CN1793169A - Sea snail toxin variant polypeptide compound, its preparation process and application thereof - Google Patents

Abstract

The invention discloses two alpha-conch toxin variant polypeptide compounds, a gene encoding the polypeptide compound, a preparation method and application of the polypeptide. belongs to the field of biology. The peptide is obtained from the mature alpha-conch toxin peptide through amino acid substitution, modification and biological activity screening. The amino acid sequence direction from the N-terminus to the C-terminus is: GCCADPRCNYDHPEIC; prepared by chemical synthesis or genetic engineering methods, it has the ability to antagonize neuronal nicotine The activity of acetylcholine-like receptors is of great value in receptor research and drug development. It is used in the preparation of analgesics and nerve injury repair and in the treatment of cancer pain, AIDS pain, neuralgia, diabetes-related pain and rheumatism. The application in pain such as pain has a good effect.

Description

Conch toxin variant polypeptide compound, preparation method and use

technical field

The invention relates to two kinds of polypeptide compounds, their preparation method and their application as medicine, belonging to the field of biology.

Background technique

For a long time, drugs such as pethidine, methadone, and morphine have been widely used clinically to relieve intractable pain such as cancer pain, arthritis, and neuralgia. In the clinical application of this kind of drugs, there will be limitations such as increasing doses, tolerance, poor drug effect persistence and even addiction, which leads to many patients’ pain not receiving appropriate analgesic treatment, especially postoperative pain and Pain in morphine-insensitive patients. Therefore, screening new analgesic drugs with high efficacy, low tolerance, and long-lasting non-addiction has become necessary.

Currently, one class of such drug candidates is represented by cysteine-rich polypeptides. They are chemically stable, have stronger analgesic effects than morphine and have long-lasting effects, and do not have various defects of opioids. According to reports, such polypeptides with analgesic activity include scorpion mammalian toxin [Chinese patent, application number CN01128235.5], scorpion insect toxin [Rong-Jin Guan, Chun-Guang Wang, Miao Wang, et al.A depressant insect toxin with a novel analgesic effect from scorpion Buthus martensii Karsch.BBA, 2001, 1549: 9-18], snake venom [Chinese patent, application number CN02114746.9], spider toxin [Wang Xianchun, Liang Songping, Luo Zemin. Huwen bird spider toxin Solid-phase synthesis and biological activity assay of -I mutant A1Y-HWTX-I. Chinese Journal of Biochemistry and Molecular Biology, 2000, 16 (3): 357-362] and conch toxin, and conch toxin MV II A has been approved by the FDA Approved for marketing, the variant polypeptide of MV II A [Chinese patent, patent number ZL00109828.4] and conch toxin SO3 have obtained Chinese patent authorization [Chinese patent, patent number ZL99106070.9].

Due to various reasons, there is no successful report on the use of scorpion venom in the development of analgesic drugs. Snake venom has strong antigenicity due to its large molecular weight, and it will cause allergic problems when used. Spider venom is a voltage-sensitive calcium channel antagonist, and its target (N-type calcium ion channel) is located in the central nervous system. Intraductal injection [Chen Jiaqin, Chen Weihua, Deng Meichun et al. Study on analgesic effect of a new N-type voltage-sensitive calcium channel antagonist Huwen spider toxin-I on rat visceral pain model, Journal of First Military Medical University, 2005, 25( 1): 10-14], inconvenient to use. Both MV II A and SO3 belong to omega-conch toxins, and both are inconvenient to administer because the drug targets are calcium channels. Although the developers of SO3 have tried to allow the drug to pass through the blood-brain barrier, there have been no reports of successful application so far. In addition, if MV II A is used improperly, it will cause obvious motor impairment in rats, and may cause central site blockage of N-type calcium ion channels and disturbance of peripheral autonomic function [Penn R D, Paice J A. Adverse effects associated with the intrathecal administration of ziconotide, Pain, 2000, 85: 291-296].

In recent years, several conch toxin peptides that are rich in cysteine but not in the Omega family have been reported to have analgesic activity superior to that of MV II A [International Patent Application No. WO0044769]. Some of them achieve analgesic effect by interfering with neuronal nicotinic acetylcholine receptors (nAChr) in the peripheral nervous system and blocking the transmission of pain sensation in the peripheral nervous system. , fat, intraperitoneal or subcutaneous injection [Satkunanathan N, Livett B, Gayler K, et al. Alpha-conotoxin Vc1.1 alleviates neuropathic pain and accelerates functional recovery of injured neurones, Brain Research, 2005, 1059(2): 149-158 ], which greatly facilitates the application.

