CN101979572A - Preparation for South China Sea conus striatus toxin S4.3 and application - Google Patents

Abstract

The invention relates to a South China Sea conus striatus toxin gene and polypeptide S4.3 coded by the same, a preparation method for the polypeptide and application thereof. The A-superfamily conotoxin gene is obtained by cloning from a conus striatus tube by a method for constructing a cDNA library, and the DNA sequence of the gene is expressed as a (400)1 sequence in a sequence table. The amino acid sequence of the polypeptide S4.3 coded by the gene is expressed as a sequence table (400)2. The preparation method for the polypeptide constructs a recombinant expression vector pTRX-S4.3, and probes and optimizes the culture method and conditions so as to improve the expression quantity of the polypeptide S4.3. The preparation method for the polypeptide S4.3 can be used for improving the conventional polypeptide production method. The polypeptide S4.3 can promote quick growth of cells, and can be used for preparing medicaments for promoting the growth of the cells.

Description

Preparation and the application of South China Sea strain line conotoxin S4.3

Technical field

The present invention relates to a kind of new South China Sea strain line conus neurotoxin gene and the technology of preparing of encoded polypeptides S4.3 and this polypeptide thereof, and the application of this polypeptide in preparation promoting growth of cell instrument medicine.

Background technology

Cone shell (Genus Conus) is the marine organisms that a class distributes extensively, kind is many.Cone shell also claims cone shell, but because its shell is conical and has beautiful decorative pattern, becomes the taro shape and gain the name " cone shell ".On taxonomy, cone shell belongs to Mollusca, Gastropoda, preceding cheek subclass, Caenogastropoda, cone shell superfamily, Conidae.The cone shell majority is lived in the shoaling water of tropic sea midoceans such as Pacific and Indian oceans, and minority is in the ballow of several meters of depth of waters rice surplus 200.Daytime or ebb back are perched under marine alga or in the coral hole, go out to look for food the spring and summer breeding night.

The cone shell of whole world discovery at present is above 700 kinds, wherein China torrid zone and marine site, subtropics had at present the cone shell of detail record have 70 surplus kind, mainly be distributed in the Nansha Islands, the Xisha Islands, southern Hainan Island and marine site, Taiwan, minority in Guangdong, Guangxi and Hainan is coastal, wherein the part kind is China's record first.

Cone shell can be divided into three major types by its feeding habits: ichthyophagy cone shell (piscivorous), food mollusk property cone shell (molluscivorous) and entomophagy cone shell (vermivorous).Wherein entomophagy cone shell most species accounts for about 70% of whole cone shell kinds, though and the ichthyophagy cone shell to account for the cone shell sum minimum, toxicity is the strongest, Bao Dao the cone shell incident that causes death is caused by such cone shell basically up to now.

(conotoxin CTX) is the active polypeptide that a class derives from the cone shell venom to conotoxin, and for cone shell self, these toxin are mainly used in predation and defence.In general, all contain the different conotoxin of 50-200 kind in each cone shell body, whole occurring in nature approximately exists 50,000-100,000 kind of different conotoxin.And up to now, having only seldom the conotoxin of a part to be found by the mankind, the toxin with definite function is 200 of less thaies then.Conotoxin generally has following characteristics: (1) sequence short molecule amount is little.Toxin sequence more than 80% is between 7-41 amino acid, and molecular weight is below 5kDa.(2) compact structure is stable.Because conotoxin is rich in halfcystine, halfcystine be paired into the basic configuration that key has determined conotoxin, and make conotoxin form densification and stable structure.(3) on primary structure, the sequence between the halfcystine is an alterable height.(4) target spot extensively but high specificity.The target spot of conotoxin mainly comprises various valtage-gated ionic channels (as sodium, potassium, calcium plasma channel) and neurotransmitter receptor (acetylcholine receptor, nmda receptor and g protein coupled receptor etc.), and a kind of toxin generally only act on certain acceptor one to several hypotypes, high specificity.

Early stage as far back as 20th century, people just begin the malicious pipe unit of cone shell is carried out the research of anatomical terms.And, it is found that more and more have different bioactive conotoxins along with the further investigation in more than 60 year afterwards.When people further are surprised to find after the research these toxin; conotoxin itself is exactly a natural huge medicine source treasure-house, and various conotoxins all have broad prospect of application aspect the diseases such as treatment chronic obstinate pain, acute pain, epilepsy, neuroprotective, cardiovascular disorder, insane, ataxia, spasm disease, cancer and apoplexy and potential exploitation is worth.Therefore, carry out the exploratory development in biochemistry, physiology, pharmacology, especially molecular biology and the genetically engineered field of China's South Sea conotoxin, and carry out conotoxin vivoexpression Study on Technology, for promoting China's polypeptide the reach of science, promote the exploitation of polypeptide drugs, have the theory that can not be ignored and be worth and using value.

Summary of the invention

The object of the present invention is to provide a kind of new South China Sea strain line conotoxin gene and encoded polypeptides S4.3 thereof.

Another object of the present invention provides a kind of new recombinant expression vector pTRX-S4.3.

A further object of the present invention provides a kind of novel polypeptide high-efficiency expression method, from the several aspects of expression, separation and purifying original expression of polypeptides method is improved.

The 4th purpose of the present invention is to provide the application of this polypeptide in preparation promoting growth of cell medicine.

The strain line cone shell that the present invention uses is picked up from Sanya, Chinese Hainan Province.

The present invention separates obtaining new A-superfamily conotoxin gene by the method for construction cDNA library and order-checking from South China Sea strain line cone shell poison pipe.

The present invention is divided into 3 pairs of complementary sequences with the cDNA sequence of above-mentioned purpose gene, use six sections primers to go out the strain line conotoxin gene of total length by pcr amplification as the template fundamental chain, and at the 5 ' end and the 3 ' restriction enzyme site (TAA) that has added restriction enzyme Kpn I and Not I respectively of this gene, added the recognition site of enteropeptidase Enterokinase in addition at 5 ' end, also added two terminator codons at 3 ' end.The nucleotide sequence of goal gene as in the sequence table＜400〉1 sequences shown in.

Said gene encoded polypeptide S4.3, its aminoacid sequence as in the sequence table＜400〉2 sequences shown in.

The present invention utilizes Kpn I and Not I double digestion method that the pTRX-IL10 plasmid is carried out the linearizing preparation, excision IL10 fragment, obtain linearizing pTRX carrier, the Oligo segment of the goal gene that the previous step pcr amplification is obtained is carried out phosphorylation, annealing and complementary pairing simultaneously, the good goal gene that will match then is connected with the pTRX carrier, constructs recombinant expression vector pTRX-S4.3 and it is changed in the bacillus coli DH 5 alpha and increase.The correct recombinant expression vector pTRX-S4.3 that afterwards checks order increasing changes in the e. coli bl21 (DE3) and carries out prokaryotic expression.By exploration and optimization to culture condition, the expression amount of the recombination fusion protein that obtains is higher, and expression amount is a 10mg/L bacterium liquid.

The present invention has also optimized the enzyme of recombination fusion protein and has cut and purification process, and the ultrasonic degradation liquid of expression product adopts the Ni-NTA Superflow filler of QIAGEN company, with Ni ²⁺For part carries out affinity chromatography, obtain fusion rotein and slightly carry product, this slightly carry product again through EK enzyme enzyme cut, chromatography filters and the RPHPLC (reversed-phase high-performance liquid chromatography) purifying, can obtain highly purified polypeptide S4.3.

