CN1594578A - Sea anemone neurotoxin gene Sr3 and application thereof - Google Patents

Abstract

The invention relates to actinocongestin gene Sr3 and its coded protein, and also the uses of the said protein in preparation of drugs for cardiovascular disease. The invention employs RT-PCR method, extracts sequence 1 from total RNA of tentacles of sagartia rosea ( sequence 1 indicates) the protein coded by the gene( sequence 2 indicates) Recombinant actinocongestin protein in the invention has cardiac and anti-arrhythmia action, can prepare the cardiovascular disease drugs.

Description

A kind of Actinia neurotoxin gene Sr3 and application thereof

Technical field

The present invention relates to a kind of Actinia neurotoxin gene Sr3 and encoded protein thereof, and the application of this albumen in preparation treatment cardiovascular disease medicine.

Background technology

Sea anemone (anthopleura) belongs to Coelenterata (soelenterata), and Anthozoa (anthozoa) is than primary animal class in the ocean.In secular organic evolution process, in order to resist harmful animal and to catch food, the stinging capsule of sea anemone tentacle can be secreted multiple congestin, is the polypeptide toxoid basically, and humans and animals is had multiple physiological activity.Physiological function according to congestin is divided into 3 classes with it: Actinia neurotoxin, sea anemone cytolysin and sea anemone potassium-channel inhibitor.

The research to Actinia neurotoxin abroad starts from the seventies.People such as the Shibata of Hawaii, America university in 1976 from yellow sea anemone (Anthopleura xanthogrammica) separation and purification Ap-A and two kinds of toxin of Ap-B, finding has the effect that strengthens myocardial contraction; 1977, Tanaka etc. measured the aminoacid sequence of Ap-A; Subsequently, there is multiple Actinia neurotoxin to be found again, and done determined amino acid sequence, and the analysis of secondary, tertiary structure.1992, Gallagher etc. are according to the aminoacid sequence of natural Ap-B, the external gene that has synthesized coding Ap-B has carried out recombinant expressedly in intestinal bacteria, and recombinant protein and natural Ap-B are identical aspect amino acid composition, sequence, secondary structure, high-pressure liquid phase collection of illustrative plates and the biological activity.The pharmacological experiments of Actinia neurotoxin shows, this toxoid single-mindedly with the 3 site combinations of sodium-ion channel membranin, can postpone the inactivation of sodium channel.On the one hand, toxin can be used for the biological study of sodium channel as molecular probe; On the other hand, toxin produces very strong positivity contraction to cardiac muscular tissue, and, to heart rate and the not influence of blood pressure of laboratory animal; In addition, congestin also has antiarrhythmic effect, and it can prolong effective refractory period and Action Potential Duration in myocardial cell's process of repolarization, belongs to 3 class antiarrhythmic effects.Therefore, the research and development Actinia neurotoxin is used for the treatment of heart failure and irregular pulse, is a job highly significant.

Sea anemone is distributed more widely in China marine site, but output is less.The sea anemone kind of China mainly contains: yellow sea anemone (Anthopleuraxanthogrammica), taeniae flesh sea anemone (Haliplanella luciae), Japan's Anthopleura (Anhopleura Japonica), Pacific Ocean Anthopleura (Anthopleura pacifica), dark Anthopleura (Anthopleura nigrescens), Qingdao Anthopleura (Aanthopleura Qingdaoensis), magnificent yellow sea anemone (anthopleura elegantissima), must capillary refer to sea anemone (Metridium dianthus), wart sea anemone (Adamsia palliata), Red sea certain herbaceous plants with big flowers (Actinia equina) etc.At present, domestic research report to Actinia neurotoxin is less.The distinctive sea anemone resource of China is studied, found new cardiac stimulant class that application prospect is arranged and anti-arrhythmia congestin, carry out gene recombination, expression, tool has very important significance.

From all purified Actinia neurotoxin primary structures, major part is 46～49 amino acid, is called long-chain neurotoxin, and molecular weight is about 5kDa; The polypeptide of other molecular weight about 3kDa is called short-chain neurotoxin.The sea anemone long-chain neurotoxin of bibliographical information has kind more than 20 at present, and aminoacid sequence its general character more as can be seen is: have 6 Cys residues; 3 pairs of disulfide linkage; Polypeptide chain art end all is a hydrophilic amino acid.These Actinia neurotoxins can be divided into two classes again according to the source: the Type-I toxoid that derives from sea anemone section is called sea anemone peptide toxoid (Actiniid); And the Type-II toxoid that derives from thorn sea anemone section is called thorn sea anemone peptide toxoid (Stichodactylid).

