CN102876683A - Conotoxin and biological preparation method and application thereof - Google Patents

Abstract

The invention discloses conotoxin and a biological preparation method and application thereof. The preparation method includes the steps of firstly, cloning conotoxin gene to an expression cassette of a baculovirus carrier to obtain a recombination transfer expression carrier; secondly, subjecting the recombination transfer expression carrier and baculovirus DNA (deoxyribonucleic acid) to co-transfection so as to obtain recombination baculovirus; and thirdly, infecting insect hosts or insect cells with the recombination baculovirus, culturing expression conotoxin of the infected insect hosts, and collecting and purifying the expression products. The invention further provides optimized conotoxin gene which is evidently improved in expression efficiency in insect hosts compared with original gene. Bioactive conotoxin is expressed efficiently and safely in individual insect bioreactor by the aid of baculovirus expression systems, safety is quite high, and expression products can be directly applied to analgesia drugs and the like. The biological preparation method is high in expression efficiency, complete in post-processing, fine in bioactivity, low in production cost, available for scale production and the like.

Description

Biology preparation method of conotoxin and products thereof and purposes

Technical field

The present invention relates to the preparation method of conotoxin, relate in particular to a kind of biology preparation method of conotoxin and the product that is prepared by this biological method and they at analgesia, the neural medicinal use of repairing or giving up in the drug addiction, belong to the biology preparation field of conotoxin.

Background technology

The ocean toxin is emerging research field of recent two decades, one of its impressive progress is to have found the great small peptide toxin of some toxicity, wherein the conotoxin (conotoxin) of mollusk cone shell (conus) secretion is a standby valued class neurotoxin, being the animal nerve toxin peptide of the minimum nucleic acid encoding found so far, also is the highest little peptide of disulfide linkage density.Conotoxin has the chemical structure novelty, and biological activity is strong, and target site selectivity high has become the new source of powerful and the new drug development of pharmacology and neuroscience.According to the site of action of conotoxin to neuromuscular system, it is divided into four classes: 1, alpha-conotoxin (α-conotoxin): it is similar to bungatotoxin, act on nerve synapse after acetylcholine receptor (AchR) play blocking effect; 2, (μ-conotoxin): it is similar to tetraodotoxin, works mutually single-minded inhibition voltage sensitivity sodium channel in activation for mu-conotoxin; 3, omega-conotoxin (ω-conotoxin), single-minded block nerves tip presynaptic voltage sensitivity calcium channel; 4, (δ-conotoxin), the over reach current potential time length is worked in the single-minded voltage sensitivity sodium channel that acts on to δ-conotoxin mutually at disactivation.Conotoxin has high-affinity and high specific to the effect of target acceptor, can distinguish different receptor subtypes.Although the aminoacid sequence of different conotoxins changes very greatly, their structural difference is little, and the process of especially expressing post-treatment is consistent substantially.

α CTx (alpha conotoxin Tx) is expected to directly be developed to medicine or as the lead compound of new drug.The conotoxin that acts on N-type VSCCs can suppress the neurotransmitter relevant with the pain sensation (such as L-glutamic acid and the Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2) release of central nervous system, thereby alleviate the neuropathic pain of animal, and caused that people wish its exploitation is become the great interest of new peptide class pain killer.α CTx is expected to the treatment for chronic pain, and it has the particular advantages of good effect, non-habituation with respect to morphine; CTx has and time length longer analgesic effect stronger than morphine drug effect, to bring into play analgesic effect by the nAChRs that blocks the periphery primary afferent neuron, its administration more convenient (but intramuscular injection or fat injection), the side reaction (such as constipation, respiration inhibition etc.) that also causes without morphine simultaneously, the utmost point are expected to exploitation becomes efficient analgesic drug product.Certain hypotype of α CTx energy selective exclusion nAChRs except analgesic effect is arranged, also is expected to be developed to the medicine of illnesss such as being used for the treatment of anxiety disorder, Parkinson's disease, muscular tone and hypertension in addition.Alpha A conotoxin Tx1 namely is the many a kind of α CTx of present research and comparison.

There is not yet so far the bibliographical information of the highly active vivoexpression of conotoxin, possible cause has following 2 points: 1, at prokaryotic expression system, can't process and remove N-end signal peptide sequence, can't solve C-end amidation problem; 2, the conotoxin molecule is little, and basic aminoacids is more, therefore is difficult to form the given activity conformation.Owing to above reason, most of laboratory have to turn to chemosynthesis for obtaining to have bioactive conotoxin.

Summary of the invention

One of purpose of the present invention provides a kind of conotoxin α ACT1 gene of optimization, and this gene can efficiently express the conotoxin with biologic activity in insect host or cell.

Two of purpose of the present invention provides a kind of biology preparation method of conotoxin.

Above-mentioned purpose of the present invention is achieved through the following technical solutions:

The present invention is with the original gene sequence (GenBank:AF146352.1 of conotoxin α ACT, its nucleotides sequence is classified as shown in the SEQ ID No.1, coded aminoacid sequence is shown in the SEQ IDNo.3) transform according to silkworm and polyhedrosis virus codon preference, GC content and unsuitable peak (to prolong the transformation period of mRNA) have been taked to adjust, remove the rare codon (reduce translation sequences even remove translating equipment) that contains series connection in restriction endonuclease sites commonly used and the original series, abolish the series of optimum means such as loop-stem structure that those affect mRNA stability and are combined with rrna, conotoxin gene after being optimized (α ACT1), its nucleotides sequence is classified as shown in the SEQ ID No.2, the present invention further adds BamHI restriction enzyme site and kozak sequence at 5 ' end of nucleotide sequence shown in the SEQ ID No.2,3 ' end adds EcoRI, obtains the nucleotide sequence shown in the SEQIDNo.4.Conotoxin α ACT1 gene after the optimization can efficiently express the conotoxin with biologic activity in the insect hosts such as silkworm or cell.

