CN100465272C - Genetic engineering for recombining scorpion venom rBmKaIT1 - Google Patents
Genetic engineering for recombining scorpion venom rBmKaIT1 Download PDFInfo
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- CN100465272C CN100465272C CNB021105812A CN02110581A CN100465272C CN 100465272 C CN100465272 C CN 100465272C CN B021105812 A CNB021105812 A CN B021105812A CN 02110581 A CN02110581 A CN 02110581A CN 100465272 C CN100465272 C CN 100465272C
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Abstract
A gene engineering for preparing the recombined scorpion venom rBmk alpha IT1 includes designing and chemically synthesizing the coded and recombined gene of said scorption venom rBmk alpha IT1, cloning it to plasmid, high expression in colibacillus, denaturing, renaturing, high efficiency liquid-phase chromatographic separation and purifying. Its advantage is high purity (more than 95%).
Description
Technical field
The present invention relates to molecular biology, relate in particular to design gene DNA order, the DNA chemistry is complete synthesis to wait genetic engineering technique with the DNA reorganization.
Background technology
Scorpio Arthropoda, Arachnida, Scorpionida are the most ancient known Lu Sheng arthropodss, the existence history in existing more than 400,000,000 year.The whole world has 800 kinds of scorpions altogether approximately, wherein human body is constituted harm, and what promptly have medical significance has 50 kinds approximately, and they nearly all belong to Buthidae.In state-owned 15 kinds of scorpions, wherein the widest with Ma Shi pincers scorpion (also claiming East Asia Ma Shi pincers scorpion, Buthus Martensii Karsch, following abbreviation BmK) distribution, it belongs to the pincers scorpion subfamily in the Buthidae, mainly is distributed in Henan, Shandong and Liaoning one band.The toxicity of Ma Shi buthotoxin a little less than, the people is not had fatal risk, in the traditional medicine treasure-house of China, important position is arranged also.Compendium of Materia Medica is pointed out scorpion " cure mainly apoplexy and hemiplegia facial hemiparalysis speak puckery spasm of hands and feet ".
Scorpion venom is by the secreted complicated mixture of composition of the poison gland of scorpion tail, its main toxic component is the protein neurotoxin of molecular weight between 5000 to 10000 dalton, wherein the overwhelming majority be a class optionally influence the voltage-sensitive sodium-ion channel by 60 to 70 single chain polypeptides that amino-acid residue is formed.These neurotoxins can be divided into Mammals toxin, insect toxins and crustacean toxin according to its effective object.Gurevitz in 1994, M. be according to the homology of primary structure and the selectivity of toxic action, and the scorpion toxin of pincers scorpion subfamily is divided into following a few class [referring to document: J.Toxicol.-Toxin Reviews (1994) 13,65-100]:
(1) α type Mammals toxin with sodium channel effect neural, muscle, makes lengthening of action potential, and depolarize slows down, and shows the inhibition depolarize;
(2) β type Mammals toxin shows and causes polarization, causes the penetrating unusually of sodium ion;
(3) excitatory insect toxins shows and improves action potential and slow down its deactivation, the neural sustained activation of feasible action;
(4) inhibition type insect toxins causes inhibition paralysis effect slowly, by increase stop sodium ion permeability and suppress the conduction of active sodium ion, realize checking to sodium ion;
(5) α type insect toxins has strong activity to insect, and Mammals then is weak toxicity, have and the similar chemical sequence structure of α type Mammals toxin, and similar inhibition sodium channel inactivation function.Electricity physiology shows that association reaction does not take place for this toxoid and rat brain synaptosomes sodium channel, and does not rely on membrane potential with combining of insect sodium channel.The reorganization scorpion venom rBmK α IT1 by engineering bacterium expression involved among the present invention promptly belongs to this type of α type insect toxins.
The specific activity and the high selectivity that are possessed just because of the scorpion neurotoxin, they and have been applied to relate in the neural bio-science evaluation and the purifying of relevant target ion channel protein composition increasingly extensively as important instrument, toxin and acceptor site bonded specificity and susceptibility, with and the receptor modulators born of the same parents in mediator discharge and the fundamental research of the biological phenomenas such as molecular mechanism of information transmission.On the other hand, they are also just demonstrating very tempting potentiality and prospect as the newtype drug in future.The long-chain scorpion toxin has high homology, and particularly the 4 pairs of specific disulfide linkage in eight positions of cysteine residues in the peptide chain amino acid sequence and formations are conserved structure factors of scorpion toxin.Scorpion toxin all has the common folding mode, promptly is made up of a α spiral and 3 antiparallel βZhe Dies.Therefore scorpion toxin also is research protein structure and emic fabulous model.
Owing to contain the very strong multiple Buthotoxin polypeptide of structural homology in the scorpion venom of natural origin, therefore certain difficulty arranged from the single Buthotoxin polypeptide gene of natural origin separation and purification.Carbonell in 1988 etc. are derived and its gene order of chemosynthesis by insect specific neurotoxin aminoacid sequence in the Buthuseupeus scorpion first, but only in baculovirus vector, obtain faint expression [referring to document: Gene (1988) 73,409-418], people's extensive experimentation is expressed the serial scorpion toxin gene recombination of each kind to expression vectors such as intestinal bacteria, yeast, baculovirus, plants since then.In the overwhelming majority's expression vector, have only the considerably less activated protein of non-activity or content can express and purifying.This be since 4 pairs of disulfide linkage that scorpion toxin contained can correctly recombinate cause.Zilberberg, people such as N. in intestinal bacteria with the inclusion body formal representation Lqh α IT scorpion venom, and obtained activated protein [referring to document: Biochemistry (1996) 35,10215-10222] in external renaturation.Tonny, people such as M.J. have adopted the coli strain of oxidized form to express the scorpion venom CssII achieving success [referring to document: Peptides (2000) 21,767-772] of β type.China expresses this field at scorpion venom and has also launched positive research.Shao, people such as F. in cereuisiae fermentum successful expression BmK M1 scorpion venom [referring to document: ProteinExpression and Purification, (1999) 17,358-365].Ma Shi buthotoxin, especially α type insect toxins still do not have report at present in the activated East Asia of expression in escherichia coli.Wu in 1999, H.-M. [referring to document: Pure Appl.Chem. (1999) 71 to wait the people to report the aminoacid sequence of BmK α IT1,1157-1162], Zhu in 2000, S.-Y. wait the people to report that the coded protein aminoacid sequence of the cDNA of Bm α TX12 and derivation thereof is [referring to document: Toxicon (2000) 38,1653-1661], what the aminoacid sequence of this sequence and BmK α IT1 related to is same objective entity.Therefore, our reasonable part of combining both is as one of foundation of design Recombined scorpion venom rBmK α IT1.