The polypeptide S4.3 that the present invention obtains has the biologic activity that promotes the cell growth.The polypeptide S4.310 μ M stimulation test tumour cell 3 strain systems of prokaryotic expression and mammary epithelial cell 1 strain system detect cells survival rate result through mtt assay and confirm that the quantity of experimental group cell is compared with control group and is significantly increased behind effect 36h in contrast.Therefore, polypeptide S4.3 of the present invention can be used for preparation promotion cell growth medicine.

Description of drawings

The preparation of Fig. 1 linear plasmid carrier pcDNA3-SfiI

The double-stranded cDNA 1.1% agarose gel electrophoresis result of Fig. 2 strain line cone shell poison pipe

Fig. 3 strain line cone shell poison pipe cDNA total plasmid pcr amplification 1% agarose electrophoresis result in library

Fig. 4 strain line cone shell poison pipe cDNA library part recon pcr analysis result

Fig. 5 strain line cone shell poison pipe cDNA library readable sequences length distribution

The distribution situation of the ESTs number that is contained in the gene cluster of Fig. 6 strain line cone shell poison pipe cDNA library

Fig. 7 strain line cone shell poison pipe cDNA library ESTs classification

Toxin sequence classification in Fig. 8 strain line cone shell poison pipe cDNA library

Physical map and the multiple clone site of Fig. 9 expression plasmid pTRX

Figure 10 recombinant expression vector pTRX-S4.3 construction procedures synoptic diagram

Figure 11 HPLC purifying South China Sea conotoxin polypeptide S4.3 lab diagram

Figure 12 strain line conotoxin polypeptide S4.3 mass spectrum

Figure 13 is the cellular form figure that 10 μ M S4.3 act on Hela cell 36h.Figure 13 a is normal Hela cell cultures 36h figure, and Figure 13 b schemes for Hela cell cultures 36h after adding 10uM S4.3.

Figure 14 MTT detects cells survival rate figure

Embodiment

Below by embodiment, technical scheme of the present invention is described further:

Experimental example 1: the structure and the evaluation in South China Sea strain line cone shell poison pipe cDNA library

1) material

11 tissue sample strain line cone shells (Conus Straitus) are picked up from Sanya, Chinese Hainan.

1.2 plasmid and bacterial strain

Plasmid vector pcDNA3 is available from Invitrogen company

Intestinal bacteria (Escherichia coli) DH5 α is preserved by medicine Molecular Biology Lab of life science institute of Zhongshan University, and its genotype is:

DH5α：supE44Δlac?U169(φ80lacZΔM15)hsdR17recA1endA1gyrA96thi-1relA1

1.3RNA extraction reagent

DEPC water: add 1ml DEPC in every 1000ml deionized water, final concentration is 0.1%, abundant mixing, 37 ℃ spend the night after, autoclaving.

1.4 damping fluid

1.4.1 the used damping fluid of alkaline lysis method of extracting e. coli plasmid dna

Solution I: 50mM glucose, 10mM EDTA, 25mM Tris-HCl (pH8.0);

Solution II: 0.2N NaOH, 1.0%SDS, preparation before using;

Solution III: 60ml 5M potassium acetate, 11.5ml glacial acetic acid, 28.5ml H ₂O, the concentration of potassium ion is 3M in the solution that is made into, the concentration of acetate moiety is 5M;

TE：10mM?Tris-HCl(pH7.4、pH7.6、pH8.0)，1mM?EDTA(pH8.0)；

STE：50mM?Tris-HCl(pH?8.0)，5mM?EDTA(pH8.0)，50mM?NaCl；

STE (lysozyme): 50mM Tris-HCl (pH8.0), 5mM EDTA (pH8.0), 50mMNaCl, 70 μ g/ml N,O-Diacetylmuramidases;

10%SDS: dissolving 100g electrophoresis level SDS in 900ml water, be heated to 68 ℃ of hydrotropies, add water and be settled to 1, the 000ml packing is standby;

1.4.2PEG method is extracted the used damping fluid of plasmid

5M LiCl:30.205g LiClH ₂O is water-soluble, is settled to 100ml, autoclaving.

13%PEG8000: contain the 1.6M NaCl solution of 13% (w/v) PEG8000, filtration sterilization.

The 10M ammonium acetate: the 770g ammonium acetate is dissolved in the 800ml water, adds water and be settled to 1, behind the 000ml, filtration sterilization.

1.4.3 agarose gel electrophoresis damping fluid

50 * TAE:242g Tris, the 57.1ml glacial acetic acid, 18.6g EDTA is settled to 1,000ml.

EB solution: add the 1g ethidium bromide in the 100ml water, magnetic agitation a few hours dissolving extremely fully, packing, room temperature keeps in Dark Place.

DNA sample loading buffer: 0.25% tetrabromophenol sulfonphthalein, 0.25% dimethylbenzene green grass or young crops, 50% glycerine (w/v).

RNA denaturing formaldehyde glue sample loading buffer: 0.25% tetrabromophenol sulfonphthalein, 0.25% dimethylbenzene green grass or young crops, 1mM EDTA (pH8.0), 50% glycerine (w/v), with the preparation of DEPC water, autoclaving is standby.

5 * denaturing formaldehyde gel electrophoresis damping fluid: 0.1M MOPS (pH7.0), the 40mM sodium acetate, 5mM EDTA (pH8.0), with the preparation of DEPC water, filtration sterilization, room temperature keeps in Dark Place.Faint yellow damping fluid can normally use, and deep yellow should be abandoned.

The preparation of denaturing formaldehyde glue: the 0.336g agarose is dissolved in the 20ml DEPC water, is cooled to 60 ℃, adds the formaldehyde of 5ml 5 * denaturing formaldehyde gel electrophoresis damping fluid and 5.5ml, pours into gel in stink cupboard, uses behind the cooling 30min.

The preparation of denaturing formaldehyde glue RNA sample: an amount of RNA, 5 * denaturing formaldehyde gel electrophoresis damping fluid, 0.5 μ l, formaldehyde 0.7 μ l, methane amide 2 μ l, 65 ℃ of heating 15min, ice bath adds 1 μ l sample-loading buffer and a small amount of EB rapidly.

1.4.4 conversion damping fluid

100mM CaCl ₂: 1.47g CaCl ₂2H ₂O is dissolved in deionized water, is settled to 100ml, filtration sterilization, 4 ℃ of preservations.

1.5 substratum

LB liquid nutrient medium: Tryptone (peptone) 10g, Yeast Extract (yeast extract) 5g, NaCl 10g adds the 800ml deionized water, regulates pH to 7.2-7.5 with 5M NaOH, is settled to 1,000ml, packing autoclaving, 4 ℃ of preservations.

The LB solid medium: adding bacterium in the LB liquid nutrient medium is 1.3-1.5% with agar powder to final concentration, autoclaving, the dull and stereotyped back 4 ℃ of preservations of perfusion.

The SOB substratum: Tryptone 20g, Yeast Extract 5g, NaCl 0.5g adds the 800ml deionized water, add 250mMKCl solution 10ml, regulate pH to 7.2-7.5, be settled to 1,000ml with 5M NaOH, autoclaving, 4 ℃ of preservations add the 2M MgCl that 5ml sterilizes before using ₂Solution.

SOB solid medium: contain 20mM MgSO ₄And the SOB substratum of the agar powder of 1.3-1.5%, autoclaving, the dull and stereotyped back 4 ℃ of preservations of perfusion.

SOC substratum: behind the SOB substratum autoclaving, when temperature is reduced to below 60 ℃, add the 1M glucose solution of 20ml filtration sterilization, 4 ℃ of preservations.

2 * YT:Tryptone 16g, Yeast Extract 10g, NaCl 5g adds the 800ml deionized water, regulates pH to 7.2-7.5 with 5M NaOH, is settled to 1,000ml, autoclaving, 4 ℃ of preservations.

