenate; C) people's whole blood; D) positive control (people's brain homogenate and the effect of anti-human brain AChE antibody).
Fig. 4 shows that the zymoprotein of mono-clonal oligonucleotide aptamers that the present invention obtains and different Pseudocholinesterases is in conjunction with mensuration.(■), human brain AChE; (▲), electric ray AChE; (●), HRBC AChE;
People BChE.n=3, Mean ± SD.
Fig. 5 shows the possible secondary structure of the mono-clonal oligonucleotide aptamers that the present invention obtains.
Embodiment
The structure in embodiment 1 ssDNA amplification library
In PCR system (10XPCR reaction buffer 10ul, dNTPs (25mM) 8ul, 5 ' primer (25pmol/ul) 2ul, 3 ' primer (25pmol/ul) 2ul, template 0.25ug, Pfu archaeal dna polymerase 1ul), 5 ' end of downstream primer is marked with vitamin H.With original ssDNA storehouse (the synthetic and purifying by Shanghai Bo Ya company) is that template is carried out pcr amplification, can obtain the dsDNA storehouse that 3 ' end has vitamin H.
Described original ssDNA storehouse (single-stranded DNA banks) is one and has 25 stochastic sequences, the random dna library of 85 bases of total length.These two ends, library are fixed sequence program, can design primer pcr amplification is carried out in the library.The sequence of ssDNA is:
5 '-TAATACGACTCACTATAGCAATGGTACGGTACTTCC (25 random nucleotides) CAAAAGTGCACGCTACTTTGCTAA-3 '
The dsDNA product is after separation and purification, add the streptavidin magnesphere combination, this moment, dsDNA combined with streptavidin on the magnetic bead by vitamin H, NaOH with 0.15N unwinds the dsDNA sex change then, and a chain that has vitamin H combines with streptavidin to be stayed on the magnetic bead, and does not dissociate out with a chain of vitamin H, through ethanol sedimentation, be dissolved in the TE damping fluid, measure the OD value, as the storehouse of ssDNA at random of screening for the first time.
The structure in embodiment 2 ssDNA level libraries
Select the 560-569 amino acids residue (NH of human brain AChE for use
2-WSSYMVHWKN-COOH) as first target molecule.It is arranged in the 11st epitope of the C-terminal of people AChE, and this decapeptide has antigenicity.At first with DC film (0.45 μ m, 0.15cm
2) with 4 ℃ of overnight incubation of target protein of 10mg/ml, then the above-mentioned biotin labeled ssDNA at random of about 0.5nmol and NC film 400 μ l contain 1%BSA (10mM Tris-HCl, pH 7.5,1mM EDTA, 1mM CaCl in conjunction with the TE damping fluid
2, 1mM MgCl
2) in hatch, jolting gently, 4 ℃ are spent the night.The NC film washs 15min * 3 time (room temperature) with 1ml TE then.Last NC film is dipped in the oligonucleotide of elution of bound in the 7M urea, 94 ℃, 10min.And then with phenol/chloroform/Virahol (25: 24: 1, pH 8) extracting.Elutriant is used ethanol sedimentation after adding 10% glycogen, carry out again PCR (behind 95 ℃ of pre-sex change 5min, 5 circulations of increasing: 94 ℃ of sex change 1min, 55 ℃ of annealing 2min, 72 ℃ are extended 1min, last 72 ℃ are extended 5min eventually.Getting the above-mentioned PCR product of 10ul is template, pcr amplification once more, and per 3 cycle samplings select PCR output big, and the cycle number of not having assorted band is the optimum cycle number, will remain the 90ul product and all increase.)。Wherein a part with [α-
32P] CTP is that substrate carries out PCR, the product that obtains and different concns decapeptide (1 μ g/5 μ g/8 μ g) combine dot blot by on the NC film, carrying out (Sorensen K, et al.Biochim Biophys Acta, 1987,912:56-62.) verify.Rest part generates biotinylation dsDNA by PCR, is split as biotinylation ssDNA again.
Dot blot is carried out in amplification library after the decapeptide of different concns and the screening of the radiolabeled first round, and the result shows the increase along with the amount that adds polypeptide, and the spot on the X-ray sheet is also deepened gradually along with the increase that adds the peptide amount.This result shows that radio-labeling library and polypeptide have keying action (Fig. 1).