Summary of the invention

The object of the present invention is to provide a kind of new Actinia neurotoxin gene Sr3 and encoded protein thereof.

Another object of the present invention is to provide the application of albumen in preparation treatment cardiovascular disease medicine of said gene.

The present invention separates the Actinia neurotoxin gene Sr3 that obtains with the method for RT-PCR from the total RNA of rose-red green sea anemone tentacle, its dna sequence dna as in the sequence table＜400〉1 sequences shown in.

The invention described above gene Sr3 encoded protein (reorganization Actinia neurotoxin, called after Sr-p3), its aminoacid sequence as in the sequence table＜400〉2 sequences shown in; This albumen iso-electric point is 7.95, and molecular weight is 4,922 dalton.

This albumen by expression vector pETTRX-Sr3 in intestinal bacteria with soluble formal representation in the born of the same parents.

Described expression vector pETTRX-Sr3 is that setting up with sulphur oxygen cyclase protein is the fusion companion body, the middle affinity labelling site of His and the efficient expression vector of protease 3 C recognition site of inserting, this system make the Sr3 gene in intestinal bacteria with soluble formal representation in the born of the same parents, expression amount can reach 150mg/L.

The selected sea anemone of the present invention belongs to rose-red green sea anemone (Sagartia rosea), picks up from Weizhou Island, the North Sea, Guangxi.

The structure in rose-red green sea anemone tentacle cDNA library: at first separate the sea anemone tentacle, extract total RNA.The Actinia neurotoxin of having found more then, their amino acid is formed and sequence has very big conservative property, conserved amino acid sequence according to the Ap-B two ends, the design degenerated primer, utilization RT-PCR method, amplification obtains the purpose band, the purpose band is connected to the T carrier, and Transformed E .coli selects recombinant clone.

The present invention has therefrom obtained the clone of 1 new rose-red green congestin of coding by to the extensive sequencing of above recombinant clone, called after Sr3 (its dna sequence dna as in the sequence table＜400〉1 sequences shown in).New 47 amino acid whose mature peptides of genes encoding (its aminoacid sequence as in the sequence table＜400〉2 sequences shown in), iso-electric point is 7.95, molecular weight is 4,922Dalton has the feature of typical Actinia neurotoxin primary structure, and intramolecularly contains 6 halfcystines, forms 3 pairs of disulfide linkage.

The present invention is by having designed a pair of primer, the new gene Sr3 of coding Anthopleura neurotoxin is come out from the amplification of T carrier with PCR method, be cloned on the prokaryotic fusion expression vector pETTRX, be built into expression plasmid and its transformed into escherichia coli BL21 (DE3).This expression vector (pETTRX-Sr3) is promotor with T7, adopt molecular chaperones TRX for merging the companion body, help recombinant protein correctly folding, with soluble formal representation, the C end of TRX has gentle sequence and 6 * His structure, is convenient to utilize immobilization metal part affinity chromatography to carry out purifying.Designed upstream primer contains protease 3 C site, so that the monomeric acquisition of foreign protein.By to incubation time, induction time, the groping and optimize of conditions such as temperature, Sr-p3 Expression of Fusion Protein amount can reach 150mg/L, and is in solvable state basically.

The present invention also gropes and has optimized the proteic purification condition of reorganization Sr-p3, and the ultrasonic degradation liquid of expression product is through Ni ²⁺Chelating Sepharose affinity chromatography obtains the higher fusion rotein of purity, and fusion rotein can obtain purity at the reorganization of the maturation more than 95% Actinia neurotoxin albumen through cutting of 3C proteolytic enzyme and further G50 gel filtration chromatography.