The present invention further provides a kind of biology preparation method of conotoxin, the method may further comprise the steps:

(1) the conotoxin gene clone is delivered to baculovirus in the expression cassette of carrier, make up and obtain restructuring transfer expression vector; (2) expression vector and baculovirus DNA cotransfection insect cell are shifted in constructed restructuring, obtain recombinant baculovirus; (3) with recombinate shape virus infection insect host or insect cell; Cultivate infected insect host and express activated conotoxin; Results and the expressed product (that is: conotoxin) of purifying, and get final product.

Wherein, the nucleotide sequence of described conotoxin gene can be the nucleotide sequence of any conotoxin gene, for example can be in alpha-conotoxin, mu-conotoxin, omega-conotoxin or the δ-conotoxin any one; Be preferably alpha-conotoxin, the conotoxin α ACT1 gene after the optimization shown in the original conotoxin α act gene shown in the SEQ ID NO:1 or the SEQ ID NO:2 more preferably most preferably is the conotoxin α ACT1 gene after the optimization shown in the SEQ ID NO:2.

The expression cassette of the baculovirus delivery carrier described in the present invention comprises promotor and enhanser; Described promotor is preferably any one in polyhedrin promotor, p10 promotor or the ie-1 promotor.

Baculovirus described in the present invention is selected from any one among BmNPV, AcMNPV, ApNPV, HaNPV, HzNPV, LdMNPV, MbMNPV, OpMNPV, SIMNPV, SeMNPV or the SpltNPV; Preferably, described baculovirus is silkworm baculovirus BmNPV, more preferably silkworm baculovirus parent plant Bm-NPV-ZJ8.

Insect host described in the present invention is selected from silkworm (Bombyx mori), wild silkworm (Bombyx mandarina), Semen Ricini silkworm (Philosamia cynthia ricim), wild silkworm (Dictyoploca japanica), Philosamia cynthia (Philosamia cynthia pryeri), tussah (Antheraea pernyi), yamama (Antheraea yamamai), wild giant silkworm (Antheraea polyphymus), autographa california (Atographa califorica), tea geometrid (Ectropis obliqua), cabbage army worm (Mamestra brassicae), prodenia litura (Spodoptera littoralis), autumn mythimna separata (Spodoptera frugiperda), cabbage looper (Trichoplusia ni), armyworm (Thaumetopoea wilkinsoni), bollworm (Heliothis armigera), U.S. bollworm (Heliothis zea), oriental tobacco budworm (Heliothis assulta), cigarette beetle (Heliothis virescens), oriental armyworm (Pseudaletia separata) or gypsymoth (Lymantria dispar); Preferably, the insect host of described baculovirus is silkworm.

Infection described in the present invention is recombinant baculovirus to be eaten to infect insect larvae or the pupal cell in 1-5 age or seen through epidermis by mouth infect 1-5 insect larvae or the pupal cell in age; Preferably, described infection is with recombinant Bombyx mori baculovirus infected silkworm cell or with recombinant Bombyx mori baculovirus percutaneous puncture-inoculation 1-5 silkworm larva or the pupal cell in age, body fluid or the tissue homogenate of collector's silkworm larva or pupa after infecting 3-6 days, to the sample ultrasonic disruption, then 5, the centrifugal 3min of 000rpm, acid or Temperature Treatment, through 0.22 micron membrane filtration etc., get the conotoxin goods; Wherein, described pupal cell is preferably 1-2 days early stage tender pupa.

The mode of the present invention by homologous recombination with the conotoxin original gene or the conotoxin gene recombination after optimizing in baculovirus, obtain recombinant baculovirus; Again with the recombinate shape virus infection silkworm, with silkworm as bio-reactor, in silkworm, efficiently express have analgesia, the neural reparation or the conotoxin product of the pharmacologically active such as solution for giving up addiction of drug addict.The inventive method adopt baculovirus expression system in the individual bio-reactor of insect safety, produce activated conotoxin efficiently, behind the prepared toxin purifying, security is high, can be directly used in the purposes such as analgesia.The advantages such as the method that the present invention prepares conotoxin has the expression efficiency height, post-treatment is complete, biological activity good, production cost is low, can accomplish scale production.

The conotoxin that the present invention of significant quantity is prepared and pharmaceutically acceptable auxiliary material or carrier compatibility can obtain to ease pain together, the neural pharmaceutical composition of repairing or giving up drug addiction.

Pharmaceutical composition of the present invention can become clinically acceptable pharmaceutical preparation with it according to the conventional preparation method of this area, such as oral preparations or injection formulations.As a kind of preferred embodiment, the conotoxin of significant quantity of the present invention can be mixed mutually with lipid acid, behind ultrasonic emulsification, be prepared into oral preparations.The final concentration of described lipid acid in oral preparations is 25-35% (g/ml) (more preferably 30%), and surplus is the conotoxin solution of purifying; Wherein, described lipid acid preferably is comprised of palmitinic acid, oleic acid and stearic acid.

Description of drawings

Fig. 1 pVL1393 synoptic diagram.