Embodiment 3 polyclone oligonucleotide aptamers
Adopt the method for " target switching " to obtain specific polyclonal oligonucleotide aptamers.Be about to initial target molecule decapeptide " switching " and be the human brain AChE in people's brain homogenate.People's brain homogenate (80 μ g) (preparation method: get human brain caudatum and cerebellum, broken at a slow speed in high-speed tissue mashing machine, make homogenate.Add sad several defoam after, add 10%Triton X-100 again and be 1%, 4 ℃ to final concentration and stir 4h, 100, the centrifugal 60min of 000g, supernatant is people's brain homogenate) carry out 12% SDS-PAGE, change behind the film with the PBS sealing that contains 5% skim-milk.NC film and biotinylated ssDNA level library incubated at room 2h in containing the 3ml TE binding buffer liquid of 1%BSA, the HRP with the streptavidin mark develops the color then.Cutting-out corresponds to the 66kD band (equally also being verified by anti-human brain AChE antibody) of human brain AChE.Bonded oligonucleotide urea-phenol method wash-out, the performing PCR of going forward side by side.The product that obtains at last is polyclone oligonucleotide aptamers.
Co-immunoprecipitation
At first with people's brain homogenate (200 μ g) and above-mentioned biotinylated polyclone oligonucleotide aptamers incubated at room 1h in TE binding buffer liquid.Add 25 μ l streptavidin magnespheres and come the mixture of separate oligonucleotides aptamers and human brain AChE.With 1ml TE damping fluid washing 4 times.Magnetic bead-oligonucleotide aptamers-human brain AChE mixture is carried out 12%SDS-PAGE, is transferred on the NC film.Then successively with anti-human brain AChE antibody and HRP-IgG two anti-colour developings.Positive control detects the AChE in people's brain homogenate with anti-human brain AChE antibody.
Co-immunoprecipitation presents a master tape (Fig. 2 A) at the 66kD place, with the single band suitable (Fig. 2 B) of anti-human brain AChE antibody (positive control).This presentation of results polyclone oligonucleotide aptamers produces specificity with human brain AChE and combines (Fig. 2).
Embodiment 4 mono-clonal oligonucleotide aptamers
Polyclone oligonucleotide aptamers is connected in pGEM-T easyvector through behind the pcr amplification, transfection competent cell then, and picking 3 clones check order.Each positive colony is a mono-clonal oligonucleotide aptamers.The co-immunoprecipitation of 3 mono-clonal oligonucleotide aptamers, western blotting, dot blot and enzyme antigen immune measurement result are similar, are the experimental result of No. 1 mono-clonal oligonucleotide aptamers (Ob1) below.
1. western blotting (western blotting)
HRBC AChE and people's whole blood sample of people's brain homogenate, purifying are carried out SDS-PAGE, hatch with No. 1 biotinylation mono-clonal oligonucleotide aptamers behind the commentaries on classics film, the HRP with the streptavidin mark develops the color then.
No. 1 mono-clonal oligonucleotide aptamers can produce specificity with human brain AChE and combine, present a master tape (Fig. 3 B) at the 66kD place, do not combine (Fig. 3 A, C) and illustrate that No. 1 mono-clonal oligonucleotide aptamers can distinguish the human brain AChE and the HRBC AChE of sex change and have with purifying HRBC AChE and people's whole blood.
2. dot blot
The Pseudocholinesterase of 0.5U is dissolved among 0.01M PBS (pH 7.4)-144mMNaCl-1ml/L Triton X-100 of 5 μ l (the sex change sample adds SDS to 1g/L concentration, boils 5min), point sample is on the NC film then.With the buffer A (0.05M Tris-HCl, pH 7.4,0.14M NaCl) of 3%BSA to NC membrane closure 2h, then once with the buffer A rinsing that does not contain BSA.Add biotinylation mono-clonal aptamers then, again the HRP colour developing that connects with streptavidin.
The result shows that the human brain AChE of No. 1 oligonucleotide aptamers and natural and sex change and electric ray AChE show the specificity combination, and does not have binding ability (table 1) with people and ORBC AChE and people BChE.
The dot blot of the purifying Pseudocholinesterase of table 1. mono-clonal oligonucleotide aptamers and different sources
3. zymoprotein is in conjunction with measuring (ECA)
The zymoprotein binding assay imitates enzyme antigen immune mensuration to carry out.Streptavidin (5g/L dilutes with PBS) is added in the microwell plate, 100 μ l/ holes, 4 ℃ are spent the night.After the PBS washing, with 1%BSA sealing, room temperature 1h.No. 1 mono-clonal oligonucleotide of biotinylation aptamers is cushioned liquid (0.01M PBS, pH 8.0,144mM NaCl) dilution back with bag and adds every hole (0.3 μ g/ hole), hatches 2h for 37 ℃.(0.01M PBS, pH 7.2,0.05%Tween-20) after the washing 2 times, are cushioned liquid with the bag that contains 1%BSA, every hole 200 μ l, 37 ℃ of sealing 1h through lavation buffer solution.After washing 2 times, every hole add enzyme reaction buffer solution (0.01PBS, pH 7.4,144mM NaCl, 0.01%Triton X-100) the AChE 100 μ l of the different genera of dilution slowly shake 2h at room temperature, after washing 5 times, add substrate reactions liquid (1mM ATChI, 0.025mM DTNB, 0.1M PBS, pH 7.4,0.1%Triton X-100), every hole 100 μ l measure extinction value at 414nm wavelength place.