The reorganization Actinia neurotoxin biologically active that the present invention obtains.This reorganization Actinia neurotoxin has intensive to the isolated rat atrium and promotes contraction.To the activity experiment that carries out of reorganization Actinia neurotoxin, detect of the effect of Sr-p3 recombinant protein to the isolated rat atrium, find that rat atrial after the medication shrinks obviously to strengthen, the highlyest strengthen 296.53%.In addition, it also has 3 class antiarrhythmic effects, can prolong rats myocardial cell effective refractory period 18%.Therefore, reorganization congestin albumen of the present invention has cardiotonic and antiarrhythmic effect, can be used for preparation treatment cardiovascular disease medicine.

By the expression plasmid pETTRX-Sr3 of Sr3 Actinia neurotoxin mature polypeptide coding sequence that contains of the present invention through Kpn I/Not I double digestion, can obtain the fragment of 191bp, be Anthopleura neurotoxin Sr3 mature polypeptide coding sequence (wherein comprising protease 3 C recognition site).

The clone method of expression plasmid of the present invention:, press CaCl with reference to Sambrook (Sambrook, et al.1989, Molecular cloing.Cold Spring Harbor Labroratory Press.USA) method ₂Method transforms plasmid in E.Coli.DH5 α or BL21 (DE3) bacterial strain, with the LB substratum transform bacteria that contains penbritin (100 μ g/mL), alkaline process extracts plasmid.

Description of drawings

Fig. 1 is the comparative result of several toxin proteins of Actinia neurotoxin albumen of the present invention and Ap-A class, and wherein " * " expression is identical; ": " expression difference is less; ". " expression obvious difference; Complete difference is represented in the space.

Fig. 2 is the total RNA electrophoresis result of rose-red green sea anemone tentacle.

Fig. 3 is rose-red green Actinia neurotoxin Sr3 Gene RT-PCR electrophoresis result.1:PCR purpose band; 2:100bp DNA marker.

Fig. 4 is that the pETTRX-Sr3 expression plasmid enzyme that contains gene Sr3 is cut evaluation figure.1:100bp DNA marker; 2:pETTRX-Sr3 Kpn I/Not I double digestion.

Fig. 5 is the protein induced expression of reorganization Sr-p3, affinity chromatography electrophorogram.1: lower molecular weight standard protein marker; 2: without inductive pETTRX-Sr3 bacterial protein; 3: the pETTRX-Sr3 bacterial protein before inducing; 4: through IPTG inductive pETTRX-Sr3 bacterial protein; 5: induce the ultrasonic supernatant total protein of bacterium; 6:Ni ²⁺Affinity chromatography percolation protein peak; 7:C liquid elution peak total protein; 8:D liquid elution peak total protein.

Fig. 6 analyzes for the SDS-PAGE (Tricine) of reorganization Sr-p3 sieve chromatography.1: lower molecular weight standard protein marker; 2: the TRX-Sr-p3 fusion rotein that the process affinity chromatography obtains; The 3:TRX-Sr-p3 fusion rotein cuts through 3C proteolytic enzyme (Prescissionprotease); The reorganization Sr-p3 of 4:G-50 molecular sieve purification.

Fig. 7 is the recombinant expression plasmid pETTRX-Sr3 design of graphics that contains gene Sr3.

Embodiment

Following examples will help those of ordinary skill in the art further to understand the present invention, but not limit the present invention in any form.

Embodiment one: the extraction of the total RNA of rose-red green sea anemone tentacle and RT-PCR amplification

The extraction of total RNA is synthetic with cDNA: separate rose-red green sea anemone tentacle, adopt guanidine isothiocyanate method to extract the total RNA of tentacle, protein is removed in phenol/chloroform extracting.Obtain the total RNA of 100 μ g poison gland, its A ₂₆₀/ A ₂₈₀=2.03, detect as seen 18s and 28s two bands clearly through 1% denaturing formaldehyde gel electrophoresis, ratio＞1 (see figure 2) shows that total RNA integrity is good.

Get the total RNA of 1ug with SMART III olignuclotide (5 '-AAGCAGTGGTATCAACGCAGAGTGGCCATTATGGCCGGG-3 ') and CDSIII/3 ' PCR primer (5 '-ATTCTAGAGGCCGAGGCGGCCGACATG-d (T) ₃₀N _-1N-3 ') carries out synthetic first chain of reverse transcription, obtain 10 μ lcDNA, the first chain product.

Design of primers and RT-PCR amplification:,, design two degenerated primers with oligo primer-design software assistant analysis according to two terminal amino acid sequences of Ap-B.