Summary of the invention
The problem to be solved in the present invention prepares Recombined scorpion venom rBmK α IT1 and mutant thereof with gene engineering method exactly.
The invention provides Recombined scorpion venom rBmK α IT1 gene, the nucleotide sequence of its DNA is as follows:
1 76
C?ATG?GTT?CGT?GAT?GCA?TAT?ATT?GCC?CAA?AAC?TAC?AAT?TGT?GTA?TAC?CAT?TGT?GCT?CGT?GAT?GCA?TAT?TGC?AAC?GAA
151
CTG?TGC?ACT?AAG?AAT?GGT?GCC?AAG?TCC?GGA?TCT?TGC?CCA?TAC?TTG?GGT?GAG?CAC?AAA?TTC?GCC?TGC?TAC?TGC?AAG
206
GAC?CTG?CCA?GAT?AAC?GTT?CCA?ATC?CGT?GTC?CCG?GGT?AAG?TGC?CAC?TAA?TAGGATC
By this synthetic gene deutero-mutant gene, can have more than 50% identical with the corresponding codon of above-mentioned synthetic gene.
The expression plasmid of Recombined scorpion venom rBmK α IT1 of the present invention can be that plasmid pE2rBmK α IT (submits China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation to November 28 calendar year 2001, preserving number is CGMCC No.0655), structure is as shown in figure 25.
The host cell of this expression plasmid can be e. coli bl21 (DE3).
At the activated Recombined scorpion venom of expression in escherichia coli, especially α type insect toxins does not still have report at present.The present invention is directed to this characteristics, when not repelling exploration separating natural Buthotoxin polypeptide gene, adopt the tactics of artificial design gene, thereby need to have guaranteed proteic structure regularity of synthetic scorpion venom and determinacy.As previously mentioned, the present invention combine the cDNA of the aminoacid sequence of BmK α IT1 and Bm α TX12 and derivation thereof coded protein aminoacid sequence reasonable part and need corresponding codon to match at expression in escherichia coli, manually designed coding rBmK α IT1 structural gene sequence, it has 51% different with the codon of the cDNA of natural report.
The present invention prepares Recombined scorpion venom rBmK α IT1 albumen with gene engineering method, in intestinal bacteria, go out except that N-end to increase the polypeptide chain that the and natural scorpion venom of a Met residue has same acid sequence, and be activated scorpion venom albumen in external renaturation with the inclusion body formal representation.It is by design and chemosynthesis Recombined scorpion venom rBmK α IT1 structural gene sequence, promptly by matching the single stranded DNA synthesis method [reference: Chen of a T4DNA ligase enzyme reaction step by step, H.-B.etal, Nucl.Acids.Res. (1990) 18,871-878], the oligonucleotide fragment of chemosynthesis is connected synthetic this gene and directly is cloned into carrier pET15b, obtain expression plasmid pE2rBmK α IT1, and this expression plasmid is transformed host e. coli BL21 (DE3) express, target protein obtains high expression level with the inclusion body form, then disulfide linkage is correctly recombinated by external renaturation, high performance liquid chromatography separates, thereby obtains the activated Recombined scorpion venom rBmK of the correct paired of disulfide linkage α IT1 albumen.
The invention provides and a kind ofly can obtain the proteic gene engineering preparation method of activated rBmK α IT1 at expression in escherichia coli and in external renaturation, by the design of Recombined scorpion venom rBmK α IT1 structure gene, the external renaturation of complete synthesis, the genetic expression of chemistry, expression product and separation and purification and determination of activity and determine to obtain Recombined scorpion venom rBmK α IT1 albumen.Detailed process is for example:
1. the design of Recombined scorpion venom rBmK α IT1 structure gene
Import computer after two ends are mixed initial code and dual stop code respectively in proper order with 64 amino-acid residues of sophisticated East Asia Ma Shi buthotoxin BmK α IT1 peptide chain, the running relevant procedures, the relative application frequency data of pressing colibacillus high expression gene degenerate codon are [referring to document: Wada, K., Nucl.Acids Res. (1992) 20 (supplement), 2111], design the requirement of single restriction endonuclease sites commonly used as much as possible and restriction enzyme NcoI and the BamHI recognition sequence that molecular cloning need be settled two ends and designed the rBmK α IT1 structure gene that contains 206 bases, as shown in Figure 1.In this synthetic gene, except the NcoI and BamHI at two ends, we also are provided with 9 single restriction endonuclease sites commonly used, and their distribution situation is seen Fig. 2, and have only 2 this sites in the corresponding BmK α IT1 natural gene.In addition, contrast BmK α IT1 natural gene has many bases different in the coding region, and this is in order to change 34 codons (account for 67 codons 50.8%) engineered rBmK α IT1 more to be adapted at expression in escherichia coli.
2. Recombined scorpion venom rBmK α IT1 structure gene is chemical complete synthesis
The present invention has finished the chemosynthesis of rBmK α IT1 structure gene with reference to Chen after people's such as H.-B. literature method [Nucl.Acids Res. (1990) 18,871-878] is improved.Become the big fragment of single stranded DNA (ssDNA) with the T4DNA ligase enzyme through the oligonucleotide fragment that the catalysis of strand method connects chemosynthesis, with the DNA recombinant technology strand large dna fragment cloning of the rBmK α IT1 that connects into is advanced plasmid vector then, and conversion host bacterium BL21 (DE3), make it in the host bacterium, to be assembled into complete rBmK α IT1 structure gene double-stranded DNA, obtain containing the cloned plasmids of this gene.Fig. 3 represents that the underlying stock of rBmK α IT1 structure gene is divided into A, B, four big fragments of C, D and connection and the required negative burst of short-movie section such as A ' b, bc, cd and D ' of clone.The agarose gel electrophoresis separating resulting demonstration of Fig. 4 has been synthesized the strand of rBmK α IT1 structure gene with dna single chain method.Recombinate them among the carrier pET15b and transform the plasmid pE2rBmK α IT1 (CGMCC No.0655) that the host bacterium obtains containing complete rBmK α IT1 structure gene, in full accord through dna sequencing analytical proof synthetic gene and design.
3. the expression of Recombined scorpion venom rBmK α IT1
Get single colony inoculation in the LB substratum behind the plasmid pE2rBmK α IT1 transformed into escherichia coli BL21 (DE3), through pre-cultivate and abduction delivering after, through SDS denaturing polyacrylamide gel electrophoresis (SDS-PAGE) Analysis and Identification occur one with expect the consistent expression product (as shown in Figure 8) of molecular weight.
