CN102242125A - BK channel blocker gene, recombinant vector thereof and cloning method thereof - Google Patents
BK channel blocker gene, recombinant vector thereof and cloning method thereof Download PDFInfo
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- CN102242125A CN102242125A CN 201110107768 CN201110107768A CN102242125A CN 102242125 A CN102242125 A CN 102242125A CN 201110107768 CN201110107768 CN 201110107768 CN 201110107768 A CN201110107768 A CN 201110107768A CN 102242125 A CN102242125 A CN 102242125A
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Abstract
The invention relates to a novel BK channel blocker martentoxin gene, a recombinant vector thereof and a recombinant expression method thereof. The gene has a base sequence represented by SEQ ID NO:1. Experimental data show that the BK channel blocker martentoxin gene which is a specific blocker of a nervous BK channel not only can be treated as a specific probe applied to scientific researches, but also can provide references for developing designs of pilot drugs for treating BK channel related nervous system diseases. A new method of martentoxin expression in vitro provides feasible technical guarantee for ulterior industrialization.
Description
Technical field
The present invention relates to a kind of novel B K channel blocker martentoxin gene, its recombinant vectors and cloning process thereof.
Background technology
Large conductance calcium activated potassium channel (being called for short the BK passage) is a kind of potassium-channel that is subjected to voltage and the dual gate of calcium ion that is distributed widely in excitability and the non-excitability cell, has the diastole of regulating neurocyte excitability, unstriated muscle; The release of control neurotransmitter, the secretion of hormone; Participate in functions such as non-specific immunity.Functional BK passage is made of four α subunits at least, and each α subunit all has 10 hydrophobicity α-Luo Xuanjiegous; Complementary β subunit is tissue specific expression usually, and each β subunit connects two transmembrane spanning hydrophobicity fragments (Lippiat, Standen et al.2003) by born of the same parents' outer shroud.α and β subunit coexpression can significantly change the kinetics and the pharmacological characteristic of BK passage.
It is worthy of note that the BK passage hypotype (α and β 4 subunit coexpressions be made of) of distribution of specific in neural system is considered to have important physical and pharmacological function effect.This passage subtype gene shortcoming can cause a series of clinical diseases.Neural BK passage has slower physiological dynamics feature, and this BK passage hypotype all presents suitable non-sensibility to the BK channel blocker that comprises classics such as classical iberiotoxin and charybdotoxin.Therefore, for physiology and the pharmacological function characteristic of accurately verifying this BK passage hypotype, specific aim medicine in order to design research and development relevant clinical BK passage disease, the specific inhibition agent of exploitation BK passage, resolve its molecular mechanism, identify new drug specificity on the BK passage, the drug effect mode, the drug effect novel targets, BK passage-blocker interaction new model, not only but the degree of depth is expanded the knowledge of relevant BK passage three-dimensional arrangement and function, for the Clinics and Practices of relevant BK passage disease provides cell and molecules foundation, also can provide beneficial reference thinking Kraft to corresponding specific drugs design, Krause et al, 2003.
The martentoxin of wild-type is one the 37 peptide blocker that is come out by purifying in the thick poison of buthus martensii Karscs, molecular weight is 4060 Da, its full-length cDNA open reading frame length is 177 bp, the precursor (including the signal peptide of 22 amino-acid residues and the mature peptide of 37 amino-acid residues) of 59 amino-acid residues of coding has been identified the calcium-activated k that can act on the rat pheochromocyte specifically
+Passage (Ji, Wang et al, 2003).With charybdotoxin is contrast, and 100 nM martentoxin can check 70% BK electric current, is equivalent to 77% activity of charybdotoxin.But on NG-108 cell and rat dorsal root ganglion cell, martentoxin is very little for the effect of Kv.Biosensor shows in conjunction with experiment, martentoxin is higher than charybdotoxin far away with the binding ability ratio of rat brain synaptosomes film, but for the BK hypotype, both pharmacological effect corresponding complementary, but 400 nM martentoxin highly selectivies blocking-up BK passage (alpha+beta 4) electric current, the possible and non-sensitive K of a kind of charybdotoxin of this prompting martentoxin
+Channel height is affine.So martentoxin can be used as research K
+An important blocker Shi of passage somatotype, electrical signal and chemical signal, He et al.2008.It is carried out gene engineering expression, be convenient to its 26S Proteasome Structure and Function is studied, can carry out mutation operation simultaneously, study the 26S Proteasome Structure and Function of specific potassium channel.Utilize prokaryotic expression system, explore simple vivoexpression program, martentoxin is able at vivoexpression, thereby be the rare property that solves natural blocker, and the research of next step blocker structure and function provides the necessary precondition condition.