Ap-B two terminal amino acid sequences: (N end) GVPCLCD-----------GWCCKK (C end);

Positive-sense strand (A1): 5 ' GG (A/C/T/G) GT (A/C/T/G) CC (A/C/T/G) TG (T/C) is T (A/C/T/G) TG (T/C) GA 3 ' (T/C);

Antisense strand (A2): 5 ' (T/C) TT (T/C) TT (A/G) CA (A/G) CACCA (A/C/T/G) CC 3 ';

Per 50 μ l PCR reaction volumes add 1 μ l cDNA strand as template, pcr amplification, and electrophoresis detection is found near the specific amplification band (see figure 3) that appearance is expected 150bp, reclaims this band.

Embodiment two: the mensuration and the analysis of reorganization Actinia neurotoxin gene sequence

The electrophoresis product that reclaims is connected to the T carrier, transforms DH5 α intestinal bacteria, select the recombinant clone order-checking.25 clones have been measured altogether, Blast homology analysis revealed, these 25 clones are the neurotoxin gene sequences, mrna length all is 141bp, each 47 amino acid whose toxin protein of encoding, wherein have 1 toxin protein to be numbered Sr-p3, it is incomplete same with the proteic aminoacid sequence of the Actinia neurotoxin of having reported, is 1 new toxin protein.

At present, the Actinia neurotoxin of having reported has kind more than 20, and amino acid number is between 46-49 (amino-acid residue that also has several toxin in addition claims short-chain neurotoxin between 27-31), and wherein 12 amino acid are conservative.Aminoacid sequence according to them can be divided into two subclass again, is representative with Ap-A, Ap-B, belongs to first subclass; With Sh I, Rp I is representative, belongs to second subclass.Between two subclass, the homology of aminoacid sequence is about 30%, and belongs to the toxin protein of same subclass, and the homology between the aminoacid sequence surpasses 60%, and this toxin protein that we found belongs to first subclass.Use the Clustalw analysis software, several toxin proteins of this toxin protein and Ap-A class are carried out multisequencing relatively, the result as shown in Figure 1.

As can be seen from Figure 1, Sr-p3 and Ap-C are closely similar, are the 21st, 22 and 47 3 amino acid whose differences between them.Show that with the Blast analytical results of Ap-C the homology of Sr-p3 and Ap-C is 95%.In addition, the sequence of it and As V is also very approaching, and the Blast analytical results shows that the homology of Sr-p3 and AsV is 91%.

In pcr amplification reaction, the mispairing of about 10-4 can appear with common Taq enzyme, because the purpose fragment is less, the cycle number of PCR is no more than 30, has reduced the wrong probability that participates in.

Embodiment three: the structure of reorganization Actinia neurotoxin expression plasmid

According to the synthetic a pair of primer of two terminal sequences of Sr3 gene, upstream primer contains Kpn I and Prescission Protease cleavage site, and downstream primer contains BamH I cleavage site.

Upstream primer (B1): 5 ' CGG GGT ACC CTG GAA GTT CTG TTC CAG GGG CCCGGG GTG

Kpn?I Precision?Protease?site

CCATGC?CTGTGC?GAC?3’；

Downstream primer (B2): 5 ' CGC GGA TCCTTA TTA CTT TTT GCA GCA CCA GCC AAT G 3 ';

BamH?I

With the pGEM-TEasy plasmid (available from Promega company) that contains the Sr3 gene is template, and B1, B2 are the primer PCR amplification, obtains the single band of specific amplified, and the product size is about 200bp.Pcr amplification product is built into BSK-Sr3 with the combination clone of Kpn I/BamHI earlier to the BSK plasmid, then with the combination clone of Kpn I/Not I to prokaryotic fusion expression vector pETTRX, obtain recombinant expression vector pETTRX-Sr3 (its building process is as shown in Figure 7).Expression vector pETTRX-Sr3 carries out double digestion through Kpn I/Not I and identifies (enzyme is cut and be the results are shown in Figure 4).Foreign gene among cloning vector BSK-Sr3 and the expression vector pETTRX-Sr3 is identified correct through order-checking.Contain Pre-scission proteolytic enzyme cutting site in the designed upstream primer, so that excision fusion partner TRX.