Embodiment

Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But these embodiment only are exemplary, scope of the present invention are not consisted of any restriction.It will be understood by those skilled in the art that lower without departing from the spirit and scope of the present invention and can make amendment or replace the details of technical solution of the present invention and form, but these modifications and replacing all fall within the scope of protection of the present invention.

Experiment material

1. the construction process of bacterial strain, virus strain and carrier e. coli strains: Bmbacmid and BmNPV is seen related documents (application for a patent for invention publication number: CNl02286534A; Denomination of invention: express insect bio-reactor and construction process and the application of many foreign genes), delivery carrier pVLl393 is available from Invitrogen company; Bombyx mori cell BmN, Bm-5 and Sf-21 cell are available from Invitrogen company; Intestinal bacteria (Top10, DH10B) etc. preserve for inventor laboratory; The pGL-3 carrier is available from Promega company; PMD-18T carrier and pMD-18T-simple carrier are available from TaKaRa company.

2. substratum: Escherichia coli culture medium is LB (1% peptone, 0.5% yeast extract, 1%NaCl, pH7.0); The insect cell substratum is TC-100.

3. laboratory animal: BALBc AnNCrlVr Strains of Mouse and Wistar strain rat are all available from Beijing Vital River Experimental Animals Technology Co., Ltd..

4. the primer:

The primer in table 1 experiment

The optimization of embodiment 1 conotoxin α act gene and the expression in silkworm

1. the clone of conotoxin α act gene and expression

1.1. preparation (Joseph et al., the molecular cloning experiment guide third edition, 2002 about solution and substratum; Ao Sibai et al., fine works molecular biology guide, 1998)

Solution I: 50mmol/L glucose, 25mmol/L Tris-HCl (pH8.0), 10mmol/LEDTA.

Solution II: 0.2mol/L NaOH, 1%SDS (now with the current).

Solution III: 100mL system, 5mol/L Potassium ethanoate 80mL, glacial acetic acid 12mL, ddH2O8mL.

TAE (50 *): 242g Tris alkali, the 57.1mL glacial acetic acid, 100mL 0.5mol/LEDTA (pH8.0), sterilized water is settled to 1000mL.

TER solution: pancreas RNAse (RNAse A) is dissolved among 10mM Tris-HCl, the 15mMNaCl, and the storage liquid-20 that is made into 10mg/mL is ℃ frozen, uses 1 * TE buffer to be diluted to the working fluid of 20 μ g/mL, 4 ℃ of preservations again.

PPt Buffer: Virahol 22mL; 5mol/mL KAc 1mL; Distilled water 2mL.

PEG solution: take by weighing the NaCl of certain mass, be made into the salts solution of 1.6M, add a certain amount of PEG, making its final concentration is 13%.

6mol/L NaI: with 0.75g Na ₂SO ₃Be dissolved in 40mL ddH ₂Among the O, add 45gNaI and be stirred to fully dissolving, 4 ℃ of storages.

Glass milk (Glassmilk): the Silica of 10g (100mg/mL, Sigma S-5631) is dissolved among the 100mL PBS, and precipitation 2h abandons supernatant, repeats this step 2～3 time; The centrifugal 2min of 2000g is dissolved in throw out among the NaI of 3mol/L, and final concentration is 100mg/mL, keeps in Dark Place under 4 ℃.

New Wash washing lotion: Tris-HCl (pH 7.4) 20mmol/L; EDTA 1mmol/L; NaCl 100mmol/L; Formulated with isopyknic dehydrated alcohol.

The LB substratum: Tryptones 10g/L, yeast powder 5g/L, sodium-chlor 10g/L adjusts pH value to 7.0 (solid medium contains 1.5% agar).

Albumen sample-loading buffer (2 *): 100mmol/L Tris-HCl (pH6.8), 200mmol/L dithiothreitol (DTT) (DTT), 4%SDS, 0.2% tetrabromophenol sulfonphthalein, 10% glycerine.

30% acrylamide soln 29g: acrylamide, 1gN, N '-methylene fork acrylamide is dissolved in 100mL water, filters.

The Xylene Brilliant Cyanine G dye liquor: the 0.24g coomassie brilliant blue R250 is dissolved in 90mL methyl alcohol: water (1: 1, v/v) and the 10mL glacial acetic acid in.

Destainer: 10% glacial acetic acid.

1.2 the acquisition of conotoxin α act gene sequence, optimization and synthetic

According to delivering document, and in conjunction with the data among the GenBank, obtain the gene order (GenBank:AF146352.1) (sequence is seen SEQ ID No.1) of α ACT.Carry out silkworm and polyhedrosis virus codon preference transformation (Codon usage biasadjustment) according to the original gene sequence, GC content is transformed (GC Content Adjustment), the series of optimum such as restriction endonuclease sites removal commonly used, α ACT1 gene order after being optimized (sequence is seen SEQ ID No.2), then 5 ' of the α ACT1 gene order after optimization end adds BamHI restriction enzyme site and kozak sequence, 3 ' end adds EcoRI, carry out synthetic (sequence is seen SEQID No.4), for next step is cloned on pVL 1393 plasmids, use the silkworm expression system expression ready.

5 ' end at conotoxin α ACT original series (SEQ ID No.1) adds BamHI restriction enzyme site and kozak sequence equally, 3 ' end adds EcoRI, carry out synthetic, so that the expression amount of conotoxin α ACT1 in silkworm behind follow-up and the Optimizing Reconstruction compares.

Said gene is connected to the pUC57 carrier after all synthesizing.