4. the external renaturation of Recombined scorpion venom rBmK α IT1 and separation and purification and evaluation
Adopt purification step as shown in Figure 7, the thalline after ultrasonication is expressed is used the damping fluid washing precipitation successively, after the denaturing soln dissolving, through dialysis, renaturation, concentrated, uses high performance liquid chromatography separation and purification (Figure 10 and 11) at last again.Purified product (Fig. 9 and 11) use successively circular dichroism, capillary electrophoresis,
1Technical evaluation such as H nucleus magnetic resonance, electrospray ionization mass spectrum, electric physiology and insect overall activity mensuration, result's (seeing Figure 12～21) shows that the reorganization rBmK α IT1 that genetically engineered produces has the complete α type anti-insect active identical with natural isolating BmK α IT1.
The present invention compared with the prior art.At present bibliographical information does not also have home-made East Asia Ma Shi pincers scorpion at e. coli expression and obtain the correct paired Buthotoxin polypeptide of complete disulfide linkage.Express acquisition Recombined scorpion venom rBmK α IT1 by Escherichia coli fermentation, convenient more feasible than having time-consuming lacking from natural separation and purification, the characteristics that economic benefit is high.Can prepare highly purified Recombined scorpion venom rBmK α IT1 by common biological fermentation.
Description of drawings:
Fig. 1: Recombined scorpion venom rBmK α IT1 synthetic gene and natural B mK α IT1 gene and amino acids coding order thereof.Illustrate among the figure: the DNA nucleotide sequence (only providing the base different) of the first behavior East Asia Ma Shi buthotoxin BmK α IT1 natural gene with design sequence; The DNA nucleotide sequence of second behavior Recombined scorpion venom rBmK α IT1 synthetic gene design; The third line is the proteic amino-acid sequence of reorganization East Asia Ma Shi buthotoxin rBmK α IT1.
Fig. 2: restriction endonuclease site distribution plan in the Recombined scorpion venom rBmK α IT1 synthetic gene.
Fig. 3: for synthesizing the oligonucleotide fragment that Recombined scorpion venom rBmK α IT1 synthetic gene is divided.
Fig. 4: sepharose (3%) electrophoretic separation is identified the rBmK α IT1 synthetic oligonucleotide fragment of reorganization and is connected product---the strand of synthetic gene.
Illustrate among the figure: swimming lane 1:DNA molecular weight standard, length is followed successively by 65,75,214,396,517,1419bp; Swimming lane 2: connect product; Swimming lane 3: fragment B; Swimming lane 4: fragment C; Swimming lane 5: fragment D.
Fig. 5: the construction procedures figure of Recombined scorpion venom rBmK α IT1 synthetic gene clone and expression plasmid pE2rBmK α IT1.
Fig. 6: the DNA sequence measurement result figure of pE2rBmK α IT1, it is in full accord that near 60 NcoI recognition sequence CCATGG begin the DNA sequence of measurement result till near 260 the HindIII recognition sequence GGATCC and design from figure.
Fig. 7: the proteic sex change of Recombined scorpion venom rBmK α IT1, renaturation and the separation and purification schema of genetic expression.
Fig. 8: the SDS-PAGE separation graph of plasmid pE2rBmK α IT1 abduction delivering in the e. coli bl21 (DE3).
Illustrate among the figure: bacterium liquid sample is through the thalline of centrifugal back collecting precipitation, thalline through the routine operation cracking after on the centrifuging and taking supernatant liquor sample to 10% gel carry out SDS-PAGE and separate.Swimming lane 1:BL21 (DE3) contains pE2rBmK α IT1 and grows to A
600Be 0.6, the whole bacterial protein when not inducing; Swimming lane 2～4: be followed successively by the whole bacterial protein after IPTG induces 4 hours, the precipitation behind cellular lysate supernatant liquor and the cellular lysate; Swimming lane 5: the inclusion body of precipitation after washing.
Fig. 9: the SDS-PAGE of the Recombined scorpion venom rBmK α IT1 behind the purifying.
Illustrate among the figure: deposition condition is identical with Fig. 8.Swimming lane 1: protein molecular weight standard is followed successively by 26.6,17.0,14.2,6.5 and 3.5kDa from top to bottom; Recombined scorpion venom rBmK α IT1 behind the swimming lane 2:HPLC purifying; The natural B mK α IT1 of swimming lane 3:HPLC purifying.
Figure 10: the C18RP-HPLC elution curve of the Recombined scorpion venom rBmK α IT1 of natural B mK α IT1 and preliminary purification.
Illustrate among the figure: gradient is set to: 0min (5% B), 5min (30% B), 35min (40% B), 40min (100% B), 45min (100% B), 50min (5% B) and 55min (5% B).
Figure 11: the HPLC elution curve of the final purifying of Recombined scorpion venom rBmK α IT1.Illustrate among the figure: gradient is set to 0min (22% B), 40min (26% B) and 45min (40% B).
Figure 12: the circular dichroism spectrogram of natural B mK α IT1 and Recombined scorpion venom rBmK α IT1.Illustrate among the figure: zero refers to the CD spectrogram of natural B mK α IT1; refers to the CD spectrogram of the rBmK α IT1 that recombinates.
Figure 13: the capillary electrophoresis behavior curve of the rBmK α IT1 of natural B mK α IT1 and reorganization.Illustrate among the figure: left figure refers to the capillary electrophoresis behavior of natural B mK α IT1; Right figure refers to the capillary electrophoresis behavior of the rBmK α IT1 that recombinates.
Figure 14: Recombined scorpion venom rBmK α IT1's
1H NMR spectrum.
Figure 15: the electrospray ionization mass spectrum figure of Recombined scorpion venom rBmK α IT1.Illustrate among the figure: the spectrogram upper right corner by mass spectrograph mark Recombined scorpion venom rBmK α IT1 molecular weight be shown as 7310.63 ± 1.07 with calculated value 7310.5 in rational limit of error.
Figure 16: the electric physiological property of Recombined scorpion venom rBmK α IT1.Illustrate among the figure: Recombined scorpion venom rBmK α IT1 is to the influence of rat dorsal root ganglion sodium current peak current in the experiment of full cell voltage sheet pincers.Last figure is control experiment, has write down the resting potential-70mV from dorsal root ganglion, increases progressively until the impulse stimulation of+30mV influence to sodium current with every 10mV; Middle figure shows that 1 μ mol/L Recombined scorpion venom rBmK α IT1 slows down the inactivationization of sodium current; Figure below be 10mV when stimulating sodium current to reach peak value Recombined scorpion venom rBmK α IT1 sodium current is lost the activatory influence.
Figure 17: Recombined scorpion venom rBmK α IT1 is to Groton bug half paralysis amount (PU
50) mensuration.Illustrate among the figure: press among the figure dotted line and calculate and get, the 50% paralysis amount (PU of Groton bug
50) be 15.6 μ g/kg, with the PU of the natural B mK α IT1 that has reported
50Be worth 24.16 μ g/kg in the limit of error that allows.