Embodiment four: reorganization Actinia neurotoxin Expression of Fusion Protein

With pETTRX-Sr3 transformed into escherichia coli BL21 (DE3).Genetic engineering bacterium ultrasonic degradation supernatant liquor shows that through the SDS-PAGE electrophoretic analysis thalline has tangible specifically expressing product band after inducing, molecular weight conform to theoretical value 18.6kD with software DNA TOOL5.1 prediction (Fig. 5).

Process is to incubation time, induced concentration, groping of conditions such as temperature: abduction delivering relatively behind the thalline enlarged culturing different time, the culture condition of genetic engineering bacterium is: the order bacterium colony is in 50ml ammonia Bian resistance LB substratum, 37 ℃, the 250mm overnight incubation, get in the ammonia Bian resistance LB substratum that overnight culture 20ml is inoculated in 2L, 37 ℃, 250mm is cultured to OD600=0.6 (the about 2h of enlarged culturing), add 100mM IPTG and 20% glucose to final concentration and be respectively 1mM and 0.2%, 25 ℃, centrifugal results thalline behind the 250rpm inducing culture 10h.Through SDS-PAGE electrophoretic analysis analysis revealed, Sr-p3 Expression of Fusion Protein amount accounts for more than 20% of bacterial protein with this understanding, is in solvable state (Fig. 5) basically.

Embodiment five: the purifying of reorganization Actinia neurotoxin fusion rotein and the acquisition of maturation protein

Total thalline of results is washed with TE (pH8.0), use Tis (50mM again, pH7.0) suspend, after the supersound process, the cracking supernatant liquor is through Ni2+Chelating Sepharose affinity chromatography single step purification, analyzes and the purity of thin layer scanning analysis revealed fusion rotein reaches 80% (Fig. 5) through SDS-PAGE.With 1L genetic engineering bacterium culture is material, the final fusion rotein that obtains about 120mg purifying.

For yield and the concentration that improves recombinant protein, simplify experimental procedure, shorten treatment time to recombinant protein, with Ni2+ affinity chromatography purification of recombinant proteins the time, we adopt single stage method to carry out purifying.According to the expressed foreign protein of vector plasmid pETTRX and the stronger characteristics of binding ability of Ni2+ affinity column, we have selected the elution requirement of simplifying for use: 50mMTris, 500mMNaCl, pH7.0 (A liquid); 50mMTris, 500mMNaCl, pH6.0 (B liquid); The 100mM imidazoles, 50mMTris, 500mMNaCl, pH6.0 (C liquid); The 300mM imidazoles, 50mMTris, 500mMNaCl, pH6.0 (D liquid).Reorganization Sr-p3 polypeptide is eluted (Fig. 5) at D liquid.Judge the separating effect The cream of the crop from SDS-PAGE result.

Utilize Folin-phenol method to measure the protein concentration of reorganization Sr-p3, the elutriant of results concentration more than 0.7mg/ml, add Precision Protease and cut, detect enzyme with SDS-PAGE and cut the result, can obviously see the bands of a spectrum (Fig. 6) of the ripe Sr-p3 of the representative of 5kD near.With reaction solution Ni2+Chelating Sepharose affinity chromatography column purification (condition is the same), results contain the percolation liquid of ripe Sr-p3, and be further purified (elutriant is PBS) with Sephadex G-50, just purity at the Actinia neurotoxin Protein S r-p3 (Fig. 6) that recombinates of the maturation 95% or more.

Embodiment six: acute toxicity test (LD ₅₀)

60 of the NIH mouse of body weight 20 ± 2g are divided into 6 groups at random, and 10 every group, male and female half and half, wherein 5 groups is the administration group, 1 group is control group.Intraperitoneal injection, toxicity symptoms such as in 1 hour, staring blankly appears in mouse, tic continue to observe 7 days, calculate the Lethal Dose 50 of mouse with the Bliss method.The result shows that reorganization Sr-p3 albumen is to the medium lethal dose (LD of mouse ₅₀) be 5.0mg/kg.

Embodiment seven: reorganization Sr-p3 albumen is to the cardiac stimulant experiment in isolated rat atrium

The biological activity of reorganization Sr-p3 adopts the isolated rat atrium to detect with reference to the method for Shibata.Found that reorganization Sr-p3 has the ability that rat heart muscle shrinks that strengthens.Myocardial contraction enhanced degree is calculated in order to following method: atrial contraction increases atrial contraction amplitude * 100% before percentage=(the atrial contraction amplitude before the atrial contraction amplitude-administration after the administration)/administration, reorganization Sr-p3 can obviously strengthen the contractility in isolated rat atrium, compare its effect stronger (data see Table 1) with cedilanid (desacetyllanatoside C).