1.3 α ACT1 sequence is connected on the pVL1393 carrier

The pVL1393 carrier figure that contains the ORF1629 homology arm sees Fig. 1.

Its main element is as follows: the upstream homology arm is Recombination sequence (ORF603+): bases 1-3997, the polyhedron upstream promoter is Polyhedrin promoter:bases 3998-4092, polyhedrosis gene is Polyhedrin gene:bases 4093-4738, multiple clone site is Multiple cloning site:bases 4128-4179, the downstream homology arm is Recombination sequence (ORF1629+): bases 4738-7002, the intestinal bacteria replication origin is ColE1 origin:bases 8029-7356, and the resistance screening gene is ammonia benzyl mycin resistant gene Ampicillin resistance gene:bases 8965-8177.

1.3.1 α ACT1 gene fragment enzyme is cut

It is as follows that enzyme is cut system:

37 ℃ were reacted 2.5 hours.To add the 3M sodium-acetate of 1/10 volume and the dehydrated alcohol of 2 times of volumes in the above-mentioned system, the centrifugal supernatant that removes of 12000rpm changes Buffer H into and adds 1 μ LEcoRI reaction 2 hours.

1.3.2 enzyme is cut the glass milk method of product and is reclaimed (Joseph et al., the molecular cloning experiment guide third edition, 2002; Ao Sibai et al., fine works molecular biology guide, 1998)

(1) after enzyme is cut product and carried out electrophoresis with ordinary gel, downcuts the DNA band of required purpose size, weigh after putting into the Eppendorf pipe;

(2) add the 6mol/L NaI solution of 3 times of volumes (v/w), gel is fully dissolved under 65 ℃;

(3) add again 10 μ L glass milk adsorption of DNA, place 5min under the room temperature behind the mixing; 10000rpm is slightly centrifugal, reaches rotating speed and gets final product, and removes supernatant;

(4) precipitation is with New Wash washing lotion washing three times, and the 10000rpm repeated centrifugation frontly reaches rotating speed twice and gets final product, last centrifugal 2min, and 37 ℃ are dried;

(5) with 10 μ L ddH ₂The O dissolving DNA is got supernatant after centrifugal, DNA is stored in-20 ℃ for subsequent use.The detected through gel electrophoresis recovering effect.

1.3.3pVL1393 the carrier enzyme is cut

It is as follows that enzyme is cut system

37 ℃ of endonuclease reactions are after 1.5 hours, take a morsel and carry out agarose gel electrophoresis, the check enzyme cuts Quan Houzai and will add the 3M sodium-acetate of 1/10 volume and the dehydrated alcohol of 2 times of volumes in the above-mentioned system, the centrifugal supernatant that removes of 12000rpm, change Buffer H into and add 1 μ L EcoRI reaction 2 hours, then 65 ℃ of temperature are bathed 15 minutes with enzyme-deactivating, obtain the carrier that enzyme cuts.

1.3.4 α ACT1 endonuclease bamhi connects the pVL1393 carrier

Linked system is as follows:

16 ℃ of reactions are spent the night.

Transform the TOP10 competent cell 1.3.5 connect product

1.3.5.1 competent preparation (Joseph et al., the molecular cloning experiment guide third edition, 2002)

The a small amount of preparation of competent cell

(1)-the TOP10 glycerol stock of 80C cold storage recovers at the LB flat board;

(2) with sterilizing toothpick picking list bacterium colony, put into 4mL LB substratum, 37 ℃ of shaking culture are spent the night, and get 100 μ L overnight culture and are inoculated in another 4mL LB substratum 37 ℃ of shaking culture 2～2.5h, OD value about 0.6;

The centrifugal 4min of (3) 5,000g collects thalline, and thalline is resuspended in the cold CaCl of 800 μ L 75mmol/L ₂In, ice bath 30min;

The centrifugal 4min of (4) 5,000g abandons supernatant, adds the cold CaCl of 200 μ L75mmol/L ₂, the tapped tube wall makes to mix, and puts on ice to be used for behind the 2h transforming.

A large amount of preparations of competent cell

(1)-80 the TOP10 glycerol stock of ℃ cold storage is recovered at the LB flat board;

(2) (diameter 2～3mm) is put into 4mL LB substratum (not containing Amp) to picking list bacterium colony, chooses in addition one and puts into the LB substratum that is added with Amp, 37 ℃ of shaking culture 8h; Do not give birth to an explanation and do not pollute the former latter that grows.The former gets the fresh LB liquid nutrient medium of 1mL inoculation 100mL, 37 ℃ of shaking culture 2～3h;

(3) culture collection is in the centrifuge tube of sterilization, and 4 ℃ of centrifugal 5min of 5,000r/min are resuspended in thalline the precooling CaCl of 20～30mL 75mmol/L ₂In the solution, ice bath 30min;

(4) 5,000r/min low-temperature centrifugation 5min abandon supernatant, add the cold CaCl of 75mmol/L that 10mL contains 10% glycerine ₂Solution, the tapped tube wall makes to mix, and puts packing tubule behind 3～4h on ice, and-80 ℃ are frozen.

Transform (Joseph et al., the molecular cloning experiment guide third edition, 2002 1.3.5.2 connect product; Ao Sibai et al., fine works molecular biology guide, 1998)

(1) plasmid DNA 20～100ng or connect mixture 3 μ L and be added in the competent cell of the above-mentioned preparation of 100 μ L, mixing gently, ice bath 30min;

(2) carry out heat-shocked (42 ℃ of insulation 90s), put rapidly 1～2min on ice, add 1mL and be incubated to 37 ℃ LB substratum, 37 ℃ of shaking culture 1h,

(3) slightly centrifugal, go resuspended precipitation behind the part supernatant, coat several and contain on the corresponding antibiotic LB flat board.Be inverted overnight incubation for 37 ℃.