Figure 18: with surface plasma body resonant vibration (SurfacePlasmon Resonance, SPR) the cockroach abdominal nerve synaptic membrane suspension of mensuration different concns combines resonance units (Resonance Unit, influence RU) with Recombined scorpion venom rBmK α IT1.Illustrate among the figure: representing concentration from top to bottom successively is 36,18,12,9 and the cockroach abdominal nerve synaptic membrane suspension of 6pmol/L and the time dependent curve of Recombined scorpion venom rBmK α IT1 bonded RU that is fixed on chip surface.
Figure 19: high density Recombined scorpion venom rBmK α IT1 suppresses the competition of immobilized Recombined scorpion venom rBmK α IT1.Illustrate among the figure: a last curve is represented 36pmol/L cockroach abdominal nerve synaptic membrane suspension and the time dependent curve of bonded RU that is fixed on the Recombined scorpion venom rBmK α IT1 of chip surface; Next bar curve represent 36pmol/L cockroach abdominal nerve synaptic membrane suspension and 30 μ mol/L Recombined scorpion venom rBmK α IT1 25 ℃ of incubations after 1 hour with the time dependent curve of bonded RU that is fixed on the Recombined scorpion venom rBmK α IT1 of chip surface, this curve shows that high density Recombined scorpion venom rBmK α IT1 has suppressed to be fixed on the combination of the Recombined scorpion venom rBmK α IT1 of chip surface to cockroach abdominal nerve synaptic membrane fully to the combination of cockroach abdominal nerve synaptic membrane in advance, does not promptly exist non-specific adsorption.
Figure 20: the cockroach abdominal nerve synaptic membrane suspension of different concns and natural B mK α IT1 combine influence to the RU value.
Illustrate among the figure: representing concentration from top to bottom successively is 36,18,12,9 and the cockroach abdominal nerve synaptic membrane suspension of 6pmol/L and the time dependent curve of bonded RU that is fixed on the natural B mK α IT1 of chip surface.
Figure 21: high density Recombined scorpion venom rBmK α IT1 suppresses the competition of immobilized natural B mK α IT1.
Illustrate among the figure: a last curve is represented 36pmol/L cockroach abdominal nerve synaptic membrane suspension and the time dependent curve of bonded RU that is fixed on the Recombined scorpion venom rBmK α IT1 of chip surface; Next bar curve represent 36pmol/L cockroach abdominal nerve synaptic membrane suspension and 30 μ mol/L Recombined scorpion venom rBmK α IT1 25 ℃ of incubations after 1 hour with the time dependent curve of bonded RU that is fixed on the natural B mK α IT1 of chip surface, this curve shows that high density Recombined scorpion venom rBmK α IT1 has suppressed to be fixed on the combination of the natural B mK α IT1 of chip surface to cockroach abdominal nerve synaptic membrane fully to the combination of cockroach abdominal nerve synaptic membrane in advance.
Figure 22: the rat brain synaptosomes film suspension of different concns and reorganization BmK α IT1 combine influence to the RU value.Illustrate among the figure: it is 36 and the cockroach abdominal nerve synaptic membrane suspension of 9pmol/L and the time dependent curve of bonded RU that is fixed on the reorganization rBmK α IT1 of chip surface that curve A, B represent concentration respectively.It is 175 and the rat brain synaptosomes film suspension of 58.3pmol/L and the time dependent curve of bonded RU that is fixed on the reorganization rBmK α IT1 of chip surface that curve C, D represent concentration respectively.This curve shows that reorganization rBmK α IT1 does not have combination to the rat brain synaptosomes film.
Figure 23: the rat brain synaptosomes film suspension of different concns and natural B mK α IT1 combine influence to the RU value.Illustrate among the figure: it is 36 and the cockroach abdominal nerve synaptic membrane suspension of 9pmol/L and the time dependent curve of bonded RU that is fixed on the natural B mK α IT1 of chip surface that curve A, B represent concentration respectively.It is 175 and the rat brain synaptosomes film suspension of 58.3pmol/L and the time dependent curve of bonded RU that is fixed on the natural B mK α IT1 of chip surface that curve C, D represent concentration respectively.This curve table right BmK α tomorrow IT1 does not have combination to the rat brain synaptosomes film.
The design cycle of Figure 24: PCR sudden change.
Figure 25: the structure of the expression plasmid pE2rBmK α IT of Recombined scorpion venom rBmK α IT1.
Embodiment
Following examples help to understand the present invention, but are not limited to content of the present invention
Materials and methods:
The phosphoramidite nucleoside monomers of four kinds of full guard and the deoxynucleoside CPG of four kinds of full guard (ControlledPore Glass, 500A or 1000A) derivative is available from ABI company (Applied Biosystem Inc.).
ATP, bovine serum albumin (BSA), acrylamide, N,O-Diacetylmuramidase, to Methyl benzenesulfonyl fluorine (PMSF), pepstatin (pepstatin) A, iodo-acid amide, 1, the 10-phenanthroline, Triton X-100, albumen lower molecular weight standard and Coomassie brilliant blue G-250 are all available from Sigma company.
The T4 dna ligase is available from Huamei Bio-Engrg Co., (SABC).
N, N '-methylene-bisacrylamide, (Ethidium Bromide is EB) from Aldrich company for ethidium bromide;
Su Shi-1,4-dimercapto-2,3-butyleneglycol (DTT) is available from BDH company;
N,N,N (TEMED) is available from E.Merck company;
Sep-pak C18 reversed-phase column is available from Waters company;
Low melting-point agarose gel (LMP Agarose) is available from Gibco BRL company;
PET15b plasmid and bacterial classification BL21 (DE3) are available from Novagen company;
Tryptones (Tryptone) and yeast extract (Yeast Extract) are available from Oxiod company;
Guanidinium hydrochloride and Tutofusin tris (Tris) are available from Amresco company;
The Bradford protein quantification uses storage liquid available from Bio-rad company;
5,5 '-dimercapto two (2-nitrobenzoic acids) is (DTNB) available from Acros company;
CsCl and sodium lauryl sulphate (SDS) are available from Boehringer Mannheim company;
Macrosep (MWCO 3k) is available from Pall-Gelman company;
The C18RP-HPLC post (
10 * 250 and 4.6 * 250mm) and pre-column Security Guard available from Phenomenex company;
CM5 chip and N-ethyl-N-(dimethylamino-propyl)-carbonyl diimine (EDC) and N-hydroxyl-succinimide (NHS) are available from Sweden BIACore company;
Sodium ampicillin (Amp) is available from Shanghai the 4th Pharmacy stock Co., Ltd;
Other inorganic salt and chemical reagent commonly used are homemade analytical reagent.
