CN1376798A - Human acetylcholinesterase isomer protein (AR-ACHE) and its gene coding sequence - Google Patents
Human acetylcholinesterase isomer protein (AR-ACHE) and its gene coding sequence Download PDFInfo
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Abstract
A novel human acetylchlorinesterase isomer protein (AR-ACHE) expressed when human tissue cells wither and its gene coding sequence is disclosed. Said cDNA has 1936 bp in length. Said protein is composed of 526 amino acid residues. The process for preparing said protein and nucleic acid sequence, the process for detecting AR-ACHE nucleic acid sequence and polypeptide in sample, and the process for promoting or inhibiting cell withering are also disclosed.
Description
The present invention relates to zymetology and molecular cytobiology, especially about participating in the enzyme in the apoptosis process.Particularly, the present invention relates to acetylcholinesteraseisomer isomer protein white (AR-ACHE) and the nucleotide sequence thereof that each histocyte of a kind of mankind is expressed when apoptosis takes place.
(EC 3.1.17, acetylcholinesterase are the lytic enzymes of neurotransmitter acetylcholine ACHE) to acetylcholinesterase, belong to glycoprotein.Mainly be present in the electric organ of electric ray, mammiferous cholinergic nerve, neuromuscular junction, erythrocyte membrane (Zakut-H; Et al:J-Clin-Invest.1990,86 (3): (A.Shafferman and B.Velan 900-8) and in the rodentine megalokaryocyte thrombocyte, Multidisplinary approaches to cholinesterase functions.1992 Plenum Press, New York).
People's acetylcholinesterasegene gene is positioned on No. 7 karyomit(e) (7q22), finds to have 3 kinds of different shear-forms at present: neuromuscular type, erythrocytic form and read over type (Soreq, H.et al.Proc Natl AcadSci U S A.1990,87,9688-92; Soreq, H.et al.Proc Natl Acad Sci U S A.1994,91,7907-11).Acetylcholinesterase is the hydrolysis neurotransmitter acetylcholine in the function of cholinergic nerve system, makes it to generate choline and acetate.Erythrocytic form is unclear with the function of reading over type, also has report to think that acetylcholinesterase has the hydrolysis of protein activity of serine protease..In recent years, since the tumor cell line of vitro culture also detect AChE mRNA transcribe with chemotherapy after the serum of ovarian cancer patients detect acetylcholine esterase active, there is the author to think that acetylcholinesterase with tumour relevant (Lev-Lehman-E takes place, et al:Blood.1997,89 (10): 3644-53).
Senile dementia (AD) is a more disease of seeing in the regression nervosa.Though the total ACHE activity in patient's AD brain reduces, in senile plaque and the ACHE activity around the senile plaque be (Javier Saez-Valero et al., J.Neurochem.1999, Vol.72, the 1600-1608 that raises; Opazo-G ﹠amp; Inestrosa-NC, Mol-Chem-Neuropathol.1998,33 (1): 39-49; MimoriY.et al., Behav Brain Res.1997,83 (1-2): 25-30.), and, irrelevant with the neurotransmitter degradation function.Proved and found that senile dementia patient affected area has apoptotic cell to exist, in the apoptosis process that studies for a long period of time, find that Mammals comprises all human cell strains, except red corpuscle, through inducing, start in the apoptotic process and express acetylcholinesteraseisomer isomer, this result can the above-mentioned phenomenon of fine note.We have applied for this zymoid preparation method (Zhang Xuejun etc., Chinese patent application numbers 97 1 25216.5, No. the 61260th, invention patent certificate) and the patent of partial function (Zhang Xuejun etc., Chinese patent application numbers 97 1 25220.3, publication number CN 1186859).The present invention adopts methods such as RACE, has obtained the cDNA complete sequence of AR-ACHE.
First purpose of the present invention just provides a kind of new people's gene acetylcholinesteraseisomer isomer (AR-ACHE) (Genbank Accession No.AF334270 is unexposed), and this gene is a human acetylcholinesteraseisomer isomer (AR-ACHE) protein gene.
Second purpose of the present invention provides a kind of recombinant technology that utilizes and produces above-mentioned new human acetylcholinesteraseisomer isomer (AR-ACHE) albumen and the method for nucleotide sequence.
The present invention also provides the application of this human acetylcholinesteraseisomer isomer (AR-ACHE) protein polypeptide and encoding sequence.