1.3.6 the evaluation of recombinant plasmid

The disrupt red cell Rapid identification

Plasmid after the restructuring and original plasmid molecule amount can detect recon (Beuken, et al., Biotechniques, 72:3827-3836,1998) with this method when certain difference is arranged.

(1) a plurality of single colony transformation of picking are inoculated in the LB substratum that 4mL contains 80 μ g/mLAmp respectively, and 37 ℃ of shaking culture are spent the night;

(2) get 300～500 μ L bacterium liquid in the Eppendorf pipe, the centrifugal 10sec of 12,000g abandons supernatant, adds 30 μ L damping fluids (6% sucrose, 0.1% tetrabromophenol sulfonphthalein), adds 20 μ L phenol/chloroforms (1: 1) again, and fully thalline is upspring in vibration;

The centrifugal 5min of (3) 12,000g gets supernatant loading electrophoresis, observations.

The further enzyme of recombinant plasmid is cut evaluation (Joseph et al., the molecular cloning experiment guide third edition, 2002; Ao Sibai et al., fine works molecular biology guide, 1998)

(1) extraction of common plasmid DNA

A. after getting top Rapid identification, show bacterium liquid 1.5mL corresponding to the plasmid of certain difference in the Eppendorf pipe, the centrifugal 5min of 5,000g collects thalline, abandons supernatant;

B. suspend with 150 μ LSolI and precipitate, place 5min on ice;

C. add 300 μ LSol II and 50 μ L chloroforms, reverse gently ice bath 5min behind the mixing;

D. add 450 μ LSol III, ice bath 5-10min behind the violent mixing;

E.4 ℃ 12, the centrifugal 10min of 000g gets supernatant, adds 450 μ L Virahols, places 20min in-20 ℃ behind the mixing;

F.12, the centrifugal 10min of 000g abandons supernatant, and precipitation is dissolved among the 250 μ LTER (TE that contains 20 μ g/mLRNaseA), 37 ℃ of digestion 20min; Add 350 μ LPPt deposit B uffer, the rearmounted 4 ℃ of 20min of mixing;

G.12, the centrifugal 10min of 000g abandons supernatant, and 70% ethanol is washed once, the vacuum-drying precipitation, and with 40 μ L0.1 * TE (pH8.0) dissolving ,-20 ℃ save backup.

(2) extraction of Bacmid plasmid DNA

A. after getting top Rapid identification, show bacterium liquid 1.5mL corresponding to the plasmid of certain difference in the Eppendorf pipe, the centrifugal 5min of 5,000rpm collects thalline, abandons supernatant;

B. suspend with 150 μ LSolI and precipitate, place 5min on ice;

C. add 300 μ LSol II and 50 μ L chloroforms, reverse gently ice bath 5min behind the mixing;

D. add 450 μ LSol III, gently ice bath 5-10min behind the mixing;

E.4 ℃ 12, the centrifugal 10min of 000rpm gets supernatant, adds 450 μ L Virahols, places 20min in-20 ℃ behind the mixing;

F.12, the centrifugal 10min of 000rpm abandons supernatant, and precipitation is dissolved among the 250 μ LTER (TE that contains 20 μ g/mLRNaseA), 37 ℃ of digestion 20min; Add 350 μ LPPt deposit B uffer, the rearmounted 4 ℃ of 20min of mixing;

G.12, the centrifugal 10min of 000rpm abandons supernatant, and precipitation is dissolved in 500 μ L ddH ₂Among the O, add the saturated phenol of isopyknic Tris, put upside down mixing;

H.12, the centrifugal 10min of 000rpm gets the upper strata water in new Eppendorf pipe, adds isopyknic phenol chloroform (phenol of 1: 1 volume and the mixed solution of chloroform) again, puts upside down mixing;

I.12, the centrifugal 10min of 000rpm gets the upper strata water in new Eppendorf pipe, adds isopyknic chloroform again, puts upside down mixing;

J.12, the centrifugal 10min of 000rpm goes the upper strata water in the Eppendorf of heart pipe, adds the 3M sodium-acetate of 1/10 volume and the dehydrated alcohol of 2 times of volumes, and ice is put 10min;

K.12, the centrifugal 10min of 000rpm abandons supernatant, and 70% ethanol is washed once, the vacuum-drying precipitation with 100 μ L0.1 * TE (pH8.0) dissolving, adds the 3M sodium-acetate of 1/10 volume and the dehydrated alcohol of 2 times of volumes again, ice is put 10min, centrifugally goes out precipitation, and-20 ℃ save backup.

(3) after the enzyme of recombinant plasmid is cut evaluation

Choose each restriction enzyme site of connection site two ends and carry out double digestion, can identify.

Identification system is as follows:

37 ℃ of enzymes are cut 1～2h, add Marker as the reference standard, detect enzyme with agarose gel electrophoresis and cut the result.

If the buffer difference of two kinds of enzymes is then used first single endonuclease digestion 1-2h, add the dehydrated alcohol precipitation DNA of the 3M sodium-acetate of 1/10 volume and 2 times of volumes, remove supernatant and carry out again second enzyme and cut.

Obtain being connected with the pVL1393-α ACT1 vector plasmid of α ACT1 sequence after identifying correctly.