The oligonucleotide fragment of the synthetic usefulness of related gene press the solid phase phosphoramidite triester method by this laboratory and is gone up at dna synthesizer (381A type, American AB I company) and synthesize among the present invention.Synthetic product spends the night through 60 ℃ of placements of strong aqua; make it to come off and excise blocking group from solid phase; it is water-soluble that head product concentrates the back; separate with urea-denatured polyacrylamide gel electrophoresis (PAGE), the gel band that contains pure products is pressed literature method [reference: Lo with Sep-pak C-18 reversed-phase column after hydrogen-carbonate triethylamine (1mol/L) leaches; K.-M.; et al, Proc.Natl.Acad.Sci.USA (1984) 81,2285-2289] obtain pure oligonucleotide fragment after the desalination.
Pure oligonucleotide fragment B, C and D carry out 5 ' phosphorylation according to a conventional method with T4 polynucleotide phosphorylating kinase and ATP.
The analysis of DNA nucleotide sequence is carried out on the ABI3700 automatic sequencer by last sea base health biotech company.
5 ' the phosphorylation and the plasmid transformation escherichia coli host cell of the polyacrylamide gel electrophoresis (SDS-PAGE) of the urea-denatured polyacrylamide gel electrophoresis (PAGE) of 8mol/L, SDS sex change, low melting-point agarose gel electrophoresis separation and purification dna fragmentation, oligonucleotide fragment carry out [reference: Sambrook according to a conventional method, J., Fritsch, E.F., Maniatis, T., 1989, Molecular Cloning, A Laboratory Manual (2
NdEd) Cold Spring Harbor Laboratory Press, New York].
Protein quantification is pressed literature method [Anal.Biochem. (1976) 72 for Bradford, M., 248] with the BSA standard water solution (A of 1mg/ml with Bradford reagent
280=0.661) makes A
595Typical curve to BSA concentration.Survey the A of protein sample then under the same conditions
595, determine the corresponding proteins sample concentration according to typical curve.
Free sulfhydryl groups quantitatively carries out [Ellman, G.L, Arch.Biochem.Biophysics (1959) 82,70-77] with Ellman reagent by literature method.
HPLC carries out on Waters 510 (Waters company).Detector is Waters 484 types.C18 RP-HPLC post (
10 * 250 and 4.6 * 250mm) and the moving phase of pre-column be A liquid: 0.1% (v/v) TFA/H
2O; B liquid: 0.1% (v/v) TFA/ acetonitrile.Flow velocity is 3ml/min.Ultraviolet detection A
280
Extreme ultraviolet CD spectrum carries out on JASCO J-715 (U.S.) spectropolarimeter.Experiment condition: the cuvette path length is 0.1cm, sweep limit 190-250nm, sweep velocity 10nm/min, time constant 0.25 second, 25 ℃ of temperature.Data reduce noise through further handling, and baseline is level and smooth, and signal averaging obtains average residue molar extinction coefficient [θ] (shown in Fig. 8 c) at last.
Capillary electrophoresis carries out on Waters Quanta 4000E capillary electrophoresis system (U.S. WATERS company).Deposition condition: moving phase is 50mml/L sodium phosphate buffer (pH2.5), voltage 25kV, and temperature is 30 ℃, and the time is 10min, and kapillary is that 30cm is long, diameter 75 μ m.The sample feeding time is 30 seconds, ultraviolet detection A
214
1H NMR gathers on Varian Inova-600 spectrometer, carries out spectrogram with VNMR software and handle on sun station.Data are gathered at 300K, with presaturation technology setting-out peak.Chemical shift is calibrated according to the water peak.Sample is dissolved in H
2O/D
2O (9:1), and be adjusted to pH4.9, sample final concentration 0.66mmol/L with HCl or NaOH.
The electrospray ionization mass spectrum of Recombined scorpion venom rBmK α IT1 is to go up at QUATTRO LC (Britain Macromass mass spectrum company) to measure.
The electricity Physiological Experiment adopts isolating neuronal cell [reference: Song, et al., J.Pharmacol.Exp.Ther. (1997) 282,707-714] from the dorsal root ganglion (DRG) of adult rat.Full cell record adopts minor diameter DRG neurone (10-25 μ m).Membrane current is connected to the EPC-9 augmentor by glass capillary suction pipe (2-5M Ω), handles by Pulse/Pulsefit software (German HEKA elektronik).Pipette solution is by 120mmol/L CsCl, 20mmol/L chlorination triethyl ammonium (TEACl), 5mmol/L Na
2ATP, 10mmol/L ethyleneglycol-bis-N,N'-tetraacetic acid, N '-tetraacethyl (EGTA), 10mmol/L HEPES, 2.5mmol/LMgCl
2With 0.4mmol/L Na
2GTP forms.Solution is adjusted to pH7.3 with CsOH.Outer liquid consists of 140mmol/L NaCl, 5mmol/L KCl, 2.5mmol/L CaCl
2, 1mmol/L MgCl
2, 10mmol/LHEPES and 10mmol/L D-glucose.Outer liquid is adjusted to pH7.3-7.4 with NaOH.The Ag-AgCl salt bridge is as reference electrode.Peak current comes out according to the relative intensity of they and contrast is selected.
The insect that anti-insect biological activity determination adopts is Groton bug (Blattella germanica, 55 ± 3mg).Albumen is dissolved in the survey damping fluid alive by different concns, surveys the damping fluid of living and consist of 0.9% NaCl and 1% BSA, abdominal injection 0.6 μ l, and use paraffin sealing, and observe paralysis symptom after 5 minutes, wing opens, back leg stretches, and faces upward and lies down at least 30 minutes, and data of every mensuration are with 8 insects.
The rat brain synaptosomes film is according to literature method preparation [reference: Dodd, P.R.etal, Brain Res. (1981) 226,107-118] and cockroach (Periplaneta americana) ventral ganglion synaptic membrane according to literature method preparation [reference: Lima, M.E.D.etal, Insect Biochem. (1989) 19,413-422].Synaptic membrane is suspended in running buffer, and [running buffer contains the 140mmol/L choline chloride 60,1.8mmol/L CaCl
2, 5.4mmol/L KCl, 0.8mmol/L MgSO
4, 10mmol/L D-glucose and 25mmol/L (pH7.4) Hepes-Tris].And to add protease inhibitor cocktail to each component final concentration be toluenesulfonyl fluoride (PMSF) 50 μ g/ml, 1 μ mol/L Pepstatin A, 1mmol/L iodo-acid amide and 1mmol/L 1,10-phenanthroline.The concentration of synaptic membrane suspension Bradford protein quantification standard measure.