Summary of the invention
One of purpose of the present invention is to provide a kind of BK channel blocker gene.
Two of purpose of the present invention is to provide the recombinant vectors of this gene.
Three of purpose of the present invention is to provide the cloning process of this BK channel blocker gene
Three of purpose of the present invention is to provide the cloning process of BK channel blocker gene recombined vector.
For achieving the above object, the present invention adopts following technical scheme:
A kind of BK channel blocker gene is characterized in that this gene is the base sequence shown in the SEQ ID NO:1.
A kind of recombinant vectors gene of above-mentioned BK channel blocker gene is characterized in that this recombinant vectors gene is the base sequence shown in the SEQ ID NO:2.
A kind of host cell is characterized in that this host cell contains the described recombinant vectors of claim 2.
A kind of method of cloning above-mentioned BK channel blocker gene, the concrete steps that it is characterized in that this method are: use the total RNA of the wild buthus martensii Karscs of 5 '-ATTGAAGCTTACGCGTCGACTATA(dT)-3 ' primer reverse transcription, obtain BK channel blocker gene.
A kind of method of cloning above-mentioned recombinant vectors is characterized in that the concrete steps of this method are:
A. with BK channel blocker gene as clone template, multiple clone site sequence according to pGEX-4T-3, select the recognition site of BamH I and two Restriction Enzymes of Sma I to do connection site, design forward primer and reverse primer, again through the PCR reaction, PCR product and carrier pGEX-4T-3 are carried out BamH I and Sma I double digestion, pass through T after enzyme is cut
4Dna ligase connects the recombinant vectors sequence that makes up BK channel blocker gene;
Described forward primer is: 5 '-TTCGGATCCTTTGGACTCATAGA-3 ';
Described reverse primer is: 5 '-CTTCCCGGGTTAATCAGTAGCAT-3 ';
B. with step a gained recombinant vectors sequence process Sepharose 4B-GSH affinity chromatography column purification, GSH elutriant wash-out obtains the protein band of a 30KD, is the recombinant vectors of BK channel blocker gene
Invention be a kind of specific inhibition agent of nervous system type BK passage, the specific probe that not only can be used as this passage applies to scientific research, and the lead drug design that can be exploitation treatment BK passage related neural systemic disease is offered reference.The novel method of Martentoxin vivoexpression provides feasible technical guarantee for industrialization in the future.
Description of drawings
Fig. 1 detects figure for the SDS-PAGE of the gene induced expression of reorganization BK channel blocker; Wherein the left side is low molecular weight protein (LMWP) Marker; The 1st swimming lane is the whole-cell protein of abduction delivering not; 2nd, 3 swimming lanes are through 3 hours whole-cell protein of IPTG abduction delivering; 4th, 5 swimming lanes are through 4 hours whole-cell protein of IPTG abduction delivering.