The present invention has used the SK-N-SH of vitro culture, and cell strains such as people's lung fibroblast (HLF), K562 are used DMEM, and 1640 substratum add 10% calf serum, at 37 ℃, and 5%CO
2, incubator is cultivated.The cell death inducing method has 3 kinds: naturally-aged method, UV-B and induced by chemotherapeutic agents method.The naturally-aged method, culturing cell did not change nutrient solution in 3-4 days, at this moment, and the cell of adherent growth, part loses adherent ability, has entered apoptotic process, collects cell at this moment.Drug-induced method is with antitumor drug or low serum or remove method cell death inducings such as cytokine.For the cell strain that cytokine relies on, go into the strain of M07e megalokaryocyte, as long as do not add GM-CSF in nutrient solution, after 2-3 days, a large amount of cell generation apoptosis are collected cell at this moment.UV-B induced 10 minutes, cultivated and can collect apoptotic cell after 18 hours.The concrete operations of whole process are as follows:
Total RNA extracts:
With the SK-N-SH cell, HLF or K562 in 1640 substratum (Gibco BRL company) are cultured to the cell state of dying outside withering under the condition of not changing liquid, use Trizol reagent (Gibco BRL company) to extract cell total rna then.About 10
6Cell adds 1ml Trizol, and piping and druming evenly back room temperature was placed 5 minutes, added the 0.2ml chloroform, acutely shook 15 seconds, and room temperature was placed 3 minutes.4 ℃ of 12000g centrifugal 15 minutes then, and the colourless water in sucking-off upper strata adds the 0.5ml Virahol, and room temperature was placed 10 minutes, centrifugal 10 minutes of 4 ℃ of 12000g.Wash precipitation with 75% ethanol again, 7500g, 4 ℃ are centrifugal 5 minutes.Be dissolved in after to be dried in the DEPC treated water ,-70 ℃ of preservations are standby.
The separation of mRNA:
Earlier that total RNA is quantitative, re-use the product operation handbook separating mRNA of the Oligotex mini mRNA Kit of Qiagen company by Kit, be dissolved in the DEPC treated water ,-70 ℃ of preservations are standby.
The full-length clone of gene:
(1)RT-PCR：
Sequence according to human ACHE gene, design 5 ' and 3 ' primer, primer sequence is R1 (SEQ IDNo.3): 5 '-cttccacggcctgcagctggct-3 ', (forward), R2 (SEQ ID No.4): 5 '-gacaagcttacaggtctgagcagcgatcctgcttgct-3 ' (oppositely), adopt the synthetic above-mentioned primer of ordinary method.MRNA with the SK-N-SH cell carries out the RT-PCR amplification.Condition is 96 ℃ after 3 minutes, with 95 ℃ 30 seconds, did 5 circulations in 2 minutes for 72 ℃, again with 95 ℃ 30 seconds, 70 ℃ 30 seconds, did 39 circulations in 1 minute for 72 ℃, last 72 ℃ were extended 10 minutes, electrophoresis detection PCR product is a 527bp length, with the product cloning order-checking, finds to exist a kind of and human normal different splicing form of ACHE gene.
(2) the terminal rapid amplifying of cDNA (Rapid Amplification of cDNA ends, RACE)
According to the sequence that has recorded, design 5 ' and 3 ' primer with the RT product of the SK-N-SH cell mRNA upstream and downstream sequence that increases respectively, is checked order the PCR product cloning.
By using above two kinds of methods, obtained the total length (seeing SEQ IDNo.1) of human AR-ACHE.
The sequence information and the homology analysis of people AR-ACHE gene:
The length of the people AR-ACHE full-length cDNA (GenBank AccessionNo.AF334270) that the present invention is new is 1936bp; Wherein open reading frame is positioned at 106-1686 position Nucleotide (seeing SEQ ID No.1).Derive the aminoacid sequence of people AR-ACHE according to full-length cDNA, totally 526 amino-acid residues (seeing SEQ ID No.2), molecular weight 58352.30D, to be 6.44. carry out Nucleotide and protein homology retrieval with the full length cDNA sequence of AR-ACHE and coded protein thereof with blast program to PI in Non-redundantGenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDStranslations+PDB+SwissProt+Superdate+PIR database, found that there is 85.7% homology at least in it and people's ACHE gene.But AR-ACHE is a kind of aberrant splicing form gene of people ACHE gene.On nucleotide level, it is mRNA whole coding sequence (GenBank Accession No.M.55040) a kind of spliceosome that from first to the 264th base forms after taking place to lack in Exon3 of people ACHE gene.On amino acid levels, it is a kind of new spliceosome albumen (see figure 1) of the 357-444 amino acids residue disappearance back formation of people ACHE albumen (GenProt Accession No.AAA68151).
People AR-ACHE of the present invention is used for further functional study except can be used as this family's a member, also can be used for producing fusion rotein with other albumen, such as producing fusion rotein with immunoglobulin (Ig).In addition, people AR-ACHE of the present invention can also merge with other members of this family or exchange fragment, to produce new albumen.For example proteic N end of people AR-ACHE of the present invention and the proteic N end of people ACHE are exchanged, to produce the albumen that new activity is higher or have new features.
At the antibody of inventor AR-ACHE, be used to screen other members of this family, perhaps be used for affinity purification associated protein (as other members of this family).
In addition, inventor AR-ACHE nucleic acid (encoding sequence or antisense sequences) can be introduced into cell, with expression level that improves people AR-ACHE or the overexpression that suppresses people AR-ACHE.People AR-ACHE albumen of the present invention or its active polypeptide fragment can be applied to tumour patient, with treatment or alleviate because of people AR-ACHE disappearance, no function or unusual cause related disorders arranged.In addition, can also be with carrying out relevant diagnosis or prognosis judgement based on nucleotide sequence of the present invention or antibody.The people AR-ACHE protein of expressing according to the cDNA sequence of people's acetylcholinesterase, and polyclonal antibody and the monoclonal antibody of utilizing this protein to produce, use this polyclone or the monoclonal antibody can be specifically and the AR-ACHE combination, so in the various diseases relevant, can detect this proteinic existence specifically with the AR-ACHE generation, also can the early stage or more proteinic existence of back detection in disease.