1.6 reagent and enzyme

LS reagent is available from Gibcol BRL company; SMART ^TMCDNA Library Construction Kit is a CLONTECH company product; Gel Extraction Kit and Plasmid Miniprep Kit are available from OMEGA BIOTEK company; Vitagene 96-easy plasmid Mini-prep Kit is available from Vitagene Biochemical Technique company; ABIPRISM Big-DyeTM Terminator v3.0Ready Reaction Cycle Sequencing Kit is available from Applied Biosystems company; Bacto tryptone and Bacto yeast extract are available from OXOID company; DNTP, RNaseA, DEPC, MOPS, re-distilled phenol and penbritin sodium salt are given birth to worker bio-engineering corporation available from Shanghai; Taq archaeal dna polymerase, various restriction enzyme and T4DNA Ligase are available from NEB company; DNA ladder is available from Promega company; Primers such as T7promoter sequencing primer, SP6promoter sequencing primer are synthetic by Shanghai Bo Ya company; Other reagent is homemade analytical reagent.

1.7 other solution

Penbritin solution: be made into 100mg/ml with aseptic deionized water, packing ,-20 ℃ of preservations, working concentration is 100 μ g/ml.

RNaseA solution: RNaseA is dissolved in 10mM Tris-HCl (pH7.5), in the 15mM NaCl solution, is made into the storage concentration of 10mg/ml, 100 ℃ of heating 15min slowly cool to room temperature, and are standby-20 ℃ of preservations after the packing.

50% glycerine:, standby behind the autoclaving with deionized water preparation 50% (w/w) glycerine.

DNTP: with the preparation of sterilization ultrapure water, contain dATP, dTTP, dCTP, the dGTP of 10mM respectively, standby after the packing-20 ℃ of preservations.

T7 and SP6 primer: with the sterilization ultrapure water primer is made into the solution of 50pmol/ μ l, standby after the packing-20 ℃ of preservations.

2) method

2.1 the processing of strain line cone shell poison tubing

Get breechblock alive, use larynx lock fragmentation spiral shell shell, expose spiral shell meat, careful on ice also sharp separation poison tubing is put into liquid nitrogen and abundant the grinding rapidly after weighing, 15 times of bulking values of adding

LS reagent (being to add 15ml in the 1g tissue), fully homogenate in the ice bath is preserved in-80 ℃ of refrigerators in order to extracting total RNA.

2.2 the extraction of total RNA

With reference to Gibcol BRL company

LS reagent specification sheets carries out.Get the 50-100mg tissue sample, use 0.75ml

The homogenate of LS reagent, 15-30 ℃ leaves standstill 5min.Add the 0.2mL chloroform then, thermal agitation is after 15 seconds, and 15-30 ℃ leaves standstill 2-15min.4 ℃, 12, the centrifugal 15min of 000rpm gets the upper strata water.Add the 0.5ml Virahol, 15-30 ℃ leave standstill 10min after, 4 ℃, 12, the centrifugal 10min of 000rpm abandons supernatant.Add 1ml 75% ethanol rinsing precipitation again, 5, the centrifugal 5min of 000rpm abandons supernatant, and the ethanol of trace in the sample is removed in vacuum-drying, adds the deionized water of an amount of no RNase.Get 1 μ l and carry out 1% denaturing formaldehyde gel electrophoresis, 1 μ l distinguishes the optical density(OD) of working sample on 260nm, 280nm wavelength and the purity of preresearch estimates RNA with ultraviolet spectrophotometer, and-20 ℃ of preservations are standby.

2.3 RNA denaturing formaldehyde gel electrophoresis

Before the application of sample, the prerunning 5min of denaturing formaldehyde glue elder generation, voltage is reduced to 5v/cm; Subsequently sample is added to gel pore, the RNA of available known dimensions makes molecular weight marker, carries out electrophoresis by the voltage of 3-4v/cm; Ultraviolet lamp is observed electrophoresis result down.

2.4 alkaline lysis method of extracting plasmid DNA

Random choose transforms the single bacterium colony of DH5 α in the LB liquid nutrient medium that contains Amp (final concentration is 100 μ g/ml) from flat board, and 37 ℃, 225rpm shaking culture spend the night.Get 1.5ml bacterium liquid in centrifuge tube, 4 ℃, 12, the centrifugal 1min of 000rpm abandons supernatant, with 1ml STE solution suspension bacterial precipitation, 4 ℃, 12, the centrifugal 1min of 000rpm abandons supernatant, the solution I that the adds 100 μ l precoolings thalline that suspends again, room temperature is placed 5min, adds the solution II that 200 μ l now join, light and slow mixing, place 5min on ice, add 150 μ l solution III again, gentleness is put upside down concussion 10s, places 3-5min on ice, 4 ℃, 12, the centrifugal 5min of 000rpm gets supernatant, uses equal-volume phenol/chloroform/primary isoamyl alcohol (25: 24: 1) and each extracting of chloroform/primary isoamyl alcohol (24: 1) once; Water adds the dehydrated alcohol of 1/10 volume 3M NaAc (pH5.2) and 2 times of volume precoolings, puts upside down mixing, and room temperature is placed 10min, 12, the centrifugal 5min of 000rpm abandons supernatant, adds 1ml 75% ethanol rinsing precipitation again, 12, the centrifugal 5min of 000rpm abandons supernatant, drying at room temperature 10min, add and contain RNaseA (20 μ g/ml) sterilization deionized water or the dissolving of TE damping fluid ,-20 ℃ of preservations in right amount.

2.5PEG precipitator method plasmid DNA purification

Picking transforms bacterium colony and is inoculated in 50ml and contains in the LB liquid nutrient medium of Amp (final concentration is 100 μ g/ml) at random, and 37 ℃ jolt overnight incubation; Get the 50ml bacterial cultures, 5,000rpm, 4 ℃ of centrifugal 5min; With the 10ml STE thalline that suspends again; 5,000rpm, 4 ℃ of centrifugal 5min; Thalline is resuspended in the solution I of 3ml, adds the solution II that 6ml newly joins, and puts upside down to mix evenly ice bath 5min; Add the 4.5ml solution III, leniently put upside down ice bath 15min 5 times; 5,000rpm, 4 ℃ of centrifugal 30min; Supernatant is transferred in the new centrifuge tube, adds the Virahol of 0.6 times of volume, room temperature is placed 10min; 5,000rpm, the centrifugal 20min of room temperature; Remove supernatant, with 75% washing with alcohol precipitation, 5,000rpm, the centrifugal 10min of room temperature; Remove the ethanol raffinate, be deposited in drying at room temperature after, with 1.5ml TE dissolving; Be transferred in the 7ml centrifuge tube, add the 5M LiCl solution of equal-volume precooling, fully mixing; 10,000rpm, 4 ℃ of centrifugal 10min; Supernatant is transferred in the new centrifuge tube, adds isopyknic Virahol, mixed evenly back room temperature is placed 10min; Room temperature 10, the centrifugal 10min of 000rpm; Remove supernatant, precipitate with 75% washing with alcohol DNA; Room temperature 10, the centrifugal 10min of 000rpm; Remove the ethanol raffinate, be deposited in drying at room temperature; The DNA precipitation is transferred in the 1.5ml centrifuge tube after dissolving with 700 μ lTE, and adding RNaseA is 20 μ g/ml to final concentration, and room temperature is placed 30min; Add isopyknic 13%PEG8000 solution, abundant mixing, room temperature is placed 10min; Room temperature 12, the centrifugal 10min of 000rpm; Remove supernatant, precipitation is fully dissolved with 500 μ l TE, through isopyknic phenol: chloroform: primary isoamyl alcohol (25: 24: 1) and chloroform: after each extracting once of primary isoamyl alcohol (24: 1), water is transferred in the new centrifuge tube, add the 10M ammonium acetate of 1/4 volume and the dehydrated alcohol of 2 times of volume precoolings, put upside down behind the mixing room temperature and place 10min; Room temperature, 12,000rpm, 4 ℃ of centrifugal 10min; Remove supernatant, precipitation 1ml 75% washing with alcohol; 4 ℃ 12, the centrifugal 5min of 000rpm; Remove the ethanol raffinate, precipitation room temperature drying sheet is carved, and adds an amount of sterilization deionized water or TE damping fluid, and-20 ℃ of preservations are standby.