Fig. 2 is reorganization BK channel blocker expression of gene and purifying SDS-PAGE detection figure; Wherein the right side is low molecular weight protein (LMWP) Marker; The 1st swimming lane is the whole-cell protein of abduction delivering not; The 2nd swimming lane is through 4 hours whole-cell protein of IPTG abduction delivering; The 3rd swimming lane is cell supernatant liquor behind ultrasonic disruption; The 4th swimming lane is the fusion rotein through the GSH affinitive layer purification; The 5th swimming lane is the fusion rotein before enzyme is cut; The 6th swimming lane is the fusion rotein after enzyme is cut;
Fig. 3 is reorganization BK channel blocker gene and the isolating gel-filtration collection of illustrative plates of GST;
Fig. 4 is a GST-martentoxin fusion rotein small intestine kinases restriction enzyme mapping; Buffer A wherein: 0.1% hydrochloric acid/deionized water, buffer B: 60% acetonitrile/ deionized water.Gradient is: the buffer B initial concentration is 15%, 2-7 minute 15-25%, 7-25 minute 25-40%, the permanent concentration of 25-30 minute 40%;
Fig. 5 identifies for reorganization rMartentoxin mass spectrum;
Fig. 6 is the wild-type and the retarding effect of rMartentoxin to BK passage (alpha+beta 4) of recombinating; Wherein A. is under 1uM concentration, and wild-type and reorganization rMartentoxin are to the restraining effect of BK channel current.(clamp down on cell in-70 mV, give+depolarize of 100 mV stimulates and brings out the BK outward current, and stimulation lengths is 100 ms); B is the statistical graph that the BK channel current is suppressed, the residual ratio of If(electric current of rMarTx)=0.3415 ± 0.05778; The If of MarTx=0.1871 ± 0.03020.
Embodiment
Embodiment one: the structure of pGEX-4T-3-martentoxin carrier
Utilize 5 '-ATTGAAGCTTACGCGTCGACTATA(dT)-3 ' primer that the total RNA of wild-type buthus martensii Karscs is carried out reverse transcription, obtain BK channel blocker gene, referring to the base sequence shown in the SEQ ID NO:1.According to multiple clone site and the BK channel blocker gene order of pGEX-4T-3, select the recognition site of BamH I and two Restriction Enzymes of Sma I to do connection site, design forward primer and reverse primer;
Described forward primer is: 5 '-TTCGGATCCTTTGGACTCATAGA-3 ';
Described reverse primer is: 5 '-CTTCCCGGGTTAATCAGTAGCAT-3 ';
Carry out the PCR reaction, PCR product and pGEX-4T-3 carrier all carry out BamH I and Sma I double digestion, pass through T after enzyme is cut
4Dna ligase is connected to BK channel blocker gene on the pGEX-4T-3 carrier, make up pGEX-4T-3-martentoxin carrier sequence, single colony inoculation of selecting to express BK channel blocker genophore send order-checking to 37 ℃ of overnight incubation of 5mL LB/Amp substratum.
PGEX-4T-3-martentoxin plasmid sequencing result is as follows:
AAATCGGATCTGGTTCCGCGTGGATCC
TTTGGACTCATAGACGTAAAATGTTTTGCATCTAGTGAATGTTGGACAGCTTGCAAAAAAGTAACAGGATCGGGACAAGGAAAGTGCCAGAATAATCAATGTCGATGCTACTGATTAACCCGGGTCGACTCGAGCGGCCGCATCGTGACTGACTGAC
The gene of martentoxin correctly has been inserted in the multiple clone site of pGEX-4T-3 carrier (line part) as can be known.
Embodiment two: expression and the purifying of reorganization martentoxin
In containing the LB liquid nutrient medium of penbritin, express the engineering bacteria of BK channel blocker with volume ratio 1:50 inoculation, 37 ℃ add the IPTG(IPTG final concentration when being cultured to the about 0.8-1.0 of OD600 is 1.0 mM), culture was induced 4 hours, and inducing temperature is 37 ℃.With compare without the inductive engineering, obviously have more the significantly protein band of a treaty 30 KD in the engineering bacteria total protein after inducing, referring to Fig. 1.