The SPR experiment is carried out on BIACore 3000 (Sweden BIACore company).Chip adopts the CM5 type.The Sensor Chip CM 5 of chip surface activates with 400mmol/L N-ethyl-N-(dimethylamino-propyl)-carbonyl diimine (EDC) and the 100mmol/L N-hydroxyl-succinimide (NHS) of 35 μ l 1:1 (v/v) earlier.Recombined scorpion venom rBmK α IT1 and natural BmK α IT1 are dissolved in fixedly damping fluid, and (this damping fluid consists of 10mmol/L NaOAc, pH6) in, final concentration 0.2mg/ml, flow through chip and Recombined scorpion venom rBmK α IT1 or natural BmK α IT1 be fixed on chip surface of this protein solution, to RU value arrival about 750, stop sample introduction.(the 1mol/L diethanolamine hydrochloride pH8.0) seals unreacted N-hydroxyl-succinimide ester with 35 μ l closed reagents.Whole fixation procedure flow velocity is 5 μ l/min.Part and acceptor interaction are to be incorporated into the Recombined scorpion venom rBmK α IT1 of chip surface and the variation and the dynamic process thereof of the RU value that natural BmK α IT1 is caused by the synaptic membrane suspension that detects different concns.Damping fluid is the above-mentioned running buffer of mentioning.Flow velocity 20 μ l/min flow through 60 μ l altogether, 25 ℃ of temperature.Competitive assay then earlier cockroach ventral ganglion synaptic membrane suspension and Recombined scorpion venom rBmK α IT1 are woven into 25 ℃ placed 1 hour or rat brain synaptosomes film suspension and Recombined scorpion venom rBmK α IT1 be woven into 37 ℃ of placements 30 minutes again with chips incorporate.
The design of Recombined scorpion venom rBmK α IT1 gene, complete synthesis and clone:
The design of A, coding Recombined scorpion venom rBmK α IT1 structure gene
Behind the amino-acid sequence input computer with Recombined scorpion venom rBmK α IT1, operation DNAStar software, according to (1) colibacillus high expression gene codon frequency of utilization, (2) evenly settle single restriction enzyme in Recombined scorpion venom rBmK α IT1 synthetic gene, (3) DNA sequence of Recombined scorpion venom rBmK α IT1 structure gene is determined in the segmentation of synthetic gene fragment with eliminating fragment self pairing to wait three manually selected each amino acid whose codons of requirement, as shown in Figure 1.
The preparation of B, oligonucleotide fragment
Oligonucleotide fragment is gone up with the solid phase phosphoramidite triester method synthetic at dna synthesizer (ABI381A type).After each fragment is synthesized separately; spend the night through 60 ℃ of placements of strong aqua; make it to come off and excise blocking group from solid phase, it is water-soluble that head product concentrates the back, separates with urea-denatured polyacrylamide gel electrophoresis (PAGE); the gel band that contains pure products is after 1mol/L hydrogen-carbonate triethylamine leaches; press literature method [Lo, K.-M., et al with Sep-pak C-18 reversed-phase column; Proc.Natl.Acad.Sci.USA (1984) 81,2285-2289] obtain pure oligonucleotide fragment after the desalination.
Pure oligonucleotide fragment B, C and D carry out 5 ' phosphorylation according to a conventional method with T4 polynucleotide phosphorylating kinase and ATP.
The clone of C, Recombined scorpion venom rBmK α IT1 each segmental connection of synthetic gene and connection product
The oligonucleotide fragment pairing also connects synthetic complete single stranded DNA: each fragment of underlying stock is respectively got 1nmol, and negative burst each fragment is respectively got 1.2nmol, matches respectively by following three groups: A+B+A ' b, C+bc+cd and D+D '.90 ℃ are incubated 3 minutes, naturally cool to 16 ℃ then, and it is 34 μ l that three groups of solution are merged cumulative volumes, adds 2 T4DNA of unit ligase enzymes, and 4 ℃ of connections are spent the night.
The clone who connects product: connect product and connect in the 9:1 ratio through the NcoI/BamHI double digestion linear fragment of 3% low melting point agarose gel electrophoresis separation and purification with pET15b, direct transformed into escherichia coli JM109, enzyme is cut and is identified correct bacterium colony, through further sequence analysis evaluation confirmation (Fig. 6), obtained corresponding clone and expression plasmid pE2rBmK α IT1 as shown in Figure 5.
Genetic expression:
Plasmid pE2rBmK α IT1, transformed into escherichia coli BL21 (DE3).Get single bacterium colony and insert 5ml LB substratum, contain Amp (100 μ g/ml) in this substratum, shaken overnight under 37 ℃ of conditions.In the above-mentioned substratum of 100ml, press the 1:100 inoculation then, 37 ℃ are cultured to A
600Be 0.6, press the 1:100 inoculation again in the above-mentioned substratum of 1L, 37 ℃ are cultured to A
600Be 0.6, add IPTG to final concentration 0.4mmol/L, continued to induce 4～6 hours, expression product detects with Tris-Tricine SDS-PAGE of 10%, and the result as shown in Figure 8.
The external renaturation and the separation and purification of expression product
The preparation of A, inclusion body: under 4 ℃ of conditions, centrifugal (5,500g) the coli somatic of collection behind abduction delivering, thalline is resuspended in that (buffer A is by 50mmol/L Tris-Cl (pH7.5) in the 20ml buffer A, 2.5mmol/L EDTA, the 20mmol/L beta-mercaptoethanol is formed), add N,O-Diacetylmuramidase and PMSF respectively to final concentration be 0.2mg/ml and 1mmol/L, ice bath was placed after 1 hour, carrying out ultrasonic bacteria breaking 10 minutes (～150W, ultrasonic 30 seconds, intermittently 30 seconds), 15, centrifugal 20 minutes of 000g, collecting precipitation.Precipitation is used 3 * 20ml buffer A (adding 1% (v/v) Triton X-100) and 20ml buffer A (including the 2mol/L Guanidinium hydrochloride) washing successively.Use the resuspended precipitation of damping fluid before each washing earlier, and at room temperature stirred 30 minutes, then centrifugal collecting precipitation.What prepare like this is purer inclusion body.