2.6 the preparation of linear plasmid carrier pcDNA3-SfiI

The pcDNA3-SfiI plasmid is the carrier of transforming through this laboratory, transforms and introduced new SfiI restriction enzyme site (Fig. 1) later in the pcDNA3 carrier.The PEG precipitator method are extracted and purifying pcDNA3-SfiI plasmid, after restriction enzyme SfiI enzyme is cut, carry out agarose gel electrophoresis.Press the Gel Extraction Kit specification sheets operation of OMEGABIOTEK company, glue reclaims above-mentioned enzyme tangent line carrier DNA.Repeat enzyme cut with reclaimer operation each once, linear carrier DNA preserves standby with 50ng/ μ l concentration.

2.7 strain line cone shell poison pipe cDNA's is synthetic

Press the SMART of CLONTECH company ^TMThe operation of cDNALibrary Construction Kit specification sheets.Its principle is to utilize the G (m that methylates of eukaryote mRNA 5 ' end ⁷G) and the characteristics design anchor primer of the PolyA tail of the special cap sequence that connects of 5 '-5 ' triphosphoric acid key and 3 ' end, it is synthetic to carry out first chain respectively, digestion is cut in the amplification of LD PCR cDNA high-fidelity, enzyme and post reclaims cDNA, thereby realizes the purpose of enrichment full-length cDNA.

2.7.1cDNA first chain is synthetic

In 5 μ l reaction systems, add following component (operation on ice): the total RNA of 1 μ g, each 1 μ l of SMART III olignucleotide and CDS III/3 ' PCR primer, with ultrapure water polishing volume, mixing, of short duration centrifugal.72 ℃ of incubation 2min, ice bath 2min, of short duration centrifugal, make mixture combine in the pipe end.Flick tube wall after adding the dNTP Mix of DTT, 1 μ l 10mmol/L of 2 μ l, 5 * First Strand Buffer, 1 μ l 20mmol/L and 1 μ l PowerScript RT (CLONTECH) more successively, mixing is also of short duration centrifugal.Put synthetic first chain of 42 ℃ of reactions of PCR instrument 1hr reverse transcription, ice bath termination reaction ,-40 ℃ of preservations.

2.7.2LD pcr amplification cDNA second chain

In the reaction system of 100 μ l, add following component: 2 μ l, the first chain product, 10 * Advantage 2PCR Buffer, 10 μ l, 50 * dNTPs Mix, 2 μ l, each 2 μ l of 20 μ mol/L upstream and downstream anchor primers, 50 * Advantage 2Polymerase Mix, 2 μ l and ultrapure water 80 μ l polishing volumes, mixing, put into the PCR instrument that is preheated to 95 ℃, move following response procedures: behind 95 ℃ of sex change 1min, continue 95 ℃ of 15sec of cycling program, 68 ℃ of 6min totally 18 circulations, get 5 μ l PCR products after being cooled to 4 ℃ to detect with 1% agarose gel electrophoresis.Residue PCR product is in-20 ℃ of preservations.

2.7.3 protease K digesting

In the PCR product (2-3 μ g dscDNA) of 50 μ l previous steps, add 2 μ l Proteinase Ks (20 μ g/ μ l) deactivation archaeal dna polymerase, 45 ℃ of incubation 20min, again once through phenol/chloroform/primary isoamyl alcohol (25: 24: 1) and each extracting of chloroform/primary isoamyl alcohol (24: 1) mixed solution, get supernatant, add 10 μ l3M sodium acetates, 1.3 μ l glycogen (20 μ g/ μ l), 95% ethanol that 260 μ l room temperatures are placed, room temperature 14, the centrifugal 20min of 000rpm, with 80% washing with alcohol precipitation, dry air adds the abundant dissolution precipitation of 79 μ l deionized waters.

2.7.4SfiI enzyme is cut digestion

Cut the following component of adding in the system at 100 μ l enzymes: the dscDNA of 79 μ l, 10 μ l, 10 * SfiI Buffer, 1 μ l 100 * BSA and 10 μ l SfiI restriction endonucleases.Fully mixing is of short duration centrifugal.Behind 50 ℃ of incubation 2hr, add the blue mixing of dimethylbenzene nitrile of 2 μ l 1%.

2.7.5cDNA the branch post reclaim

The dscDNA that it is good that the previous step enzyme is cut digestion is gone up sample to the good CHROMASPIN-400 post of pre-treatment, be in charge of and collect dscDNA not of uniform size, 1 of every pipe after having collected 16, covers the pillar loam cake; From every collection tube, take out the 5ul sample and carry out electrophoresis detection, 1% agarose gel electrophoresis is determined satisfactory collection tube number, the 20mg/ml glycogen of 3M NaAc (pH4.8), the 1.3 μ l of 0.1 times of volume of adding and 95% ethanol of 2.5 times of volumes after concentrating are placed precipitation for-20 ℃ and are spent the night; Room temperature 14, the centrifugal 20min of 000rpm; Supernatant is removed in suction, and precipitation is used 80% washing with alcohol, room temperature 14, and the centrifugal 5min of 000rpm inhales and removes supernatant, is deposited in about air drying 10min; With the dried dscDNA of 7 μ l deionized water dissolvings ,-20 ℃ of preservations are standby, also can directly be connected with carrier.

2.7.6dscDNA with being connected of carrier

Set up following three ligation systems: respectively get 1 μ l through the pcDNA3-SfiI carrier DNA (50 μ g/ μ l) that the SfiI enzyme is cut, dscDNA gets 0.5,1.0,1.5 μ l respectively, 10 * connection damping fluid, 1 μ l, T4DNA ligase enzyme (100units) 0.5 μ l, adding aseptic deionized water to volume is 10 μ l, set from connecting the control reaction system, 16 ℃ connect 16hr, to determine optimum proportion.

2.7.7 the electricity in strain line cone shell poison pipe cDNA library transforms

The preparation of DH5 α electricity transformed competence colibacillus cell: 37 ℃ of line activatory E.coli DH5 α, 1 single bacterium colony of picking is inoculated into 37 ℃ of concuss overnight incubation in the LB substratum.Get fresh LB liquid nutrient medium next day, in 1: 100 ratio enlarged culturing to OD ₆₀₀=0.6.With the rapid ice bath of bacterium liquid, behind the 30min, 4 ℃, 4, the centrifugal 10min of 800rpm receives bacterium, abandons supernatant, and usefulness waits bacteria liquid ultrapure water long-pending and half volume to wash thalline once respectively, and per 1 liter of bacterium liquid is resuspended with 300 μ l, 10% glycerine, by every pipe 40 μ l packing.