1.3.7 original gene connects the pVL1393 carrier

The conotoxin original gene α ACT sequence (SEQ ID No.1) of not passing through Optimizing Reconstruction is cloned on the pVL1393 carrier with above-mentioned same method, obtains being connected with the pVL1393-α ACT vector plasmid of conotoxin original gene α ACT after evaluation is correct.

1.4pVL1393-α ACT1 vector plasmid and BmNPV DNA cotransfection bombyx mori cell obtain recombinant virus

With pVL1393-α ACT1 plasmid with BmNPV DNA with liposome embedded cotransfection bombyx mori cell recombinate because BmNPV DNA carries out the pVL1393 plasmid of homology arm, the cell culture that obtains without the baculovirus that is in the polyhedron plaque after the restructuring.

Inoculate about 0.5～1 * 10 ⁶The Bm-N cell is in 15cm ²In the culturing bottle, adherent culture spend the night (being that cell density is about 80%).Remove the substratum that contains FBS, wash the cell secondary with serum free medium, add again the 1mL serum free medium.The DNA1 μ g that in 50 μ L reaction systems, adds above-mentioned BmNPV, pVL1393-α ACT1 plasmid DNA 2ug, liposome 5uL mixes, and add water and supply volume, mixing gently, 27 ℃ of incubation 15min dropwise add in the culturing bottle, and the limit edged shakes up.Cultivate the serum free medium that the 4-6h hypsokinesis removes to contain transfection liquid for 27 ℃, add the 4.5mL serum free medium and add the FBS of 500 μ L, making its final concentration is 10%, sealing, cultivate 4-5d for 27 ℃, after floating to cell, collect screening and expression that supernatant liquor is used for recombinant virus.

1.5 screening, the purification and amplification of recombinant virus Bm-α ACT1

Inoculate an amount of Bm cell (plate bottom area 80%) in the little dish of 35mm, cultivate 16h to cell attachment for 27 ℃, suck substratum, with the cotransfection supernatant liquor by 10 ^-3-10 ^-5After doing suitably dilution, get the 1mL diluent and be added in the attached cell, be evenly distributed.27 ℃ are infected 1h, 2% low melting point sepharose is melted in 60 ℃ of water-baths, being chilled to 40 ℃ mixes through the 2X of 40 ℃ of preheatings TC-100 substratum (containing 20%FBS) with equal-volume, adding X-gal makes its final concentration reach 150 μ g/mL, along little dish edge glue is slowly poured into, solidify afterwards with Parafilm sealing, be inverted for 27 ℃ and cultivate 4-7d.Microscopically is chosen without polyhedron recombinant virus plaque, get the recombinant virus spot with the Tip choicest in the super clean bench, be dissolved in the 400 μ LTC-100 substratum, placing 4h for 4 ℃ discharges virus particle, get 100 μ L virus liquids and infect cell in 24 orifice plates, behind 27 ℃ of cultivation 3d, get 150uL cells infected supernatant and prepare fast virus genom DNA by alkaline denaturation, carry out the pcr amplification analysis, the virus of finding 33 spots all is the recombinant virus Bm-α ACT1 that contains conotoxin α ACT1 gene, the viral supernatant liquor that can amplify the purpose fragment spreads spot and carries out the screening first time, the final pure recombinant virus that obtains to contain goal gene.Get respectively the Bm-5 attached cell of the pure recombinant virus infection normal growth of 0.1uL, about 5d is after cell floats afterwards in infection, and 4 ℃ of preservations are in order to injection.

1.6 the expression of conotoxin α ACT1 in silkworm

Above-mentioned 33 recombinant virus Bm-α ACT1 nutrient solutions are pressed 10 ⁵The pfu/ head is injected children silkworm in five ages, after the silkworm morbidity, cuts foot, collects silkworm blood, and-20 ℃ frozen, obtains the expression product of conotoxin α ACT1 in silkworm.According to the comparison of the molecular weight of other anodyne (similar peptide) and analgesia equivalent, determine the content of α ACT1 in every milliliter of hemolymph between the 40-50 microgram, and its conformation and modified forms conform to natural α ACT1.

1.7 conotoxin original series α ACT recombinates on the expression vector and the expression in silkworm

Equally, the α act gene of Optimizing Reconstruction (nucleotides sequence is classified SEQ ID No.1 as) does not utilize aforesaid method to express in silkworm biological reactor, according to its molecular weight and analgesia equivalent, determine that its content in every milliliter of silkworm blood is between the 10-15 microgram.Can find out and compare conotoxin α ACT1 gene behind the Optimizing Reconstruction not the expression amount of α act gene in silkworm biological reactor of Optimizing Reconstruction improved 3-5 doubly.

Experimental example 1 conotoxin α ACT1 analgesic activities is estimated

With embodiment 1 expressed improved conotoxin α ACT1 product in silkworm, (preliminary experiment shows to carry out the analgesic activities evaluation, with the not conotoxin α act gene expression product of the Optimizing Reconstruction processing of easing pain among the embodiment 1, its consumption will exceed 5-10 than improved conotoxin α ACT1 gene product doubly just can have same analgesic effect; So adopt the expression product of improved conotoxin α ACT1 gene in silkworm to carry out the analgesic activities evaluation in this experimental example).

According to the pathological pain that at utmost keeps peripheral nerve injury and cause, the standard of operation simplification, reliability, repeatability, validity and practicality, selected classical formaldehyde acute pain model, estimated the analgesic activities of conotoxin α ACT1 (the conotoxin α ACT1 product that embodiment 1 expresses) in silkworm.