Because people AR-ACHE albumen of the present invention has the natural acid sequence that is derived from the people, therefore, compare with the albumen of the same clan that derives from other species, estimate to have higher active and/or lower side effect (for example in the intravital immunogenicity of people lower or do not have) being applied to man-hour.
The preparation and the purification of people AR-ACHE polypeptide
In this embodiment, the people AR-ACHE encoding sequence of total length or fragment are built into commercial protein merge among the expression vector, to express and purification of recombinant proteins.
People AR-ACHE polypeptide is carried out prokaryotic expression with the form of gst fusion protein in intestinal bacteria.Construction of prokaryotic expression vector, and transformed into escherichia coli.Complete encoding sequence according to people AR-ACHE, design amplifies complete coding and reads the primer of frame (correspond respectively to encoding sequence 5 ' and about 20 above Nucleotide of 3 ' end), and on positive anti-primer, introduce restriction endonuclease sites (this decides according to the pGEX-2T carrier of selecting for use) respectively, so that construction of expression vector.With the product that obtains among the embodiment 1 is template, behind pcr amplification, with people AR-ACHE gene guarantee to read be cloned under the correct prerequisite of frame the pGEX-2T carrier (Pharmacia, Piscataway, NJ).Identify that good expression vector utilizes CaCl
2Method changes bacillus coli DH 5-α over to, and Screening and Identification obtains containing the engineering bacteria DH5-α-pGEX-2T-AR-ACHE of pGEX-2T-AR-ACHE expression vector.
Express the isolation identification of the engineering bacteria of GST-AR-ACHE recombinant protein
The engineering bacteria DH5-α-pGEX-2T-AR-ACHE of picking list bacterium colony contains jolting overnight incubation in the LB substratum of 100 μ g/ml acillins in 3ml, drawing nutrient solution by 1: 100 concentration cultivated about 3 hours in new LB substratum (containing 100 μ g/ml acillins), after reaching 0.5 to OD 600, adding IPTG continues at 37 ℃ to final concentration 1mmol/L and cultivates 0 respectively, 1,2,3 hours.It is centrifugal to get the different 1ml bacterium liquid of incubation time, in the bacterial precipitation thing, add lysate (2XSDS sample-loading buffer 50 μ l, distilled water 45 μ l, 3-mercaptoethanol 5 μ l), the suspendible bacterial precipitation, boiled in the boiling water bath 5 minutes, centrifugal 1 minute of 10000rpm, supernatant adds electrophoresis in the 12%SDS-PAGE glue.The bacterial strain that the protein content of dyeing back observation expection molecular weight size increases with the IPTG induction time is the engineering bacteria of expressing the GST-AR-ACHE fusion rotein.
The extraction purifying of GST-AR-ACHE fusion rotein
Bacterium centrifugation after the proteic engineering bacteria DH5-of abduction delivering GST-AR-ACHE amalgamation and expression α-pGEX-2T-AR-ACHE induces as stated above adds the resuspended bacterium of 20ml PBS, ultrasonication bacterium by every 400ml bacterium.The ultrasonic completely liquid of broken bacterium adds 50% saturated Triptide Sepharose 4B of PBS by every milliliter of amount that adds 20 microlitres, 37 ℃ of joltings were in conjunction with 30 minutes, 10000rpm precipitated the Triptide Sepharose 4B that combines GST-AR-ACHE in centrifugal 10 minutes, abandoned supernatant.Clean twice by every milliliter of amount that plays sound liquid gained precipitation adding 100 μ l PBS, then add 10 μ l reduced glutathione elutriants by every milliliter of ultrasonic liquid gained precipitation, room temperature was put 10 minutes, centrifugal 10 minutes of 10000rpm, and supernatant is the fusion rotein of wash-out.Repeat twice of wash-out.The supernatant of wash-out is stored in-80 ℃, and carries out the SDS-PAGE electrophoresis, detects purification effect.Protein band at the 58kDa place promptly is a people AR-ACHE albumen (see figure 2).
People's acetylcholinesterasegene gene is positioned on No. 7 karyomit(e) (7q22), finds to have 3 kinds of different shear-forms at present: neuromuscular type, erythrocytic form and read over type.The present invention is the isomer (AR-ACHE) of being cloned into acetylcholinesterase.Make acetylcholinesterase have the 4th type.It is expressed when any karyocyte apoptosis of people, in apoptosis, plays an important role, and for the medicinal design and the tumour medicine design of regression diseases such as treatment AD provides new approaches and method, all be first in the world.
The present invention has following advantage:
1. utilize AR-ACHE to can be used for further functional study as a member of this acetylcholinesterase family.
2. AR-ACHE can merge with other members of this family or exchange fragment, and to produce new albumen, it is higher and have new features albumen to be expected to obtain activity.
3. AR-ACHE nucleic acid of the present invention can be introduced into cell, with expression level that improves people AR-ACHE or the overexpression that suppresses people AR-ACHE.
4. medicinal design and the tumour medicine design for diseases such as treatment AD provides new approaches and method.
Description of drawings
Fig. 1, AR-ACHE and human brain type ACHE aminoacid sequence compare, few 88 amino-acid residues of AR-ACHE.