Table 1 reorganization Sr-p3 strengthens the isolated rat atrium and shrinks result (n=6 x ± s)

After the dosage grouping administration isolated atria is shunk increase percentage (%)

(mol/L) 1min 3min 5min

Cedilanid (20 μ g/ml) 3.75 ± 4.12 10.85 ± 6.23 15.16 ± 10.04

High dosage Sr-p3 (2.0 * 10 ^-8) 60.46 ± 22.32 138.02 ± 42.45 168.65 ± 59.88

Middle dosage Sr-p3 (2.0 * 10 ^-9) 13.69 ± 15.55 35.84 ± 26.36 40.97 ± 26.14

Low dosage Sr-p3 (2.0 * 10 ^-10) 7.12 ± 6.74 10.33 ± 12.38 13.37 ± 12.53

Sequence table

＜110〉Zhongshan University

＜120〉a kind of Actinia neurotoxin gene Sr3 and application thereof

<160>2

<210>1

<211>141

<212>DNA

＜213〉rose-red green sea anemone (Sagartia rosea)

<220>

<221>mat?peptide

<222>(1)…(141)

<400>1

ggg?gtg?cca?tgc?ctg?tgc?gac?agt?gac?ggt?ccg?agc?gtg?aga?ggc?aac?48

Gly?Val?Pro?Cys?Leu?Cys?Asp?Ser?Asp?Gly?Pro?Ser?Val?Arg?Gly?Asn

1 5 10 15

acc?ttg?tca?gga?aca?tac?tgg?ttg?gcc?ggc?tgc?cca?tcc?ggt?tgg?cat?96

Thr?Leu?Ser?Gly?Thr?Tyr?Try?Leu?Ala?Gly?Cys?Pro?Ser?Gly?Try?His

20 25 30

aac?tgc?aag?gcc?cat?gga?ccg?acc?att?ggc?tgg?tgc?tgc?aaa?aag?141

Asn?Cys?Lys?Ala?His?Gly?Pro?Thr?Ile?Gly?Try?Cys?Cys?Lys?Lys

35 40 45

<210>2

<211>47

<212>PRT

＜213〉rose-red green sea anemone (Sagartia rosea)

<220>

<221>mat?peptide

<222>(1)…(47)

<400>2

Gly?Val?Pro?Cys?Leu?Cys?Asp?Ser?Asp?Gly?Pro?Ser?Val?Arg?Gly?Asn

1 5 10 15

Thr?Leu?Ser?Gly?Thr?Tyr?Try?Leu?Ala?Gly?Cys?Pro?Ser?Gly?Try?His

20 25 30

Asn?Cys?Lys?Ala?His?Gly?Pro?Thr?Ile?Gly?Try?Cys?Cys?Lys?Lys

35 40 45

Claims (5)

1. with the method for RT-PCR, from the total RNA of rose-red green sea anemone tentacle, separate the Actinia neurotoxin gene Sr3 that obtains, its dna sequence dna as in the sequence table＜400〉1 sequences shown in.

2. the Protein S r-p3 of the described genes encoding of claim 1, its aminoacid sequence as in the sequence table＜400〉2 sequences shown in; This albumen iso-electric point is 7.95, and molecular weight is 4,922 dalton.

3. Protein S r-p3 as claimed in claim 2, it is characterized in that this albumen by expression vector pETTRX-Sr3 in intestinal bacteria with soluble formal representation in the born of the same parents.

4. Protein S r-p3 as claimed in claim 3, it is characterized in that described expression vector pETTRX-Sr3 is that setting up with sulphur oxygen cyclase protein is the fusion companion body, the middle affinity labelling site of His and the efficient expression vector of protease 3 C recognition site of inserting, this system make the Sr3 gene in intestinal bacteria with soluble formal representation in the born of the same parents, expression amount can reach 150mg/L.

5. the application of the described albumen of claim 2,3 or 4 in preparation treatment cardiovascular disease medicine.

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