Connect the product electricity and transform DH5 α competent cell: per 40 μ l bacterium liquid add 1 μ l and connect product or control plasmid solution, and mixing changes electricity over to and transforms cup, ice bath is no more than 5min, dry outer wall with coil paper, place the BioRad electroporation apparatus to carry out electricity at once and transform, electricity transforms parameter: voltage 2.0kV; Electric current 25 μ F; Resistance 200 Ω; Electricity swashs time 4.5mS.Mix with the SOB substratum of 37 ℃ of preheatings of 800 μ l at once after the electric shock, use the rifle mixing, bacterium liquid in the sucking-off cup is loaded in the clean aseptic centrifuge tube as far as possible.37 ℃, 225rpm recovery cell 1hr coat Amp ⁺The LB flat board on, be inverted to cultivate 12-16hr in 37 ℃ of incubators.Converted product then is divided into two portions, and wherein a part is stored in 25% glycerine as cDNA library Zong Ku, and-80 ℃ frozen; Another part is coated with flat board, deposits standby for 4 ℃.

2.7.8cDNA cloned sequence is measured

Picking mono-clonal bacterium (is added with the substratum 1ml that contains Amp) in the hole in 96 well culture plates at random, and the sticking paper of plastics is sealed the plate mouth, 37 ℃ of concussion overnight incubation in shaking table.Get glycerine that 100 μ l bacterium liquid add 100 μ l 50% next day and in 96 porocyte culture plates, protect kind, two parts of every plates.Portion is used for order-checking, and-20 ℃ of preservations of another part are standby.

Utilize Vitagene 96-easy plasmid Mini-prep Kit to extract strain line cone shell poison pipe cDNA library plasmid.Operating process is carried out according to the test kit specification sheets.Agarose gel electrophoresis detects plasmid concentration, (5 '-TAATAC GAC TCA CTA TAG GGA-3 ') is sequencing primer with the T7 sequence, and the ABI PRISM Big-DyeTM Terminator v3.0Ready Reaction Cycle Sequencing Kit specification sheets that provides according to Applied Biosystems company carries out 5 ' and holds unidirectional sequencing.In the reaction system of 10 μ l, add following component: 5 * Sequencing Buffer, 2 μ l, Reaction Mix 1 μ l, plasmid template 200ng, T7 primer (3.2pmol/ μ l) 1 μ l and sterilization deionized water polishing volume, mixing reactant, put into the PCR instrument that is preheated to 96 ℃, move following response procedures: 96 ℃ of 10s, 50 ℃ of 5s, 60 ℃ of 4min, circulate 30 times, take out reaction product after being cooled to 4 ℃.Change reaction product over to 96 hole PCR purifying plates, add 70% ethanol, 90 μ l, seal up mucous membrane, shake mixing on the oscillator, the room temperature lucifuge is placed 20min, 4 ℃, 4, the centrifugal 40min of 000rpm abandons supernatant, is inverted PCR plate (being lined with paper handkerchief under the PCR plate) again, 4 ℃, the centrifugal 30sec of 300rpm remove trace ethanol.Lucifuge drying at room temperature 30min is with 10 μ l sterilization deionized water dissolving precipitation.Adopt ABI PRISM 3700 automatic sequencers of Perkin Elmer company to carry out sequencing.

2.7.9 sequential analysis and homology retrieval

Sequencing result input laboratory data base utilizes computer program to remove the contained carrier sequence of each sequencing result, the sequence of determining spliced, and with identical est sequence cluster.Then, use nucleic acid and the protein sequence database of BLASTX and BLASTN (http://www.ncbi.nlm.nih.gov/BLAST) search National Center for Biotechnology Information (NCBI) respectively, carry out sequence similarity analysis and retrieval homologous sequence.Utilize CLUSALW to carry out sequence relatively, and DNATOOLS 6.0 analytical sequence opening code-reading frames.By website http://www.cbs.dtu.dk/services/SignalP and in conjunction with document analysis predicted protein signal peptide sequence.

3) result and analysis

3.1 the extraction of strain line cone shell poison pipe RNA

With

The total RNA of strain line cone shell poison pipe that LS reagent extracts, 1% denaturing formaldehyde agarose gel electrophoresis detects, and the total RNA integrity of show sample is good.Record OD ₂₆₀=0.100, OD ₂₈₀=0.056, according to formula: total rna concentration (μ g/ml)=A ₂₆₀* 30 (extension rate) * 40, the concentration that calculates total RNA is about 120 μ g/ml; The A of total RNA sample ₂₆₀/ A ₂₈₀Ratio is 1.78, between 1.7-2.0, illustrates that the purity of total RNA is higher.

3.2cDNA synthetic

Get the total RNA of about 1 μ g and directly carry out synthetic first chain of reverse transcription, the first chain product of getting 2 μ l is with LD PCR method amplification enrichment cDNA, and half of cDNA cut digestion and the post recovery obtains double-stranded cDNA 7 μ l through enzyme.LD PCR product electrophoresis result presents smear distribution uniformly from big to small, and is big or small in the 0.3-2.0kb scope, and mainly concentrates on more than the 500bp, but do not see characteristic bright band (see figure 2).Show that the composition of strain line cone shell poison pipe is various and mRNA that transcribe is comparatively complicated.

3.3 the structure and the evaluation in strain line cone shell poison pipe cDNA library

Get cDNA1 μ l and be used for ligation, reaction system 5 μ l transform wherein 1 μ l, get 1/100 volume transformed bacteria liquid coated plate, and all the other are used to shake total storehouse bacterium colony; The dull and stereotyped average single colony number of next day numeration is 949.

By counting dull and stereotyped single colony number, calculate the obtainable cDNA library clone of the 7ul strain line cone shell poison double-stranded cDNA of pipe number and be: 949 * 100 * 7 * 5/3=1.107 * 10 ⁶

By library total plasmid pcr analysis result, show that the uniform distribution (see figure 3) of 0.3-2.0kb also appears presenting in the PCR product electrophoresis of the T7 that utilizes carrier multiple clone site two ends and SP6 primer amplification, the size of this and cDNA is coincide mutually.50 mono-clonals of picking identify that from connecting segmental special primer pcr amplification recombination fraction surpasses 95% (48/50) by carrier at random.

To fixed recon, find that most insertion fragment is on average inserted fragment length and is about the 600bp (see figure 4) greater than 250bp (22/24) after utilizing the T7 at carrier multiple clone site two ends and SP6 primer amplification.Deduct the irrelevant sequence of upstream and downstream, it is 405bp that strain line cone shell poison pipe cDNA on average inserts sub-length in the library.

Consider the length less (7-41 amino acid) of conotoxin molecule, so the cDNA insertion sequence that is comprised in the library also can be shorter relatively.Above result and analyze can show that the aspects such as subnumber, recombination fraction and insertion clip size of recombinating in the library, strain line cone shell poison pipe cDNA library that we make up all meet the quality standard in good library.

3.4 the sequencing and the distribution in strain line cone shell poison pipe cDNA library

Use the T7 primer to carry out unidirectional sequencing to 500 random cdna clones.Sequencing result is imported the database of the information biology platform of setting up in this laboratory, remove carrier sequence and uncertain fuzzy sequence by quality inspection, and the sequence of determining spliced, further the similarity cluster according to sequence is cluster, and carry out sequence homology with public database and compare, its result has tentatively shown the gene information readable sequences that is contained in the strain line cone shell poison pipe.

According to the result of quality inspection, effectively EST adds up to 429, and length distribution is seen Fig. 5.429 est sequences that obtain are gathered into 137 gene clusters (cluster), wherein, have 21 gene clusters to comprise plural est sequence (contig), and 116 gene clusters are formed (singleton) by unique est sequence.Because strain line cone shell poison pipe cDNA library is the original library of heterogenize, thus its gene cluster be distributed in the diversity that can reflect relevant mRNA to a certain extent effectively.