Fig. 2. SDS-PAGE electrophoresis, Western blot, ECL dyeing.1 lane is an apoptosis SK-N-SH lysis sample; 2 lane are normal SK-N-SH lysis samples.Normal SK-N-SH cell expressing is cerebral acetylcholinesteraseobtained, the about 65Kda of monomer whose molecular weight; The AR-ACHE monomer molecule amount of apoptosis SK-N-SH cell expressing is less than 58Kda.
The present invention is further elaborated by following drawings and Examples, but does not place restrictions on scope of the present invention.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, " molecular cloning " laboratory manual (NeW York:Cold Sprng Harbor Laboratory Press of people such as Sambrook for example, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1:
1. total RNA extracts;
SK-N-SH cell (Gibco BRL company) in 1640 substratum is cultured to the cell state of dying outside withering under the condition of not changing liquid, uses Trizol reagent (Gibco BRL company) to extract cell total rna then.About 10
6Cell adds 1ml Trizol, and piping and druming evenly back room temperature was placed 5 minutes, added the 0.2ml chloroform, acutely shook 15 seconds, and room temperature was placed 3 minutes.4 ℃ of 12000g centrifugal 15 minutes then, and the colourless water in sucking-off upper strata adds the 0.5ml Virahol, and room temperature was placed 10 minutes, centrifugal 10 minutes of 4 ℃ of 12000g.Wash precipitation with 75% ethanol again, 7500g, 4 ℃ are centrifugal 5 minutes.Be dissolved in after to be dried in the DEPC treated water ,-70 ℃ of preservations are standby.
The separation of mRNA:
Earlier that total RNA is quantitative, re-use the operational manual separating mRNA of the Oligotex mini mRNA Kit of Qiagen company by the Kit product, be dissolved in the DEPC treated water ,-70 ℃ of preservations are standby.
2. the full-length clone of gene:
(1).RT-PCR：
Sequence according to human ACHE gene, design 5 ' and 3 ' primer, primer sequence is R1 (SEQ IDNo.3): 5 '-cttccacggcctgcagctggct-3 ', (forward), R2 (SEQ ID No.4): 5 '-gacaagcttacaggtctgagcagcgatcctgcttgct-3 ' (oppositely).MRNA with the SK-N-SH cell carries out the RT-PCR amplification.Condition is 96 ℃ after 3 minutes, with 95 ℃ 30 seconds, did 5 circulations in 2 minutes for 72 ℃, again with 95 ℃ 30 seconds, 70 ℃ 30 seconds, did 39 circulations in 1 minute for 72 ℃, last 72 ℃ were extended 10 minutes, electrophoresis detection PCR product is a 527bp length, with the product cloning order-checking, finds to exist a kind of and human normal different splicing form of ACHE gene.
(2) the terminal rapid amplifying of .cDNA (Rapid Amplification of cDNA ends, RACE)
According to the sequence that has recorded, design 5 ' and 3 ' primer with the RT product of the SK-N-SH cell mRNA upstream and downstream sequence that increases respectively, checks order the PCR product cloning.By using above two kinds of methods, obtained the total length (seeing SEQ ID No.1) of human AR-ACHE.
The sequence information and the homology analysis of embodiment 2 people AR-ACHE genes:
The length of the people AR-ACHE full-length cDNA (GenBank AccessionNo.AF334270) that the present invention is new is 1936bp; Wherein open reading frame is positioned at 106-1686 position Nucleotide.Derive the aminoacid sequence of people AR-ACHE according to full-length cDNA, totally 526 amino-acid residues (seeing SEQ ID No.2), molecular weight 58352.30 D, to be 6.44. carry out Nucleotide and protein homology retrieval with the full length cDNA sequence of AR-ACHE and coded protein thereof with blast program to PI in Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDS translations+PDB+SwissProt+Superdate+PIR database, found that there is 85.7% homology at least in it and people's ACHE gene.But AR-ACHE is a kind of aberrant splicing form gene of people ACHE gene.On nucleotide level, it is mRNA whole coding sequence (GenBank Accession No.M.55040) a kind of spliceosome that from first to the 264th base forms after taking place to lack in Exon3 of people ACHE gene.On amino acid levels, it is a kind of new spliceosome albumen (see figure 1) of the 357-444 amino acids residue disappearance back formation of people ACHE albumen (GenProt Accession No.AAA68151).
People AR-ACHE of the present invention is used for further functional study except can be used as this family's a member, also can be used for producing fusion rotein with other albumen, such as producing fusion rotein with immunoglobulin (Ig).In addition, people AR-ACHE of the present invention can also merge with other members of this family or exchange fragment, to produce new albumen.For example proteic N end of people AR-ACHE of the present invention and the proteic N end of people ACHE are exchanged, to produce the albumen that new activity is higher or have new features.At the antibody of inventor AR-ACHE, be used to screen other members of this family, perhaps be used for affinity purification associated protein (as other members of this family).
In addition, inventor AR-ACHE nucleic acid (encoding sequence or antisense sequences) can be introduced into cell, with expression level that improves people AR-ACHE or the overexpression that suppresses people AR-ACHE.People AR-ACHE albumen of the present invention or its active polypeptide fragment can be applied to patient, with treatment or alleviate because of people AR-ACHE disappearance, no function or unusual cause related disorders arranged.In addition, can also be with carrying out relevant diagnosis or prognosis judgement based on nucleotide sequence of the present invention or antibody.