The dissolving of B, inclusion body and external renaturation: the inclusion body 100mg that will from the 1L substratum, obtain, (the sex change damping fluid contains 50mmol/L Tris-Cl (pH7.5) to add 5ml sex change damping fluid, the 6mol/L Guanidinium hydrochloride, 0.2mol/L DTT), 4 ℃ of placements are spent the night, centrifugal (15,000g) collect supernatant.Supernatant with the 1:50 ratio to 4 ℃ of dialysed overnight of dialysis buffer liquid (dialysis buffer liquid contains 50mmol/L Tris (pH7.5), 4mol/L Guanidinium hydrochloride) after, centrifugal collection dialysis supernatant liquor.It is quantitative that the dialysis supernatant liquor carries out protein concentration with the Bradford method, and diluting this albumen to final concentration with renaturation buffer rapidly then is 0.1mg/ml (renaturation buffer contains 50mmol/LTris-Cl (pH7.5), 50mmol/L KCl).Place this renaturation solution and follow the tracks of free sulfhydryl groups until the reaction that is negative for 4 ℃ then with the Ellman reaction.
The separation and purification of C, expression product: above-mentioned renaturation solution is concentrated into 3ml with the centrifugal ultrafiltration device, and last sample is to C18 RP-HPLC post, gradient elution.Moving phase is A liquid: 0.1% (v/v) trifluoroacetic acid (TFA)/H
2O; B liquid: 0.1% (v/v) TFA/ acetonitrile.Flow velocity is 3ml/min.Ultraviolet detection A
280Gradient is 0min (5% B) during the initial gross separation purifying, 5min (30% B), 35min (40% B), 40min (100% B), 45min (100% B), 50min (5% B), 55min (5% B).The HPLC wash-out behavior of Recombined scorpion venom rBmK α IT1 and natural BmK α IT1 is consistent as shown in figure 10.The gradient of final purifying is 0min (22% B), 40min (26% B), 45min (40% B).Purity as the Recombined scorpion venom rBmK α IT1 of Fig. 9 and final purifying shown in Figure 11 reaches more than 95%.
The purity of expression product and determination of activity
A, SDS-PAGE: carry out purity detecting by 10%Tris-Tricine SDS-PAGE, Fig. 9 shows Recombined scorpion venom rBmK α IT1, and not only the BmK α IT1 electrophoresis behavior with natural is identical, the protein band size shows with expection molecular weight unanimity, and purity also reaches more than 95%.
B, circular dichroism (CD): Recombined scorpion venom rBmK α IT1 and natural BmK α IT1 all are dissolved in 20mmol/L sodium phosphate buffer (pH7.0).Figure 12 has shown that Recombined scorpion venom rBmK α IT1 has identical circular dichroism behavior with natural BmK α IT1, has illustrated that Recombined scorpion venom rBmK α IT1 has secondary structures such as the α spiral identical with natural BmK α IT1, βZhe Die.
C, capillary electrophoresis (CE): Recombined scorpion venom rBmK α IT1 and natural BmK α IT1 all are dissolved in 50mmol/L sodium phosphate buffer (pH2.5), and the sample final concentration is 0.1mg/ml.Recombined scorpion venom rBmK α IT1 has identical appearance time with natural BmK α IT1 as can be seen from Figure 13, all is 8.25 minutes, illustrates that their proteic surface propertieies are identical.
D,
1HNMR composes ():
1HNMR spectrum (Figure 14) demonstration peak shape is disperseed finely, has shown that Recombined scorpion venom rBmK α IT1 has good space tertiary structure, and promptly disulfide linkage is correct assembling rather than unordered.
E, mass spectrum: the molecular ion peak of Recombined scorpion venom rBmK α IT1 is shown as 7310.63 ± 1.07 (Figure 15), and is consistent with the molecular weight 7310.5 that calculates.
F, electric physiology: Figure 16 have shown that under 1 μ mol/L concentration Recombined scorpion venom rBmK α IT1 has significantly to slow down to the sodium-ion channel of rat dorsal root ganglion and have lost the activatory influence, illustrated that Recombined scorpion venom rBmK α IT1 has the typical electrical physiological property of α type scorpion toxin.
G, anti-insect biological activity determination: Recombined scorpion venom rBmK α IT1 is dissolved in the damping fluid of survey living, and the sample final concentration is respectively 1,1.5, and 2 and 2.5ng/ μ l, every little Lian injects 0.6 μ l, and 3 groups of independent experiments are done in 8 of each concentration injections altogether.According to paralysis percentage ratio concentration is mapped, survey the slip-knot fruit as shown in figure 17, can obtain the 50% paralysis amount (PU of Recombined scorpion venom rBmK α IT1 Groton bug
50) be 15.6 μ g/kg, with the PU of the natural B mK α IT1 that has reported
50Be worth 24.16 μ g/kg in the limit of error that allows.Illustrate that Recombined scorpion venom rBmK α IT1 has had the identical anti-insect active of natural B mK α IT1.
The test that combines of H, Recombined scorpion venom rBmK α IT1 and natural B mK α IT1 and insect sodium-ion channel: the CM5 chip carries out coupling with Recombined scorpion venom rBmK α IT1 and the natural B mK α IT1 of 0.2mg/ml respectively after NHS and EDC activation, seal with thanomin then.The RU value of chip is respectively 768 and 737 in its final coupling, has identical coupling level.Cockroach ventral ganglion synaptic membrane suspension is quantitative through the Bradford protein concentration, and its concentration is 0.45mg/ml (≈ 0.9nmol/L sodium-ion channel).Dilution obtains the cockroach ventral ganglion suspension of a series of concentration in proportion, and the concentration of sodium-ion channel is respectively 36,18,12,9,6pmol/L.By concentration order from low to high sample introduction successively, the variation that the sodium-ion channel that can obtain different concns and the Recombined scorpion venom rBmK α IT1 (as shown in figure 18) and the natural B mK α IT1 (as shown in figure 20) of chip surface combine caused RU value.Carry out kinetics by BIAevaluation3.1 software by the Langmuir pattern of 1:1 and fit, the dissociation equilibrium constant that can obtain Recombined scorpion venom rBmK α IT1 and insect sodium-ion channel is 1.3 * 10
-9Mol/L, the dissociation equilibrium constant of natural B mK α IT1 and insect sodium-ion channel is 1.3 * 10
-8Mol/L.The Recombined scorpion venom rBmK α IT1 of chip surface and natural B mK α IT1 to the non-specific adsorption of cockroach ventral ganglion synaptic membrane suspension be Recombined scorpion venom rBmK α IT1 (30 μ mol/L) by high density with cockroach ventral ganglion synaptic membrane suspension (36pmol/L) earlier at 25 ℃ of incubations after 1 hour, sample introduction is measured and is got again.Show that from Figure 19 and Figure 21 high density Recombined scorpion venom rBmK α IT1 can suppress to be fixed on the Recombined scorpion venom rBmK α IT1 of chip surface or the natural B mK α IT1 combination to cockroach abdominal nerve synaptic membrane fully to the combination of cockroach abdominal nerve synaptic membrane in advance, i.e. there is not non-specific adsorption in explanation.