According to the quantity of contained EST, gene cluster is mainly divided five big classes (as shown in Figure 6):

(1) there are 2 gene clusters to comprise est sequence more than 30, account for 1.46% (2/137) of total gene number of clusters, always clone 20.74% (89/429) of number.They are two kinds of the highest genes of expression amount in the strain line cone shell poison pipe cDNA library, and two kinds of precursor sequence of conotoxin A-superfamily are gone up in comparison respectively.

(2) there are 2 gene clusters to comprise 20 to 29 est sequence, account for 1.46% (2/137) of total gene number of clusters, always clone 11.42% (49/429) of number.Precursor and strain line conotoxin κ A-SIVA precursor with strain line conotoxin ω-SVIA mutant 1 has higher similarity respectively.

(3) there are 7 gene clusters to comprise 10 to 19 est sequence, account for 5.11% (7/137) of total gene number of clusters, always clone 22.14% (95/429) of number.The precursor of strain line conotoxin ω-SVIA mutant 2, strain line conotoxin α-SII precursor, strain line conotoxin SO6 precursor, strain line conotoxin SO5 precursor, and 18S ribosome-RNA(rRNA) and 16S ribosome-RNA(rRNA) sequence are gone up in comparison respectively.

(4) there are 10 gene clusters to comprise 2 to 9 est sequence, account for 7.30% (10/137) of total gene number of clusters, always clone 6.06% (26/429) of number.They compare the precursor of strain line conotoxin ω-SVIB respectively, the picture-weaving in silk conotoxin.

(5) there are 116 gene clusters to comprise unique est sequence, promptly are called as singletons, account for 84.67% (116/137) of total gene number of clusters, always clone 27.04% (116/429) of number.

3.5 the sequence classification of strain line cone shell poison pipe cDNA

The sequence of 137 gene clusters is submitted to GenBank carries out BLAST X and BLAST N homology analysis.The result shows, has 63 gene clusters corresponding to 300 clones (account for total gene cluster 45.98%) and known functional protein gene to have homology (E value＜10 of height ^-4).Said gene bunch mainly is divided into three class (see figure 7)s.The first kind is the toxin gene class, and its member is maximum, has accounted for the over half of total gene cluster.Second class is a house-keeping gene, for example, and mitochondrial RNA(mt RNA), ribosomal protein gene or the like.Other has 129 clones and any known protein all not to have obvious similarity, lacks enough information and determines its function, is listed in the 3rd genoid.These unknown genes may be the genes of specifically expressing in the strain line cone shell poison pipe, and their diversity has disclosed the complicacy that mRNA in the strain line cone shell poison pipe transcribes the result.

Because strain line cone shell poison pipe is the tissue of altitude response, the library mainly study to as if in its expression amount, occupy the toxin protein sequence (amount to 221 clones) of dominant position, consider again in the order-checking of library and more tumor-necrosis factor glycoproteins occurs, therefore, existing order-checking quantity can be so that we have had a understanding roughly to the genetic expression overview of toxin in the strain line cone shell poison tubing.We can see that A-superfamily member conotoxin and O-superfamily member conotoxin occupy sizable ratio in the strain line conotoxin sequence from Fig. 8.Wherein, the former has 132 clones, accounts for 59.7% of toxin sequence clone sum, and the latter has 80 clones, and shared ratio is 36.2% in the toxin sequence sum.Other also has discovery (seeing Table 1) as T-superfamily, M-superfamily, S-superfamily and the conotoxin members such as Contryphan of containing a pair of disulfide linkage in the library.

Toxin sequence is mainly represented in the table 1 strain line cone shell poison pipe cDNA library

Embodiment 2: the structure of South China Sea strain line toxin polypeptide S4.3 fusion expression vector

1) plasmid and bacterial strain

Intestinal bacteria (Escherichia coli) DH5 α is preserved by this laboratory available from Invitrogen company, and its genotype is:

DH5α：supE44Δlac?U169(φ80lacZΔM15)hsdR17recA1endAl?gyrA96thi-1relA1

Escherichia coli expression host bacterium BL21 (DE3) is available from Stratagene company, and genotype is:

BL21(DE3)：F-ompT?hsdSB(r-B，m-B)dcm?galλ(DE3)

2) reagent and other materials

Restriction enzyme KpnI, NotI and T4DNA ligase enzyme are available from TaKaRa company; Taq archaeal dna polymerase, 10 * PCR Buffer and dNTP are available from ancient cooking vessel state company; Gel Extraction Kit and Plasmid Miniprep Kit are OMEGA BIOTEK company product; BCATM Protein Assay Kit is a PIERCE company product; Trans2K DNA Marker is available from TransGen company; Lower molecular weight standard protein 14,400～108,000kD and 14,300～97,200kD be the biological and TaKaRa company available from triumphant base respectively; Enteropeptidase (EK enzyme) inspires Bioisystech Co., Ltd available from Shanghai; Chromatographic grade TFA (trifluoroacetic acid) is a Sigma company product; The acillin sodium salt is North China Pharmaceutical Factory's product; Tryptone and Yeast Extract are Oxoid company product; T7promoter sequencing primer (T7).The oligonucleotide primer of goal gene is synthetic by Invitrogen company in the South China Sea strain line conotoxin polypeptide gene synthesis step; Other reagent is homemade analytical reagent.

3) experimental technique

3.1 South China Sea strain line conotoxin gene is synthetic

Obtain target gene sequences in the cDNA library that from embodiment 1, makes up, determine that through checking order its total length is 102 Nucleotide, nucleotide sequence as in the sequence table＜400〉1 shown in.According to Nucleotide quantity the cDNA sequence of goal gene is divided into 3 pairs of complementary sequences, and carries out pcr amplification as the template fundamental chain.

Primer is synthetic: the method that designs six sections primers process PCR amplifies the strain line conotoxin gene of total length, holds the restriction enzyme site that has added restriction enzyme Kpn I and Not I respectively at the 5 ' end and 3 ' of this gene.Added the recognition site of enteropeptidase Enterokinase in addition at 5 ' end, also added two terminator codons (TAA) at 3 ' end, to prevent ribosomal jump, the primer composition sequence is as follows:

(a)5’ C?GAT?GAT?GAT?GAT?AAA?CAG?AAA?GAA?CTG?GTG?CCG?AGC?AAA?ACC?3’

(b)3’ CATGG?CTA?CTA?CTA?CTA?TTT?GTC?TTT?CTT?GAC?CAC?GGC?TCG?TTT?TGG?TGG?TGG?AC?5’

(c)5’ACC?ACC?TGC?TGC?GGC?TAT?AGC?CCG?GGC?ACC?ATG?TGC?CCG?AGC?T?3’

(d)3’G?ACG?CCG?ATA?TCG?GGC?CCG?TGG?TAC?ACG?GGC?TCG?ACG?TAC?ACG?5’

(e)5’GC?ATG?TGC?ACC?AAC?ACC?TGC?CCG?CCG?CAG?TAA?TAA? GC?3’

(f)3’TGG?TTG?TGG?ACG?GGC?GGC?GTC?ATT?ATT? CGCCGG?5’

Behind synthetic and pcr amplification, obtain the Oligo segment of goal gene.