ECA shows that No. 1 mono-clonal oligonucleotide aptamers of human brain AChE and human brain and electric ray AChE all show tangible specific binding reaction, and with HRBC AChE and the no fixation phenomenon of people's pseudocholinesterase (BChE) (Fig. 4).From No. 1 mono-clonal oligonucleotide of another side illustration aptamers human brain AChE there is specificity.
The primary structure and the secondary structure analysis of embodiment 5 oligonucleotide aptamers
3 clones in the positive hole of being obtained are checked order difference called after Ob1, Ob2, Ob3.Use Clustal W and DNA folding server software respectively these 3 aptamers to be carried out primary structure and secondary structure analysis.Primary structure is as follows.Wherein Ob1 and Ob2 have certain homology, contain conserved sequence, and Ob3 does not have same Feed Discovery.
Ob1：CTCTCGTGCT
AAACATA
GGCCCGTA(SEQ?ID?NO：1)
Ob2：CTTCGA
AAACACCCT
GCCCCTCACA(SEQ?ID?NO：2)
Conserved sequence: AAACA G (G/C) CCC
Ob3：CATTAGAATCTGTGACAATAACGTT(SEQ?ID?NO：3)
Secondary structure analysis:
The secondary structure of 3 oligonucleotide aptamers molecules is all carried out the minimum energy secondary structure analysis with DNA Folding Server.The result shows that the secondary structure of 0b1-3 has similar framework, and nearly 5 ' end fixed sequence program forms an identical stem ring, and there is a little stem ring (Fig. 5) at the stochastic sequence middle part.
Claims (19)
1. the oligonucleotide aptamers of specific combination human brain acetylcholinesterase comprises following nucleotide sequence:
A) comprise sequence A AACAX
nThe nucleotide sequence of the long 15-50nt of GYCCC, wherein, each X is respectively A, T, C or G independently, and n is the integer of 0-20, and Y is G or C;
B) sequence of SEQ ID NO:3;
C) under stringent condition with a) or b) the sequence of sequence hybridization; Or
D) a) or b) or complementary sequence c).
2. the oligonucleotide aptamers of claim 1, wherein a) described in the length of nucleotide sequence be 15-40nt.
3. the oligonucleotide aptamers of claim 1, wherein a) described in the length of nucleotide sequence be 20-30nt.
4. the oligonucleotide aptamers of claim 1, wherein a) described in the length of nucleotide sequence be 23-28nt.
5. the oligonucleotide aptamers of claim 1, wherein a) described in the length of nucleotide sequence be 25nt.
6. the oligonucleotide aptamers of claim 1, wherein a) described in n be the integer of 0-15.
7. the oligonucleotide aptamers of claim 1, wherein a) described in n be the integer of 1-10.
8. the oligonucleotide aptamers of claim 1, wherein a) described in n be the integer of 2-4.
9. the oligonucleotide aptamers of claim 1, wherein a) described in n be 2 or 4.
10. the oligonucleotide aptamers of claim 1, it is DNA.
11. the oligonucleotide aptamers of claim 10 has the sequence of SEQ ID NO:1, SEQ IDNO:2 or SEQ ID NO:3.
12. the oligonucleotide aptamers that claim 1-11 is any, its connection or mark other functional group or molecule.
13. the oligonucleotide aptamers of claim 12, wherein said other functional group or molecule are sulfydryl, amino, radio isotope, fluorescein, vitamin H or enzyme.
14. comprise the carrier of any one the oligonucleotide aptamers of claim 1-13.
15. comprise the cell of the carrier of claim 14.
16. comprise the diagnostic reagent or the medicine of any one the oligonucleotide aptamers of claim 1-13.
17. the diagnostic reagent of claim 16 or medicine, the disease that it is used to diagnose or treatment is relevant with human brain acetylcholinesterase.
18. the diagnostic reagent of claim 17 or medicine, the wherein said disease relevant with human brain acetylcholinesterase is selected from neural tube defect and nervous dysfunction.
19. any one the oligonucleotide aptamers of claim 1-13 preparation claim 16-18 each diagnostic reagent or the purposes in the medicine.
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| 张兴梅.抗乙酰胆碱酯酶基因表达的反义寡核苷酸的研究进展.Foreign Medical Sciences Section on Pharmacy30 2.2003,30(2),94-97. |
| 张兴梅.抗乙酰胆碱酯酶基因表达的反义寡核苷酸的研究进展.Foreign Medical Sciences Section on Pharmacy30 2.2003,30(2),94-97. * |
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