Through Sepharose 4B-GSH affinity chromatography column purification, GSH elutriant wash-out obtains the protein band of a treaty 30KD.Because in the expression plasmid carrier, merged a Thiadiazolidine isomerase gene before the cDNA of BK channel blocker martentoxin, the about 26KD of Thiadiazolidine isomerase adds that the martentoxin of 4 KD is about 30KD.Judge that from the affinity of molecular weight of albumen size and GSH medium gained 30KD protein band is fusion rotein GST-martentoxin.Fusion rotein under the wash-out is carried out enzyme cut, by the GST of the digested one-tenth of the visible GST-martentoxin fusion rotein of SDS-PAGE protein electrophorese 26KD and the rMartentoxin of about 4KD, referring to Fig. 2.Through Sephadex G-50 gel-filtration, GST can be separated with martentoxin, referring to Fig. 3, obtain the reorganization martentoxin of thick purifying, yield is about 1 mg/L culture.
Embodiment three: HPLC separation and purification and the mass spectrum of reorganization martentoxin are identified
Product sample introduction after enzyme is cut and the HPLC system reverse chromatography column of C18 detect.The result shows that the absorption peak that occurs is the absorption peak of GST about 2 minutes; A higher absorption peak occurred at 15 minutes,, inferred that it is the absorption peak of purpose product (micromolecule polypeptide of reorganization martentoxin) probably referring to Fig. 4.The absorption peak of collecting at 15 minutes is carried out mass spectroscopy.The result shows that its molecular weight is 4059.0 Da, and is close with the molecular weight 4060 of natural martentoxin, referring to Fig. 5.
Embodiment four: the Function Identification of reorganization martentoxin
In behind the BK passage plasmid transfection HEK293 cell 24-72 hour, carry out electric Physiological Experiment.HEK293T cell resulting channel current under+60 mV stimulate is consistent with the feature that (Lippiat et al. 2003) reported, referring to Fig. 6.
Sequence table
＜110〉Shanghai University
＜120〉BK channel blocker gene, its recombinant vectors and cloning process thereof
<160> 2
<210> 1
<211> 118
<212> DNA
＜213〉artificial sequence
<400> 1
TTTGG?ACTCA?TAGAC?GTAAA?ATGTT?TTGCA?TCTAG?TGAAT?GTTGG?ACAGC?TTGCA?AAAAA 60
GTAAC?AGGAT?CGGGA?CAAGG?AAAGT?GCCAG?AATAA?TCAAT?GTCGA?TGCTA?CTGAT?TAA 118
<210> 2
<211> 184
<212> DNA
＜213〉artificial sequence
<400> 2
AAATC?GGATC?TGGTT?CCGCG?TGGAT?CCTTT?GGACT?CATAG?ACGTA?AAATG?TTTTG?CATCT 60
AGTGA?ATGTT?GGACA?GCTTG?CAAAA?AAGTA?ACAGG?ATCGG?GACAA?GGAAA?GTGCC?AGAAT 120
AATCA?ATGTC?GATGC?TACTG?ATTAA?CCCGG?GTCGA?CTCGA?GCGGC?CGCAT?CGTGA?CTGAC 180?TGAC
Claims (5)
1. a BK channel blocker gene is characterized in that this gene is the base sequence shown in the SEQ ID NO:1.
2. the recombinant vectors gene of a BK channel blocker gene according to claim 1 is characterized in that this recombinant vectors gene is the base sequence shown in the SEQ ID NO:2.
3. a host cell is characterized in that this host cell contains the described recombinant vectors of claim 2.
One kind clone BK channel blocker gene according to claim 1 method, the concrete steps that it is characterized in that this method are: use the total RNA of the wild buthus martensii Karscs of 5 '-ATTGAAGCTTACGCGTCGACTATA(dT)-3 ' primer reverse transcription, obtain BK channel blocker gene.