Because people AR-ACHE albumen of the present invention has the natural acid sequence that is derived from the people, therefore, compare with the albumen of the same clan that derives from other species, estimate to have higher active and/or lower side effect (for example in the intravital immunogenicity of people lower or do not have) being applied to man-hour.
Embodiment 3
The proteic 26S Proteasome Structure and Function research of people AR-ACHE:
1. with the proteic aminoacid sequence input database of people AR-ACHE, and the software analysis that utilizes database relatively (network address is: http://www.ncbi.nlm.nih.gov/) obtain following result:
1) amino acid sequence of our input: 1 MRPPQCLLHTPSLASPLLLLLLWLLGGGVGAEGREDAELLVTVRGGRLRGIRLKTP GGPV, 61 SAFLGIPFAEPPMGPRRFLPPEPKQPWSGVVDATTFQSVCYQYVDTLYPGFEGTEM WNPN121 RELSEDCLYLNVWTPYPRPTSPTPVLVWIYGGGFYSGASSLDVYDGRFLVQAERTV LVSM181 NYRVGAFGFLALPGSREAPGNVGLLDQRLALQWVQENVAAFGGDPTSVTLFGESAG AASV241 GMHLLSPPSRGLFHRAVLQSGAPNGPWATVGMGEARRRATQLAHLVGCPPGGTGGN DTEL301 VACLRTRPAQVLVNHEWHVLPQESVFRFSFVPVVDGDFLSDTPEALINAGDFHGLQ LAGR361 LAAQGARVYAYVFEHRASTLSWPLWMGVPHGYEIEFIFGIPLDPSRNYTAEEKIFA QRLM421 RYWANFARTGDPNEPRDPKAPQWPPYTAGAQQYVSLDLRPLEVRRGLRAQACAFWN RFLP481 KLLSATDTLDEAERQWKAEFHRWSSYMVHWKNQFDHYSKQDRCSDL
2) processing. as a result after the analysis
QueryID:UNKNOW is a sequence of the present invention
QueryLength length: 526aa
The most probable action site pattern of Most Probable ProSite Patterns:
PS00122 CARBOXYLESTERASE_B_1 Procaine esterase B1 type EC3.1。1。1
PS00941 CARBOXYLESTERASE_B_2 Procaine esterase B2 type EC3.1。1。7
PS00014 ER_TARGETGLOBAL MOTIF BKID KLEN BLAST SKID ％IDN RATIO PSFLAG 9.0 10.0 ACES_HUMAN 614 0.0 ACES_HUMAN 85.7 1.17 T-PS00122 9.0 10.0 ACES_HUMAN 614 0.0 ACES_HUMAN 85.7 1.17 T-PS00941 6.5 0.0 EST4_RAT 561 1e-46 EST4_RAT 32.6 1.06 ？-PS000143 ) Most Probable PIR Superfamilies：SFA00837 cholinesteraseSFA01749 thyroglobulinSFA05547 juvenile-hormone esteraseSFA00831 triacylglycerol lipase GL0BAL BKID KLEN BSF# BLAST SKID SSF# ％IDN RATIO 9.0 ACES_HUMAN 614 SFA00837 0.0 ACES_HUMAN SFA00837 85.7 1.17 6.5 THYG_BOVIN 2769 SFA01749 2e-44 THYG_BOVIN SFA01749 31.9 1.03 5.0 ESTJ_HELVI 564 SFA05547 8e-40 ESTJ_HELVI SFA05547 34.7 0.60 5.0 LIP2_CANRU 548 SFA00831 8e-38 LIP2_CANRU SFA00831 36.7 0.604 ) ProSite Pattern Match and Clustal W Multiple Motif Alignment：AC PS00122ID CARBOXYLESTERASE_B_1； PATTERN.DE Carboxylesterases type-B serine active site.PA F-[GR]-G-x (4)-[LIVN]-x-[LIV]-x-G-x-S-[STAG]-G
ACES_BOVIN+S10712 PST 190 FGGDPTSVTLFGESAG
ACES_HUMAN+A39256 PST 221 FGGDPTSVTLFGESAG
QueryID GFT 221 FGGDPTSVTLFGESAG
ACES_RAT+JH0811 PST 221 FGGDPMSVTLFGESAG
LIP2_CANRU+S32615 PST 210 FGGDPSKVTIYGESAG
ESTJ_HELVI+A34325 PST 207 FGGDPSDITIAGQSAG
* * * * .:*:*:***5) the similar site of Pseudocholinesterase AC PS00941ID CARBOXYLESTERASE_B_2; PATTEPN.DE Carboxylesterases type-B signature 2.PA [ED]-D-C-L-[YT]-[LIV]-[DNS]-[LIV]-[LIVFYW]-x-[PQR]
ACES_HUMAN+A39256 PST 125 EDCLYLNVWTP
ACES_MOUSE+JH0314 PST 125 EDCLYLNVWTP
ACES_RAT+JH0811 PST 125 EDCLYLNVWTP
QueryID GFT 125 EDCLYLNVWTP
LIP2_CANRU+S32615 PST 109 EDCLTINVIRP
THYG_BOVIN+UIB0 PST 2280 EDCLYLNVFVP
ESTJ_HELVI+A34325 PSn 107 EACIYANIHVP
*: *: *
Embodiment 4
The preparation and the purification of people AR-ACHE polypeptide
The people AR-ACHE encoding sequence of total length or fragment are built into commercial protein merge among the expression vector, to express and purification of recombinant proteins.