Then the formaldehyde of model Mouse and rat (2.5%) acute inflammation pain model feeds and subcutaneous injection α ACT1 by filling with.Study of behaviour detects, and observes its posture, shakes, licks the behaviors such as pawl, limping, gives a mark the variation of test administration group and control group threshold of pain according to standard.Detection of biological changes, because pain can cause the variation of a series of inflammatory factors in the nervous tissue, such as TNF α, IL-1 etc. so detect the variation of TNF α mRNA in this model nervous tissue, tentatively obtain the analgesic effect of α ACT1 albumen.

1. the preparation that contains α ACT1 protein sample is processed

Be the sample of 10ug/ml with α ACT1 concentration, dilute with 1: 3 with 1 * PBS, ultrasonic disruption, then 5, the centrifugal 3min of 000rpm, acid or temperature are bathed and are processed (pH2.0 acidity is processed, and perhaps 65 ℃ to 70 ℃ temperature are bathed and processed 15-30min), through 0.22 micron membrane filtration, for subsequent use as injection preparation.

Be mixed to get mixture with (the α ACT1 concentration behind the purifying being adjusted into 10ug/ml) behind the α ACT1 purifying with lipid acid, wherein the final concentration of lipid acid in mixture is 25-35% (g/ml), and optimum concn is that (lipid acid consists of palmitinic acid, oleic acid, other unsaturated fatty acids to lipid acid 30%; It is 3% that their final concentrations in mixture are respectively palmitinic acid 8%, oleic acid 19%, other unsaturated fatty acids composition); Mixture is obtained oral preparations (fill with and feed mouse) behind ultrasonic emulsification.

2 mouse group

2.1 formaldehyde in mice models treated method

Get 40 mouse (BALB/c AnNCrlVr strain), 10 every group, press table 2 division operation:

Table 2

Each group of each operation is got 1 simultaneously operation, carry out the formaldehyde injection behind the α ACT1 administration 30min, observe it to reaction and the record of pain, dissect mouse after the record end, get its dorsal root ganglion and spinal cord and detect the wherein expression amount variation of TNF α (primer is as TNF-F and TNF-R) take β-actin (primer is as ACT-F and ACT-R) as confidential reference items with the RT-PCR method.

2.2 methods of marking

Carry out the study of behaviour scoring according to the different physiological responses in the table 3 after the formaldehyde injection:

Table 3

	Score
		Mouse licks, stings, trembles the injection pawl	0.3
The injection pawl lifts and does not contact any surface	0.2
		Inject the pawl surface in contact but not load-bearing	0.1
Normal walking	0

Score finished to record altogether score in 30 minutes from injecting beginning in rear 5 minutes to 35 minutes.

2.3 experimental result

Mouse shows obvious mental relaxation state in injection with after fill with feeding the prepared α ACT1 of embodiment 1, blink behavior and torpescence occur.

Mouse all occurs licking foot, lifts the reaction such as foot after the formaldehyde treated, wants manifest symptom to alleviate but inject or fill with the treatment group (fill with and feed group and subcutaneous injection group) of feeding with the α ACT1 of embodiment 1 preparation than control group, and respectively organizing marks sees Table 4:

Table 4

	The scoring of mouse group study of behaviour
		The blank group	0
Formaldehyde injection control group	6.62±0.82
		α ACT1 fills with and feeds group	3.20±0.69
α ACT1 subcutaneous injection group	4.36±0.73

RT-PCR result shows: the mRNA content of TNF α has improved about 4 times than the blank group in the reaction formaldehyde injection control group, and corresponding mRNA and content has improved respectively about 2.8 times and 3.3 times in hello group and the α ACT1 subcutaneous injection group and α ACT1 fills with.The result is presented in the mouse the oral and subcutaneous injection of conotoxin α ACT1 all better analgesic activity.

3 rats group

Described with mouse group, used dosage is according to body weight, suitably dosage.Getting 40 rats (Wistar strain) tests.

Result and mouse category seemingly, the study of behaviour score sees Table 5.

Table 5

	The scoring of mouse group study of behaviour
		The blank group	0
Formaldehyde injection control group	6.33±0.75
		α ACT1 fills with and feeds group	3.42±0.72
α ACT1 subcutaneous injection group	4.02±0.53

Formaldehyde experiment contrast group, α ACT1 fill with feed TNF α in group and the α ACT1 subcutaneous injection group mRNA content respectively blank group improved about 4.5 times, 2.3 times and 2.6 times.