3.2 the structure of recombinant expression vector pTRX-S4.3

The pTRX-IL10 plasmid is that (the pTRX plasmid makes up and apply for Chinese patent voluntarily for the applicant, and patent name is: a kind of efficient prokaryotic expression carrier with people IL10 gene insertion pTRX plasmid; Patent No. CN00124832.4, Granted publication CN1189565) Kpn I and Not I enzyme are cut window and are made up and form, and the physical map of pTRX plasmid as shown in Figure 9.Now utilize Kpn I and Not I double digestion method that the pTRX-IL10 plasmid is carried out the linearizing preparation, excision IL10 fragment obtains linearizing pTRX carrier.Carrying out glue after the linearizing of pTRX carrier reclaims and purification process, the Oligo segment of the goal gene that step (1) is obtained is carried out phosphorylation, annealing and complementary pairing simultaneously, the good goal gene that will match then is connected with pTRX carrier after the linearizing, and changes in the bacillus coli DH 5 alpha and increase.Sequencing result is correct, illustrates that recombinant expression vector pTRX-S4.3 successfully constructs.Reaction process is as follows:

3.2.1 the pTRX-IL10 plasmid DNA of extracting is through restriction enzyme Kpn I and Not I double digestion.

The endonuclease reaction system is (30 μ L):

Plasmid DNA	2.5μL?
		10x?K?Buffer	3.0μL?
Kpn?I?	2.0μL?
		Not?I?	2.0μL?

0.1％BSA?	3.0μL?
		ddH2O?	20.0μL?

Place 37 ℃ of enzymes to cut 12hr in sample, then enzyme is cut product and carried out 1% agarose gel electrophoresis, cutting-out contains the sepharose piece of purpose nucleic acid belt, adopts the Gel Extraction Kit (Cat.No.D2500-02) of OMEGA BIOTEK company and carries out the glue reclaimer operation according to its specification sheets.The carrier pTRX of pTRX-IL10 plasmid DNA after restriction enzyme Kpn I and Not I double digestion obtain linearizing.

3.2.2 the structure of recombinant expression vector pTRX-S4.3

Ten sections 6 primers of synthetic among the step 3.2.1 (Oligo) segment is become the solution of 10 μ mol/L with the atom level water dissolution.Primer is divided into three groups of complementary pairing carries out phosphorylation and annealing respectively.

The phosphorylation system is (10 μ L):

10μM?Oligo?	1.0μL?
		10 * T4 Starch phosphorylase damping fluid	1.0μL?
10mM?ATP?	1.0μL?
		T4 polynucleotide kinase (10U/ μ L)	0.5μL?
ddH2O?	6.5μL?

Complementary pairing: the complementary Oligo fragment that phosphorylation is good adds together, and 94 ℃ of reaction 2.5min naturally cool to 45 ℃, the goal gene that obtains matching then in water-bath.

Using the T4DNA ligase enzyme will match good goal gene then is connected with the pTRX carrier.

PTRX-S4.3 ligation system is (20 μ L):

Oligo?	4.0μL?
		2×T4DNA?ligase?Buffer	10.0μL?
pTRX?	4.0μL?
		T4DNA ligase enzyme (10U/ μ L)	2.0μL?

With the sample mixing, 16 ℃ of connections are spent the night, and its construction procedures as shown in figure 10.To connect product adding DH5 α competent cell and transform, order-checking, the result is correct, and S4.3 gene clone success is described, obtains recombinant expression vector pTRX-S4.3.The correct recombinant expression vector pTRX-S4.3 of order-checking is converted into e. coli bl21 (DE3) is built into engineering strain.

Embodiment 3: the efficient prokaryotic expression of South China Sea strain line conotoxin polypeptide gene

1) recombination fusion protein is slightly proposed the acquisition of product

Engineering strain list colony inoculation after the picking previous step transforms is in Amp+LB liquid enriched medium, and 37 ℃ of shaking culture are spent the night as planting daughter bacteria.Getting kind of daughter bacteria is inoculated in the fresh Amp+LB rich medium by 1: 100 volume ratio, 37 ℃ of thermal agitation amplification culture are about 1.0 to OD600, add IPTG to final concentration be 0.2mmol/L, add 20% glucose simultaneously to final concentration 0.2%, in 18 ℃ of abduction delivering 12hr.Induce and finish back 4 ℃, 10, the centrifugal 10min of 000rpm receives bacterium, and the gained thalline washs thalline once with the PBS damping fluid (pH7.4) of precooling, and is centrifugal, uses the Lysis Buffer (50mmol/LNaH of precooling again ₂PO ₄, 300mmol/L NaCl, the 10mmol/L imidazoles, pH 8.0) resuspended with 1: 10 ratio.With 300W power, the broken bacterial cell of ultrasonic 1.5h under the ice bath.Ultrasonic end back 4 ℃ centrifugal twice: 10, the centrifugal 10min of 000rpm, 12,000 leave heart 20min, collect supernatant, what obtain recombination fusion protein slightly carries product.

2) purifying of recombination fusion protein

2.1 the affinity chromatography of recombination fusion protein

Because recombination fusion protein has 6 * His label between fusion partner and target protein, with Ni ²⁺, Cu ²⁺, Zn ²⁺Deng metal ion special binding ability is arranged.Therefore can utilize Ni ²⁺Metal ion chromatography purification fusion rotein.

Adopt the Ni-NTA Superflow filler of QIAGEN company, with Ni ²⁺Be part, the treatment process before and after the perfusion of chromatography column and the use is seen specification sheets.

Ni-NTA Superflow post blanking aperture is 60-160 μ m, and the entire operation process is kept the constant flow rate of 2.0ml/min.Lysis Buffer with 5 times of column volumes cleans the Ni-NTA Superflow affinity column of sealing up for safekeeping, remove ethanol and balance chromatography column, then, wash post to ultraviolet absorption value with Lysis Buffer and reach baseline, leave and take percolation peak sample in the process the centrifugal supernatant upper prop after ultrasonic.Use imidazole concentration to carry out the foreign protein wash-out then, use the Elution Buffer wash-out target protein of imidazole concentration at last as 150mmol/L as the Wash Buffer of 20mmol/L.Obtain the recombination fusion protein behind the preliminary purification.

2.2 the enzyme of recombination fusion protein is cut

Adopt the Sephadex G25 post of Pharmacia company to change sds sample buffer.The perfusion and the treatment process of chromatography column see specification sheets for details.The entire operation process is kept the constant flow rate of 2.0ml/min.Earlier cut Buffer balance chromatography column, then with Ni with the EK enzyme enzyme of 2 times of volumes ²⁺The purified target protein upper prop continues to use EK enzyme enzyme to cut Buffer then and carries out wash-out and collect the target protein peak.Sample is after Sephadex G25 gel permeation chromatography is enzyme cutting buffering liquid with buffer exchange, and the EK enzyme enzyme of adding 2% is cut 18h.

2.3 enzyme is cut product G50 gel permeation chromatography

Sephadex G50Fine molecular sieve chromatography specification is that (5.0cm * 100cm), use the AKTAexplorer system to carry out medium pressure chromatography, constant flow rate is 2ml/min to Pharmacia XK50.Use 50mM NH ₄HCO ₃1～1.5 column volume of balance Sephadex G50 chromatography column, last sample 50ml enzyme is cut the product sample.Set automatic collection procedure, collect uv-absorbing target protein peak.Obtain product polypeptide S4.3.

2.4 the HPLC of polypeptide S4.3 detects and purifying

Target protein after the freeze-drying is directly gone up the C18 reversed-phase column with small amount of deionized water dissolving back and is detected also purifying.Linear gradient elution, program as shown in figure 11,215nm and 280nm place dual wavelength detect, and collect a small amount of elution peak sample and prepare to carry out mass spectrometric detection, detect mass spectrum and see Figure 12.All the other samples carry out vacuum lyophilization once more and are stored in-20 ℃.Example 4:MTT method detects the effect of polypeptide S4.3 pair cell survival rate

MTT full name 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, Chinese chemistry 3-(4,5-dimethylthiazole-2)-2 by name, 5-phenylbenzene tetrazole bromine salt, trade(brand)name: tetrazolium bromide.