1. people AR-ACHE polypeptide is carried out prokaryotic expression with the form of gst fusion protein in intestinal bacteria.Construction of prokaryotic expression vector, and transformed into escherichia coli
Complete encoding sequence according to people AR-ACHE, design amplifies complete coding and reads the primer of frame (corresponding respectively to about 2O above Nucleotide of encoding sequence 5 ' and 3 ' end), and on positive anti-primer, introduce restriction endonuclease sites (this decides according to the pGEX-2T carrier of selecting for use) respectively, so that construction of expression vector.With the product that obtains among the embodiment 1 is template, behind pcr amplification, with people AR-ACHE gene guarantee to read be cloned under the correct prerequisite of frame the pGEX-2T carrier (Pharmacia, Piscataway, NJ).Identify that good expression vector utilizes CaCl
2Method changes bacillus coli DH 5-α over to, and Screening and Identification obtains containing the engineering bacteria DH5-α-pGEX-2T-AR-ACHE of pGEX-2T-AR-ACHE expression vector.
Express the isolation identification of the engineering bacteria of GST-AR-ACHE recombinant protein
The engineering bacteria DH5-α-pGEX-2T-AR-ACHE of picking list bacterium colony contains jolting overnight incubation in the LB substratum of 100 μ g/ml acillins in 3ml, drawing nutrient solution by 1: 100 concentration cultivated about 3 hours in new LB substratum (containing 100 μ g/ml acillins), after reaching 0.5 to OD 600, adding IPTG continues at 37 ℃ to final concentration 1mmol/L and cultivates 0 respectively, 1,2,3 hours.It is centrifugal to get the different 1ml bacterium liquid of incubation time, in the bacterial precipitation thing, add lysate (2XSDS sample-loading buffer 50 μ l, distilled water 45 μ l, 3-mercaptoethanol 5 μ l), the suspendible bacterial precipitation, boiled in the boiling water bath 5 minutes, centrifugal 1 minute of 10000rpm, supernatant adds electrophoresis in the 12%SDS-PAGE glue.The bacterial strain that the protein content of dyeing back observation expection molecular weight size increases with the IPTG induction time is the engineering bacteria of expressing the GST-AR-ACHE fusion rotein.
The extraction purifying of GST-AR-ACHE fusion rotein
Bacterium centrifugation after the proteic engineering bacteria DHS-of abduction delivering GST-AR-ACHE amalgamation and expression α-pGEX-2T-AR-ACHE induces as stated above adds the resuspended bacterium of 20ml PBS, ultrasonication bacterium by every 400ml bacterium.The ultrasonic completely liquid of broken bacterium adds 50% saturated Triptide Sepharose 4B of PBS by every milliliter of amount that adds 20 microlitres, 37 ℃ of joltings were in conjunction with 30 minutes, 10000rpm precipitated the Triptide Sepharose 4B that combines GST-AR-ACHE in centrifugal 10 minutes, abandoned supernatant.Clean twice by every milliliter of amount that plays sound liquid gained precipitation adding 100 μ l PBS, then add 10ul reduced glutathione elutriant by every milliliter of ultrasonic liquid gained precipitation, room temperature was put 10 minutes, centrifugal 10 minutes of 10000rpm, and supernatant is the fusion rotein of wash-out.Repeat twice of wash-out.The supernatant of wash-out is stored in-80 ℃, and carries out the SDS-PAGE electrophoresis, detects purification effect.Protein band at the 58kDa place promptly is a people AR-ACHE albumen (see figure 2).