The test that combines of I, Recombined scorpion venom rBmK α IT1 and natural B mK α IT1 and Mammals sodium-ion channel: rat brain synaptosomes film suspension is quantitative through the Bradford protein concentration, and its concentration is 0.35mg/ml.Dilution obtains the rat brain synaptosomes film suspension of a series of sodium-ion channel concentration in proportion, is respectively 350,175,116.7,87.5,70,58.3pmol/L.By concentration order from low to high sample introduction successively, the variation that the sodium-ion channel that can obtain different concns and the Recombined scorpion venom rBmK α IT1 and the natural B mK α IT1 of chip surface combine caused RU value.By East Asia Ma Shi buthotoxin rBmK α IT1 and natural B mK α IT1 and cockroach ventral ganglion synaptic membrane and the rat brain synaptosomes film (shown in Figure 22 and 23) of relatively recombinating, no matter Recombined scorpion venom rBmK α IT1 or natural B mK α is IT1 as can be seen, its to the combination of rat brain synaptosomes film far below cockroach ventral ganglion synaptic membrane, its RU value all is lower than 20RU under the acceptor density of 175pmol/L, and illustrating does not have combination to the rat brain synaptosomes film.
Comprehensive above-mentioned experimental result, Recombined scorpion venom rBmK α IT1 has significantly to slow down to the sodium-ion channel of rat dorsal root ganglion and loses the activatory influence, with cockroach ventral ganglion synaptic membrane tangible the combination arranged, but do not combine with the rat brain synaptosomes film, illustrated that it has the feature of the anti-insect toxins of α type.
The example of artificial gene sudden change, the l-asparagine (Asn) that rBmK α IT1 is the 30th is mutated into aspartic acid (Asp), the i.e. structure of N30D mutant.
The design of A, PCR mutant primer
The PCR mutation method is with reference to " modern gene manipulation techniques ", and Hu Fuquan edits, the People's Medical Officer Press, and the 31st chapter carries out transgenation and gene fusion with Megaprimer PCR.
The design cycle of PCR sudden change is referring to accompanying drawing 24.
Upstream primer A5 ' GCGATGG
CCATGGTTCGTGA3 ' (including the NcoI site)
Mutant primer M5 ' (being the downstream primer that contains mutating alkali yl in the accompanying drawing 24)
CTTGGCACCATCCTTAGTGCACAGTTCGTTG3’
Downstream primer B5 ' GAGTGC
GGCCGC
AAGCTTGCATGCC3 ' (containing BamH I and HindIII site)
The structure of B, plasmid pE2BmK α IT1-N30D
PCR1: with pE2BmK α IT1 is template, adds upstream primer A and mutant primer M, carries out first round PCR, and the PCR condition is identical with regular-PCR.The PCR product reclaims separation and purification through Agarose glue, obtains the fragment of 108bp, as the big primer Megaprimer of next step PCR.
PCR2: add downstream primer B, upstream primer then adopts the product Megaprimer of previous step PCR.Have in the 100 μ l PCR reaction volumes: template DNA (with pE2BmK α IT1 identical among the PCR1) 400ng, downstream primer B25pmol, Megaprimer 18 pmol and general PCR reactant.60 ℃ of annealing temperatures, 30 circulations of increasing.Get 5 μ l reaction solutions and separate (the TAE damping fluid that the electrophoretic buffer employing was revised through Agarose low melting point glue, EDTA concentration is 0.1mM), cutting-out contains the segmental glue of required size (257bp) and put into Ultrafree-DA (Millipore Co.) centrifugal 10 minutes, obtains 20 μ l DNA extraction liquid.Get the template that 5 μ l solution promptly react as next step PCR.
Contain template 5 μ l (about 25ng) in the PCR3:100 μ l reaction volume, upstream primer A 100 pmol, downstream primer B 100 pmol and general PCR reactant, 30 circulations of increasing.
The PCR product carries out enzyme with Nco I/BamH I and cuts the linear plasmid of cutting with the NcoI/BamH I enzyme of pET15b and be connected after Agarose glue reclaims separation and purification, has made up pE2BmK α IT1-N30D plasmid.
Separation and purification and the determination of activity of C, rBmK α IT1-N30D
Plasmid pE2BmK α IT1-N30D transformed into escherichia coli BL21 (DE3).Microbial culture and aftertreatment separation and purification operation are referring to embodiment 2,3.But do not occur unimodally when HPLC separates, crude product mainly shows " random coil " when measuring CD,
1The HNMR spectrum also shows " random coil ", anti-insect biological activity determination PU
50Show that its activity is about natural about 4%.After sporting Asp by 30 residues of above description of test by Asn, under our renaturation condition, can not obtain correctly folding product, 30 residues of this explanation be Asn for this scorpion venom albumen form correct folding be very important.
Claims (7)
1, a kind of Recombined scorpion venom rBmK α IT1 gene is characterized in that the dna nucleotide sequence of described gene is as follows:
1 75
C?ATG?GTT?CGT?GAT?GCA?TAT?ATT?GCC?CAA?AAC?TAC?AAT?TGT?GTA?TAC?CAT?TGT?GCT?CGT?GAT?GCA?TAT?TGC?AAC?GAA
150
CTG?TGC?ACT?AAG?AAT?GGT?GCC?AAG?TCC?GGA?TCT?TGC?CCA?TAC?TTG?GGT?GAG?CAC?AAA?TTC?GCC?TGC?TAC?TGC?AAG
206
GAC?CTG?CCA?GAT?AAC?GTT?CCA?ATC?CGT?GTC?CCG?GGT?AAG?TGC?CAC?TAA?TAGGATC
2, a kind of Recombined scorpion venom rBmK α IT1 expression of gene plasmid is characterized in that described plasmid contains gene as claimed in claim 1, and the preserving number of described plasmid is CGMCC No.0655.
3, the e. coli host cell of a kind of express recombinant East Asia Ma Shi buthotoxin rBmK α IT1 gene is characterized in that described host cell contains the described expression plasmid of claim 2.
4, a kind of method with the described host cell expression Ma Shi of claim 3 buthotoxin, comprise the expression plasmid carrier is gone in the described gene clone of claim 1, be built into expression plasmid pE2rBmK α IT1 as shown in Figure 5, in e. coli host cell, express then, obtain Recombined scorpion venom by external renaturation and separation and purification.
5, method as claimed in claim 4 is characterized in that described expression plasmid carrier is pET15b as shown in Figure 5.
6, the application of the Ma Shi buthotoxin of genes encoding as claimed in claim 1 in preparation α type insect toxins medicine.
7. the application of the Ma Shi buthotoxin of the described expression plasmid generation of claim 2 in preparation α type insect toxins medicine is characterized in that four pairs of disulfide linkage that described Ma Shi buthotoxin is contained have correct covalent linkage connection.
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