Natural alpha-conch toxin (Vc1.1) is a polypeptide composed of sixteen amino acids, which has the activity of antagonizing neuronal nicotinic acetylcholine receptors (nAChr). This receptor is related to the conduction of pain sensation. Appropriate inhibition of this receptor can play an analgesic effect, and can accelerate the recovery of nerve damage after nerve cells are injured [Sandall D W, Satkunanathan N, Keays DA, et al. A novel R-Conotoxin identified by gene sequencing is active in suppressing the vascular response to selective stimulation of sensory nerves in vivo, Biochemistry, 2003, 42: 6904-6911]. 5 years before the discovery of Vc1.1, Quiram et al. Another alpha-conch toxin (ImI) has been subjected to different forms of amino acid substitutions and modifications. These different molecular modifications make the binding constants of ImI and nAChr show different times of change (from 0.798 times to 1907 times). Among them, the two changes of S4A and amidation can increase the binding constant more appropriately, and they increase the binding constant of the variant ImI and nAChr by 58.4% and 78.6%, respectively, which is the same alpha-conch toxin and homologous to ImI. The property optimization of Vc1.1 provides good theoretical basis and experimental evidence.

Vc1.1 can be directly extracted from conch, but the source of raw materials is insufficient; it can also be obtained by chemical synthesis, but the cost is extremely high. Therefore, for polypeptide compounds, genetic engineering methods may be a good preparation or production scheme.

Contents of the invention

Technical Problems The purpose of the present invention is to disclose a variant polypeptide AP2.1 with stronger analgesic activity than the natural alpha-conch toxin Vc1.1 and its non-amide The molecule AP2.0 in the optimized form discloses its preparation method and its application in the preparation of analgesic and nerve injury repair medicines.

Technical solutions

Two kinds of alpha-conch toxin variant polypeptide compounds are characterized in that the amino acid sequence direction from the N-terminus to the C-terminus is:

AP2.1 GCCADPRDNYDHPEIC*

AP2.0 GCCADPRDNYDHPEIC

Among them, AP2.1 has amidated C-terminus, *—indicates amidation.

In addition, there are proteins or fusion proteins comprising the polypeptide sequence, and compounds obtained on the basis of them by means of amino acid deletion, substitution, addition, and covalent and non-covalent modification alone or in combination.

Method for preparing AP2.1 and AP2.0

1 chemical synthesis method

The preparation method of AP2.1 and AP2.0 can adopt the method of direct synthesis or genetic engineering. For direct synthesis, solid-phase chemical synthesis can be used: the peptide-resin complex is obtained through a multi-step reaction, the complex is cleaved, washed and then subjected to gel chromatography to obtain the target peptide, in Tris-HCl buffer at pH 8.0 Refolding was performed by air oxidation.

2 Genetic engineering preparation method

According to the codon preference of E. coli, design the nucleotide sequence that can be inserted in the correct reading frame between the KpnI and EcoRI restriction sites of the pET-32a(+) vector: ggtacc gac gac gac gac aag GGTTGC TGC GCT GAC CCG CGT GAC AAC TAC GAC CAC CCG GAA ATC TGC taa. gaattc . (order 5' to 3'). Wherein, the underlined part is the restriction enzyme cleavage site, the italic part is the enterokinase cleavage site, and the capital letter part is the coding region. This design enables the AP2.1 gene coding region to be inserted between the enterokinase site and the EcoRI restriction site in the correct reading frame. According to the conventional techniques in molecular cloning, synthesize the double strand of the above sequence, and make sticky ends of KpnI and EcoRI, and integrate the resulting nucleotide sequence into the pET-32a(+) vector KpnI and EcoRI restriction enzyme site . Sequencing verified the correctness of the reading frame and the correctness of the insertion, thereby obtaining the recombinant vector pET-32-AP2.1. The steps above are shown in Figure 1. The recombinant vector was transformed into Escherichia coli BL21(DE3) to obtain recombinant genetic engineering strain EBAP2.1. Under the opening and expression of the T7 strong promoter, after induction at low temperature (22°C) for a long time (24h), the expressed Trx-AP2.1 fusion protein (thioredoxin-AP2.1 fusion protein) can occupy the bacterial cell More than 50% of total protein. After the bacterium is broken, the fusion protein can be adsorbed on the metal ion affinity chromatography column, refolded and cut by enterokinase to obtain biologically active AP2.0, which can be used as an analgesic drug.

Finally, the C-terminus of the product can also be amidated to obtain AP2.1 with higher activity and stronger stability. Beneficial effect

Natural Vc1.1 and AP2.1 act on neuronal nAChr. The difference in chemical structure and properties between the two lies in (1) the C-terminus of the latter is amidated; (2) the fourth amino acid of the former is Ser, The latter is Ala; (3) the molecular weight of Vc1.1 is 1806.6, AP2.1 is 1794.7; (4) the isoelectric point of the former is 4.29, and the latter is 4.37. The sequences of natural ImI and Vc1.1 and their variants are shown below (* indicates amidation). These differences in chemical structure and basic properties determine that the latter has outstanding advantages in analgesic effect and stability.