Sequence table
(1) information of SEQ ID NO.1
(i) sequence signature:
(A) length: 1936bp
(B) type: nucleic acid
(C) chain: two strands
(D) topological framework: linearity
(ii) molecule type: cDNA
( iii ) ：SEQ ID NO.1Sequence：NewSequence1 CAGAGTCGGC TCAGCCTGCG CCGGGGAACA TCGGCCGCCT CCAGCTCCCG GCGCGGCCCG GCCCGGCCCG71 GCTCGGCCGC CTCAGACGCC GCCTGCCCTG CAGCCATGAG GCCCCCGCAG TGTCTGCTGC ACACGCCTTC141 CCTGGCTTCC CCACTCCTTC TCCTCCTCCT CTGGCTCCTG GGTGGAGGAG TGGGGGCTGA GGGCCGGGAG211 GATGCAGAGC TGCTGGTGAC GGTGCGTGGG GGCCGGCTGC GGGGCATTCG CCTGAAGACC CCCGGGGGCC281 CTGTCTCTGC TTTCCTGGGC ATCCCCTTTG CGGAGCCACC CATGGGACCC CGTCGCTTTC TGCCACCGGA351 GCCCAAGCAG CCTTGGTCAG GGGTGGTAGA CGCTACAACC TTCCAGAGTG TCTGCTACCA ATATGTGGAC421 ACCCTATACC CAGGTTTTGA GGGCACCGAG ATGTGGAACC CCAACCGTGA GCTGAGCGAG GACTGCCTGT491 ACCTCAACGT GTGGACACCA TACCCCCGGC CTACATCCCC CACCCCTGTC CTCGTCTGGA TCTATGGGGG561 TGGCTTCTAC AGTGGGGCCT CCTCCTTGGA CGTGTACGAT GGCCGCTTCT TGGTACAGGC CGAGAGGACT631 GTGCTGGTGT CCATGAACTA CCGGGTGGGA GCCTTTGGCT TCCTGGCCCT GCCGGGGAGC CGAGAGGCCC701 CGGGCAATGT GGGTCTCCTG GATCAGAGGC TGGCCCTGCA GTGGGTGCAG GAGAACGTGG CAGCCTTCGG771 GGGTGACCCG ACATCAGTGA CGCTGTTTGG GGAGAGCGCG GGAGCCGCCT CGGTGGGCAT GCACCTGCTG841 TCCCCGCCCA GCCGGGGCCT GTTCCACAGG GCCGTGCTGC AGAGCGGTGC CCCCAATGGA CCCTGGGCCA911 CGGTGGGCAT GGGAGAGGCC CGTCGCAGGG CCACGCAGCT GGCCCACCTT GTGGGCTGTC CTCCAGGCGG981 CACTGGTGGG AATGACACAG AGCTGGTAGC CTGCCTTCGG ACACGACCAG CGCAGGTCCT GGTGAACCAC1051 GAATGGCACG TGCTGCCTCA AGAAAGCGTC TTCCGGTTCT CCTTCGTGCC TGTGGTAGAT GGAGACTTCC1121 TCAGTGACAC CCCAGAGGCC CTCATCAACG CGGGAGACTT CCACGGCCTG CAGCTGGCTG GGCGACTGGC1191 TGCCCAGGGT GCCCGGGTCT ACGCCTACGT CTTTGAACAC CGTGCTTCCA CGCTCTCCTG GCCCCTGTGG1261 ATGGGGGTGC CCCACGGCTA CGAGATCGAG TTCATCTTTG GGATCCCCCT GGACCCCTCT CGAAACTACA1331 CGGCAGAGGA GAAAATCTTC GCCCAGCGAC TGATGCGATA CTGGGCCAAC TTTGCCCGCA CAGGGGATCC1401 CAATGAGCCC CGAGACCCCA AGGCCCCACA ATGGCCCCCG TACACGGCGG GGGCTCAGCA GTACGTTAGT1471 CTGGACCTGC GGCCGCTGGA GGTGCGGCGG GGGCTGCGCG CCCAGGCCTG CGCCTTCTGG AACCGCTTCC1541 TCCCCAAATT GCTCAGCGCC ACCGACACGC TCGACGAGGC GGAGCGCCAG TGGAAGGCCG AGTTCCACCG1611 CTGGAGCTCC TACATGGTGC ACTGGAAGAA CCAGTTCGAC CACTACAGCA AGCAGGATCG CTGCTCAGAC1681 CTGTGACCCC GGCGGGACCC CCATGTCCTC CGCTCCGCCC GGCCCCCTAG CTGTATATAC TATTTATTTC1751 AGGGCTGGGC TATAACACAG ACGAGCCCCA GACTCTGCCC ATCCCCACCC CACCCCGACG TCCCCCGGGG1821 CTCCCGGTCC TCTGGCATGT CTTCAGGCTG AGGTCCTCCC CGCGTGCCTT CGCCCTCTGG CTGCAAATAA1891 ACTGTTACAG GCCAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAA
(2) information of SEQ ID NO.2
(i) sequence signature:
(A) length: 526aa
(B) type: amino acid
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: polypeptide
(iii) sequence description: SEQ ID NO.2 1 MRPPQCLLHTPSLASPLLLLLLWLLGGGVGAEGREDAELLVTVRGGRLRGIRLKTP GGPV 61 SAFLGIPFAEPPMGPRRFLPPEPKQPWSGVVDATTFQSVCYQYVDTLYPGFEGTEM WNPN121 RELSEDCLYLNVWTPYPRPTSPTPVLVWIYGGGFYSGASSLDVYDGRFLVQAERTV LVSM181 NYRVGAFGFLALPGSREAPGNVGLLDQRLALQWVQENVAAFGGDPTSVTLFGESAG AASV241 GMHLLSPPSRGLFHRAVLQSGAPNGPWATVGMGEARRRATQLAHLVGCPPGGTGGN DTEL301 VACLRTRPAQVLVNHEWHVLPQESVFRFSFVPVVDGDFLSDTPEALINAGDFHGLQ LAGR361 LAAQGARVYAYVFEHRASTLSWPLWMGVPHGYEIEFIFGIPLDPSRNYTAEEKIFA QRLM421 RYWANFARTGDPNEPRDPKAPQWPPYTAGAQQYVSLDLRPLEVRRGLRAQACAFWN RFLP481 KLLSATDTLDEAERQWKAEFHRWSSYMVHWKNQFDHYSKQDRCSDL
(3) information of SEQ ID NO.3
(i) sequence signature:
(A) length: 22bp
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: oligonucleotide
(iii) sequence description: SEQ ID NO.3
5′-cttccacggcctgcagctggct-3′
(4) information of SEQ ID NO.4
(i) sequence signature
(A) length: 37bp
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: oligonucleotide
(iii) sequence description: SEQ ID NO.45 '-gacaagcttacaggtctgagcagcgatcctgcttgct-3 '
Claims (8)
1. the dna molecular of an isolated human acetylcholinesteraseisomer is characterized in that, it includes the human acetylcholinesteraseisomer isomer of the cDNA sequence shown in the SEQ ID No.1.