One kind clone recombinant vectors according to claim 2 method, it is characterized in that the concrete steps of this method are:
A. with BK channel blocker gene as clone template, multiple clone site sequence according to pGEX-4T-3, select the recognition site of BamH I and two Restriction Enzymes of Sma I to do connection site, design forward primer and reverse primer, again through the PCR reaction, PCR product and carrier pGEX-4T-3 are carried out BamH I and Sma I double digestion, pass through T after enzyme is cut
4Dna ligase connects the recombinant vectors sequence that makes up BK channel blocker gene;
Described forward primer is: 5 '-TTCGGATCCTTTGGACTCATAGA-3 ';
Described reverse primer is: 5 '-CTTCCCGGGTTAATCAGTAGCAT-3 ';
B. with step a gained recombinant vectors sequence process Sepharose 4B-GSH affinity chromatography column purification, GSH elutriant wash-out obtains the protein band of a 30KD, is the recombinant vectors of BK channel blocker gene.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106498066A (en) * | 2016-11-10 | 2017-03-15 | 张代民 | A kind of fluorescence quantification PCR primer of detection human macrophage calcium activated potassium channel nucleic acid, probe and test kit |
| CN107304452A (en) * | 2016-04-25 | 2017-10-31 | 复旦大学 | BK channel proteins are preparing the purposes during osteoblastic proliferation intervenes medicine extremely |
| CN109384852A (en) * | 2018-11-05 | 2019-02-26 | 中国科学院上海有机化学研究所 | Recombinate preparation, characterization and the application of Martentoxin |
Citations (3)
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| CN1344745A (en) * | 2001-10-12 | 2002-04-17 | 吉永华 | Martentoxin as one great-conductance calcium-activating potassium channel blocker and its prepn and use |
| WO2006116456A1 (en) * | 2005-04-25 | 2006-11-02 | Cargill, Incorporated | Polyurethane foams comprising oligomeric polyols |
| CN101647999A (en) * | 2009-06-26 | 2010-02-17 | 上海大学 | Application of sodium channel modulator BmK1 in building induction pain model |
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2011
- 2011-04-28 CN CN 201110107768 patent/CN102242125A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1344745A (en) * | 2001-10-12 | 2002-04-17 | 吉永华 | Martentoxin as one great-conductance calcium-activating potassium channel blocker and its prepn and use |
| WO2006116456A1 (en) * | 2005-04-25 | 2006-11-02 | Cargill, Incorporated | Polyurethane foams comprising oligomeric polyols |
| CN101647999A (en) * | 2009-06-26 | 2010-02-17 | 上海大学 | Application of sodium channel modulator BmK1 in building induction pain model |
Non-Patent Citations (2)
| Title |
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| 《上海第二医科大学学报》 20001231 焦哲等 K＋通道在门脉高压鼠动脉NE低反应中的作用 412-417 1-5 第22卷, 第5期 * |
| 《沈阳药科大学学报》 20081231 袁树荣等 东亚钳蝎神经毒素基因的序列相似性分析 321-330 1-5 第25卷, 第4期 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107304452A (en) * | 2016-04-25 | 2017-10-31 | 复旦大学 | BK channel proteins are preparing the purposes during osteoblastic proliferation intervenes medicine extremely |
| CN106498066A (en) * | 2016-11-10 | 2017-03-15 | 张代民 | A kind of fluorescence quantification PCR primer of detection human macrophage calcium activated potassium channel nucleic acid, probe and test kit |
| CN109384852A (en) * | 2018-11-05 | 2019-02-26 | 中国科学院上海有机化学研究所 | Recombinate preparation, characterization and the application of Martentoxin |
| WO2020094005A1 (en) * | 2018-11-05 | 2020-05-14 | 中国科学院上海有机化学研究所 | Preparation, characterization and use of recombinant martentoxin |
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Application publication date: 20111116 |