1) reagent and other materials

MTT concentration is 5mg/ml, takes by weighing MTT 0.5 gram, is dissolved in the phosphoric acid buffer (PBS) of 100ml or does not have in the phenol red substratum,, puts 4 ℃ and keeps in Dark Place and get final product to remove the bacterium in the solution with 0.22 μ m membrane filtration.

2) MTT purposes and principle

MTT mainly contains two purposes:

(1) medicine (also comprising other processing modes such as radiation exposure) is to the Cytotoxic mensuration of vitro culture;

(2) cell proliferation and cytoactive are measured.

Detect principle and be succinodehydrogenase in the viable cell plastosome and can make exogenous MTT be reduced to water-insoluble bluish voilet crystallization first a ceremonial jade-ladle, used in libation (Formazan) and be deposited in the cell, and dead cell does not have this function.First a ceremonial jade-ladle, used in libation in dimethyl sulfoxide (DMSO) (DMSO) the energy dissolved cell is measured its absorbance value with microplate reader at 490nm wavelength place, and in certain cell count scope, the amount that the MTT crystallization forms is directly proportional with cell count.According to the absorbance that records (OD value), judge viable cell quantity, the OD value is big more, cytoactive strong more (if survey drug toxicity, representing that then drug toxicity is more little).

3) mtt assay experimental procedure

(1) collect the logarithmic phase cell, adjust concentration of cell suspension, every hole adds 160ul, and bed board makes cell to be measured transfer density to the 1000-10000 hole;

(2) 5%CO2 is hatched 12h for 37 ℃, gets final product dosing to cell attachment, and every hole adds 40ul, establishes 3 multiple holes;

(3) 5%CO2 was hatched 24 hours for 37 ℃, and inverted microscope is observed down;

(4) every hole adds 20ulMTT solution (5mg/ml, i.e. 0.5%MTT), continues to cultivate 4h.Stop cultivating, the careful suction removed nutrient solution in the hole;

(5) every hole adds the 100ul dimethyl sulfoxide (DMSO), and crystallisate is fully dissolved.Measure the light absorption value in each hole with dual wavelength OD570nm/630nm place at enzyme-linked immunosorbent assay instrument;

(6) zeroing hole (substratum, MTT, dimethyl sulfoxide (DMSO)), control wells (the medicine dissolution medium of cell, same concentrations, nutrient solution, MTT, dimethyl sulfoxide (DMSO)) are set simultaneously

The 4 strain clones that this experiment is chosen are respectively: mammary epithelial cell system (MCF-10A), breast cancer cell line (MB-MDA-231), breast cancer cell line (MCF-7) and cervical cancer cell system (Hela).

4) interpretation of result

Figure 13 is for just to put the cellular form figure that 10 captured μ M polypeptide S4.3 of fluorescent microscope (100X) act on Hela cell 36h with Carl Zeiss-ImagerZ1, Fig. 5 a is normal Hela cell cultures 36h figure, and Fig. 5 b schemes for Hela cell cultures 36h after adding 10uM S4.3.Can find out intuitively that by Fig. 5 the experimental group cell speed of growth that adds 10 μ M polypeptide S4.3 is obviously faster than the cellular control unit that does not add polypeptide S4.3 normal growth.

Figure 14 detects cells survival rate figure for MTT, and this experiment uses the method for 1/2 doubling dilution to detect the influence of polypeptide S4.3 to four kinds of different cell strain growth situations.Setting the experimental group cell growth rate that does not add the S4.3 polypeptide is 100%, the mean value of the mean value/cellular control unit MTT absorbancy of experimental group cell growth rate=experimental group cell MTT light absorption value.As seen from the figure, the MCFl0A cell strain is [0 in polypeptide S4.3 concentration, 0.5] there is not to occur obviously promoting the phenomenon (p＞0.05) of growth during mg/ml, but in concentration is under the S4.3 polypeptide effect of 1.0mg/ml, growth rate is 136% (p＜0.01), proof is under high density, and polypeptide S4.3 has obvious promoter action to the MCF10A cell strain growth.The MB-MDA-231 cell strain is under the condition of 0.0039mg/ml in the add-on of S4.3, the effect of cell growth occurred promoting, and growth rate is 154% (p＜0.05); And when S4.3 concentration reached 0.5mg/ml, growth rate reached the maximum 185% (p＜0.01) in the experiment, illustrated that under this concentration, S4.3 has the effect of significant stimulation MB-MDA-231 cell strain growth.Equally, Hela cell strain and MCF-7 cell strain also demonstrate the effect of tangible quickening growth under the effect of S4.3 different concns.

Sequence table

＜110〉Zhongshan University

＜120〉preparation and the application of South China Sea strain line conotoxin S4.3

<160>2

<210>1

<211>105

<212>DNA

＜213〉South China Sea strain line cone shell (Conus Striatus)

<220>

<221>precursor?peptide

<222>(1)…(102)

<400>1

cag?aag?gag?ctg?gtc?cct?tcg?aaa?acc?acg?act?tgc?tgt?ggt?tat 45

Gln?Lys?Glu?Leu?Val?Pro?Ser?Lys?Thr?Thr?Thr?Cys?Cys?Gly?Tyr

1 5 10 15

agt?cca?ggg?aca?atg?tgc?cct?tct?tgc?atg?tgc?aca?aat?acc?tgt 90

Ser?Pro?Gly?Thr?Met?Cys?Pro?Ser?Cys?Met?Cys?Thr?Asn?Thr?Cys

20 25 30

ccc?ccc?caa?taa?taa 105

Pro?Pro?Gln *

<210>2

<211>33

<212>PRT

＜213〉South China Sea strain line cone shell (Conus Striatus)

<220>

<221>precursor?peptide

<222>(1)…(33)

<400>2

Gln?Lys?Glu?Leu?Val?Pro?Ser?Lys?Thr?Thr?Thr?Cys?Cys?Gly?Tyr

1 5 10 15

Ser?Pro?Gly?Thr?Met?Cys?Pro?Ser?Cys?Met?Cys?Thr?Asn?Thr?Cys

20 25 30

Pro?Pro?Gln

Claims (5)

1. one kind is separated the A-superfamily conotoxin gene that obtains from South China Sea strain line cone shell poison pipe, and its nucleotide sequence is as sequence table＜400〉in 1 shown in the sequence.

2. polypeptide S4.3 by genes encoding described in the claim 1, it is characterized in that: this amino acid sequence of polypeptide is as sequence table＜400〉shown in 2.

3. recombinant expression vector pTRX-S4.3, it is characterized in that: formed by the described gene fusion reorganization of pTRX carrier and claim 1, the IL10 fragment of wherein said pTRX carrier in Kpn I and Not I double digestion method excision pTRX-IL10 plasmid gone forward side by side and obtained after the line linearity preparation.

4. the preparation method of the described polypeptide S4.3 of claim 2, carry out according to following steps:

(1) with gene clone described in the claim 1 to prokaryotic fusion expression vector pTRX, construct recombinant expression vector pTRX-S4.3 and with its transformed into escherichia coli BL21 (DE3);

(2) e. coli bl21 (DE3) after transforming is cultivated;

(3) e. coli bl21 (DE3) after cultivating is carried out ultrasonic degradation, the lysate that comprises expression product that centrifugation is gone out carries out affinity chromatography, obtains recombination fusion protein;

(4) recombination fusion protein through EK enzyme enzyme cut, chromatography filters and the RPHPLC (reversed-phase high-performance liquid chromatography) purifying, obtains polypeptide S4.3.

5. the application of the described polypeptide S4.3 of claim 2 in preparation promoting growth of cell medicine.

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