2. the dna molecular of human acetylcholinesteraseisomer according to claim 1, it is characterized in that, the homology of cDNA sequence at least 85.7% shown in the cDNA of described human acetylcholinesteraseisomer isomer and people's the acetylcholinesterase, and be the formed new acetylcholinesterase dna molecular of from first to the 264th base deletion among the people ACHE gene Exon3.
3. the dna molecular of human acetylcholinesteraseisomer according to claim 1 is characterized in that it includes the white polypeptide of human acetylcholinesteraseisomer isomer protein of the polypeptid acid sequence shown in the SEQ ID No.2.
4. a generation has human acetylcholinesteraseisomer isomer (AR-ACHE) method of protein, is characterised in that its step is as follows;
(1) nucleotide sequence that coding is had a polypeptide of human acetylcholinesteraseisomer isomer (AR-ACHE) protein-active operationally is connected in expression regulation sequence, forms human acetylcholinesteraseisomer isomer (AR-ACHE) protein expression vector;
(2) change the expression vector in the step (1) over to host cell, form the proteic reconstitution cell of acetylcholinesteraseisomer isomer (AR-ACHE);
(3) under the condition that is fit to expressing human acetylcholinesteraseisomer isomer (AR-ACHE) protein polypeptide, the reconstitution cell in the culturing step (2);
(4) isolate the polypeptide of (AR-ACHE) protein-active that has human acetylcholinesteraseisomer isomer.
5. human acetylcholinesteraseisomer isomer according to claim 4 (AR-ACHE) method of protein, it is characterized in that complete encoding sequence according to people AR-ACHE, design amplifies complete coding and reads the frame primer, on positive anti-primer, introduce restriction endonuclease sites respectively, with the amplified production is template, through behind the pcr amplification people AR-ACHE gene being guaranteed to be cloned into the PGEX-2T carrier under the correct prerequisite of reading frame.
6. application that comprises the human acetylcholinesteraseisomer isomer of the cDNA sequence shown in the SEQ ID No.1, it is characterized in that people AR-ACHE protein according to the cDNA sequence expression of SEQ ID No.1, and polyclonal antibody and the monoclonal antibody of utilizing this protein to produce, use this polyclone or the monoclonal antibody can be specifically and the AR-ACHE combination, so in the various diseases relevant, can detect this proteinic existence specifically with the AR-ACHE generation, also can the early stage or more proteinic existence of back detection in disease.
7. use according to the described human acetylcholinesteraseisomer isomer that comprises cDNA sequence shown in the SEQ ID No.1 of claim 6, it is characterized in that the overexpression that is applied to improve the expression level of people AR-ACHE or suppresses people AR-ACHE.
8. use according to the described human acetylcholinesteraseisomer isomer that comprises cDNA sequence shown in the SEQ ID No.1 of claim 6, it is characterized in that utilizing AR-ACHE and other members of this family to merge or exchange fragment, obtain active higher and albumen with new features.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 01105781 CN1244700C (en) | 2001-03-23 | 2001-03-23 | Human acetylcholinesterase isomer protein (AR-ACHE) and its gene coding sequence |
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| Application Number | Priority Date | Filing Date | Title |
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| CN 01105781 CN1244700C (en) | 2001-03-23 | 2001-03-23 | Human acetylcholinesterase isomer protein (AR-ACHE) and its gene coding sequence |
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| CN1244700C CN1244700C (en) | 2006-03-08 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006050667A1 (en) * | 2004-11-12 | 2006-05-18 | Shanghai Institutes For Biological Sciences, Chinese Academy Of Sciences | MONOCLONAL ANTIBODIES AGAINST APOPTOSIS-RELATED ACETYLCHOLINESTERASE (AR-AChE) AND USE THEREOF |
| WO2013020366A1 (en) * | 2011-08-08 | 2013-02-14 | 中国科学院上海生命科学研究院 | The use of ache as nuclease |
| CN103087154A (en) * | 2012-12-05 | 2013-05-08 | 江苏澳格姆生物科技有限公司 | Polypeptide for preparing monoclonal antibody for detecting acetylcholin esterase and application of polypeptide |
-
2001
- 2001-03-23 CN CN 01105781 patent/CN1244700C/en not_active Expired - Fee Related
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006050667A1 (en) * | 2004-11-12 | 2006-05-18 | Shanghai Institutes For Biological Sciences, Chinese Academy Of Sciences | MONOCLONAL ANTIBODIES AGAINST APOPTOSIS-RELATED ACETYLCHOLINESTERASE (AR-AChE) AND USE THEREOF |
| WO2013020366A1 (en) * | 2011-08-08 | 2013-02-14 | 中国科学院上海生命科学研究院 | The use of ache as nuclease |
| CN103087154A (en) * | 2012-12-05 | 2013-05-08 | 江苏澳格姆生物科技有限公司 | Polypeptide for preparing monoclonal antibody for detecting acetylcholin esterase and application of polypeptide |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1244700C (en) | 2006-03-08 |
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