Sequence table

＜110〉Biological Technology institute, Chinese Academy of Agricultural Sciences

＜120〉biology preparation method of conotoxin and products thereof and purposes

<130> DQXL--0078

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 201

<212> DNA

<213> conus

<400> 1

atgggcatgc ggatgatgtt caccgtgttt ctgttggttg tcttggcaac cactgtcgtt 60

tcctccactt caggtcgtcg tgaatttcgt ggcaggaatg ccgcagccaa agcgtctgac 120

ctggtcagtt tgaccgacaa gaagcgagga tgctgtagtg atcctcgctg taactatgat 180

catccagaaa tttgtggttg a 201

<210> 2

<211> 201

<212> DNA

<213> artifical sequence

<400> 2

atgggcatgc gcatgatgtt taccgtgttt ttgttggttg tcttggcaac cactgtcgtt 60

tcgtcgactt cgggtcgtcg tgaatttcgt ggcagaaatg ccgcagccaa agcgtctgac 120

ttggtcagtt tgaccgacaa gaagcgagga tgctgtagtg atcctcgctg taactatgat 180

catccagaaa tttgtggcta a 201

<210> 3

<211> 66

<212> PRT

<213> conus

<400> 3

Met Gly Met Arg Met Met Phe Thr Val Phe Leu Leu Val Val Leu Ala

1 5 10 15

Thr Thr Val Val Ser Ser Thr Ser Gly Arg Arg Glu Phe Arg Gly Arg

20 25 30

Asn Ala Ala Ala Lys Ala Ser Asp Leu Val Ser Leu Thr Asp Lys Lys

35 40 45

Arg Gly Cys Cys Ser Asp Pro Arg Cys Asn Tyr Asp His Pro Glu Ile

50 55 60

Cys Gly

65

<210> 4

<211> 216

<212> DNA

<213> artifical sequence

<400> 4

ggatccaata tgggcatgcg catgatgttt accgtgtttt tgttggttgt cttggcaacc 60

actgtcgttt cgtcgacttc gggtcgtcgt gaatttcgtg gcagaaatgc cgcagccaaa 120

gcgtctgact tggtcagttt gaccgacaag aagcgaggat gctgtagtga tcctcgctgt 180

aactatgatc atccagaaat ttgtggctaa gaattc 216

Claims (10)

1. the conotoxin α ACT1 gene of optimizing, it is characterized in that: its nucleotides sequence is classified as shown in the SEQID No.2.

2. the recombinant expression vector that contains the conotoxin α ACT1 gene of the described optimization of claim 1; Preferably, described recombinant expression vector is that recombinant baculovirus shifts expression vector.

3. the host cell that contains the described expression vector of claim 2; Preferably, described host cell is insect host or insect cell; Preferred, described insect host is silkworm, and described insect cell is bombyx mori cell.

4. conotoxin α ACT1 gene claimed in claim 1 is in the purposes of preparation in the conotoxin.

5. the biology preparation method of a conotoxin is characterized in that, comprising: (1) in the expression cassette of baculovirus delivery carrier, makes up the conotoxin gene clone to obtain restructuring transfer expression vector; (2) expression vector and baculovirus DNA cotransfection insect cell are shifted in constructed restructuring, obtain recombinant baculovirus; (3) with recombinate shape virus infection insect host or insect cell; Cultivate infected insect host, results and the expressed product of purifying, and get final product.

6. according to biology preparation method claimed in claim 5, it is characterized in that: described conotoxin is any one in alpha-conotoxin, mu-conotoxin, omega-conotoxin or the δ-conotoxin; Be preferably alpha-conotoxin; The conotoxin gene after the optimization shown in the original conotoxin α act gene shown in the SEQ ID NO:1 or the SEQ ID NO:2 more preferably most preferably is the conotoxin α ACT1 gene after the optimization shown in the SEQID NO:2.

7. according to biology preparation method claimed in claim 5, it is characterized in that: the expression cassette of described baculovirus delivery carrier comprises promotor and enhanser, and described promotor is preferably any one in polyhedrin promotor, p10 promotor or the ie-1 promotor;

Described baculovirus is selected from any one among BmNPV, AcMNPV, ApNPV, HaNPV, HzNPV, LdMNPV, MbMNPV, OpMNPV, SIMNPV, SeMNPV or the SpltNPV; Preferably, described baculovirus is silkworm baculovirus BmNPV, more preferably silkworm baculovirus parent plant Bm-NPV-ZJ8;

Described insect host is selected from silkworm (Bombyx mori), wild silkworm (Bombyxmandarina), Semen Ricini silkworm (Philosamia cynthia ricim), wild silkworm (Dictyoplocajapanica), Philosamia cynthia (Philosamia cynthia pryeri), tussah (Antheraea pernyi), yamama (Antheraea yamamai), wild giant silkworm (Antheraea polyphymus), autographa california (Atographa califorica), tea geometrid (Ectropis obliqua), cabbage army worm (Mamestra brassicae), prodenia litura (Spodoptera littoralis), autumn mythimna separata (Spodoptera frugiperda), cabbage looper (Trichoplusia ni), armyworm (Thaumetopoea wilkinsoni), bollworm (Heliothis armigera), U.S. bollworm (Heliothis zea), oriental tobacco budworm (Heliothis assulta), cigarette beetle (Heliothis virescens), oriental armyworm (Pseudaletia separata) or gypsymoth (Lymantria dispar); Preferably, the insect host of described baculovirus is silkworm;

Described infection is that recombinant baculovirus is eaten to infect 1-5 insect larvae or the pupal cell in age by mouth, or infects 1-5 insect larvae or the pupal cell in age through epidermis; Preferably, described infection is with recombinant Bombyx mori baculovirus infected silkworm cell, or with recombinant Bombyx mori baculovirus percutaneous puncture-inoculation 1-5 silkworm larva or the pupal cell in age; Wherein, described pupal cell is preferably 1-2 days early stage tender pupa.

8. state the conotoxin that the biology preparation method prepares by claim 6 or 7.

9. an analgesia, the neural pharmaceutical composition of repairing or giving up drug addiction is characterized in that: the conotoxin claimed in claim 8 and the pharmaceutically acceptable auxiliary material that comprise significant quantity.

10. inject or oral preparations an analgesic, it is characterized in that: the conotoxin claimed in claim 8 and the final concentration that comprise the upper significant quantity for the treatment of are the lipid acid of 25-35%.

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