ImI GCCSDPRDAWRC

ImI variant 1 GCCCADPRDAWRC

ImI variant 2 GCCSDPRDAWRC*

AP2.1 GCCADPRDNYDHPEIC*

AP2.0 GCCADPRDNYDHPEIC

Vc1.1 GCCSDPRDNYDHPEIC

1. The Vc1.1 variant has a stronger nAChr binding constant than the natural compound Vc1.1, but the binding constant is increased to a moderate degree

According to the background of this specification: the two variants of S4A and amidation of conch toxin ImI can increase its binding constant to nAChr by 58.4% and 78.6% compared with the natural product. In addition to the above two variants, the binding constants of other variants of ImI to nAChr either decreased, increased slightly, or were too strong (the binding constant increased by up to 1907 times) compared with natural ImI. If the decrease or increase is too small, the purpose of biological activity optimization cannot be achieved, and if it is too strong, it will completely cause nAChr to lose its normal function, and it will show great toxicity in application, so it cannot be used as a medicine.

Vc1.1 is homologous to ImI, so we optimized the properties of Vc1.1 based on the principle of "strengthening the binding, but not excessively". We prepared a variety of variants that may be suitable for pharmaceutical use, and used analgesic models and toxicology experiments to screen out the most suitable compounds. In the end, two variants were invented: AP2.1 and AP2.0, a form of the molecule without amidation at the C-terminus. They have better analgesic activity and longer analgesic duration than natural molecules. According to the method in the aforementioned reference 15 of this specification, we performed the binding constant experiment. The experimental results show that the combination constants of the two make it increase with nAChr by 80.9% and 53% respectively compared with Vc1.1. This is because amidation makes the negatively charged C-terminal carboxyl group become an uncharged amide group, thereby reducing the repulsion on the negative charge of the conch toxin and nAChr binding surface, on the contrary, it strengthens the hydrophobic interaction; the fourth amino acid Ser to The replacement of Ala is to remove the hydroxyl group in Ser, which reduces the steric hindrance, so that the combination is properly tight. However, these two changes are appropriate and mild modifications, which do not greatly change the overall electrical distribution and spatial structure of the molecule, so that these variants still present a better reversible combination. Compared with natural molecules, the molecular weight of AP2.1 in the present invention is slightly lower, the isoelectric point is basically unchanged, the molecular properties are not greatly changed, and the binding constant is appropriately increased. This just right change at the key site makes the essence of the pharmacological effect of the Vc1.1 variant still antagonize nAChr, while the medicinal effect can be significantly improved.

2. AP2.1 and AP2.0 have better analgesic activity than Vc1.1

Because the variant molecules AP2.1 and AP2.0 both appropriately increase the binding constant with nAChr, appropriately increase the activity of antagonizing nAChr, and appropriately increase the blocking effect on pain conduction, so that the variant molecules have Better analgesic activity.

Animal experiments showed that after subcutaneous injection of the same dose of Vc1.1, AP2.1 and AP2.0, the analgesic activity of AP2.1 and AP2.0 was significantly better than the former at 1h, 4h, and 16h.

3. AP2.1 and AP2.0 have better chemical stability than Vc1.1

Because the modification may appropriately reduce the charge of the molecule and enhance the hydrophobic interaction, the molecules will be agglomerated more tightly under the action of the hydrophobic force, thereby improving the chemical stability.

Experiments showed that Vc1.1 basically lost its analgesic activity (only 13.5% of the original activity) at 37°C for 48 hours, while AP2.1 and AP2.0 maintained 85.8% and 81.7% of the original activity respectively under the same experimental conditions.

4. Compared with Vc1.1, AP2.1 and AP2.0 have longer analgesic time

Due to the reasons described in 1.1 and 1.3: stronger nAChr binding activity and better chemical stability, the combination of these two factors enables the variant molecule to exert a longer analgesic duration.

Animal experiments showed that after subcutaneous injection of the same dose of Vc1.1, AP2.1 and AP2.0, the analgesic activity of AP2.1 and AP2.0 was significantly better than the former at 16 hours, and at 24 hours, Vc1. 1 has basically lost its analgesic activity, while AP2.1 and AP2.0 still maintain certain analgesic activity.

5. The preparation cost of AP2.1 and AP2.0 is low

Compared with chemical synthesis, the method of genetic engineering to prepare these two variant polypeptides has the following advantages:

Compared with the polypeptide synthesis preparation method and the general genetic engineering fermentation method, the preparation process in the present invention has fewer product purification steps because the main core operations are all performed on the affinity chromatography column. The entire purification process only needs four steps of adsorption, renaturation, cutting and elution, which greatly reduces the investment in equipment, personnel, raw materials and energy.

The core component of this process is the metal ion affinity chromatography column. This is a chromatographic column that can be regenerated repeatedly and has excellent effect, and its regeneration cost is very low.

In addition, the product obtained by using the preparation process of the present invention is detected by reverse-phase HPLC, and the peak area is integrated and calculated, and the purity of one-time purification can reach 95.4%, which shows that it has a relatively high purity. The detection wavelength of this experiment was 215 nm, and the sample volume was 100 μL. Acetonitrile was eluted from 0% to 100%. The column type is SOURCE 5RPC ST. Compared with the chemical synthesis preparation method and general genetic engineering fermentation, the product produced by this process has high purity, so the downstream purification cost can be reduced.

After economic calculation, the cost of producing alpha-conch toxin (Vc1.1) and its variant polypeptides (AP2.1 and AP2.0) by the method involved in the present invention is 1/20 of the production cost by chemical synthesis.

6. Application of Vc1.1 variants in analgesia

1) High analgesic activity

According to literature reports, the analgesic activity of conch toxin MVIIA is more than 1000 times higher than that of morphine, and lasts for a long time. Its mechanism is to exert its analgesic effect by inhibiting the N-type calcium channel in the central nervous system. The analgesic mechanism of Vc1.1 is to antagonize the activity of neuronal nicotinic acetylcholine receptor (nAChr). Appropriate inhibition of this receptor can prevent the transmission of pain signals, thereby playing an analgesic effect. And after nerve cells are injured, Vc1.1 can accelerate the recovery of nerve damage. Experiments on mouse acetic acid writhing model showed that the analgesic activity of Vc1.1 was superior to that of MVIIA, and the analgesic effect and duration of Vc1.1's variant polypeptide AP2.1 were significantly better than natural Vc1.1. Therefore, the variant polypeptide of Vc1.1 described in this patent can be used as another highly active analgesic drug.

2) Convenient administration

The drug target of conch toxin MVIIA is located in the central nervous system, so intracranial injection, subarachnoid injection or intraspinal injection is required. In terms of clinical anesthesia and analgesia, the principles of central nervous system administration are: (1) the corresponding risk knowledge must be mastered; (2) it must be a case that is ineffective by other surgical methods; (3) it must be oral or conventional treatment invalid case. This obviously limits its use considerably.

Vc1.1, AP2.1 and AP2.0 are much more convenient to use than MVIIA. Animal experiments have shown that these three compounds can exert their analgesic effects through subcutaneous injection, intravenous injection, intramuscular injection and intraperitoneal injection. This is because its drug target neuronal nAChr is located in the peripheral nervous system. Vc1.1 and its variants can block the transmission of pain sensation to the central nervous system.

In addition to the above several administration routes, the administration route referred to in the present invention is any method that can achieve curative effect, but subcutaneous injection, intravenous injection, intramuscular injection and intraperitoneal injection are the most simple and feasible.

3) As a drug, it has low tolerance and no addiction

AP2.1 and AP2.0 do not have the tolerance and addiction caused by opioid analgesics. In order to achieve a satisfactory analgesic effect, the dose of morphine must be continuously increased, resulting in the accumulation of side effects. At the same time, morphine has no obvious curative effect on some neuralgia and the pain of patients with advanced tumors. Morphine use is also known to lead to dependence. Animal experiments have shown that AP2.1 and AP2.0 are the same as their natural peptides. Under the premise of achieving the same analgesic effect, there is no need to increase the dose during the experiment. It can be seen that continuous use will not produce obvious tolerance, nor will it will be dependent on it.

4) Small side effects

AP2.1 and AP2.0 have very small side effects due to their precise drug target, specific action site, just right binding strength to the drug target, low dosage, and no need to continuously increase the dosage.

5) Wide range of uses

Since the drug targets are different from opioids, AP2.1 and AP2.0 are applicable to all opioid analgesic-sensitive and insensitive pain. The application of AP2.1 or AP2.0 in the present invention refers to their application as candidate drugs or lead drugs for relieving pain and repairing nerve damage. Specifically, it refers to the application in the treatment of pain such as cancer pain, AIDS pain, neuralgia, diabetes-related pain, and pain caused by rheumatism. It also covers the application of Trx-AP2.1 fusion protein as a drug intermediate in drug production.

6) Long effective time

Experiments have shown that the analgesic effect of AP2.1 can be maintained for 24 hours with an appropriate dose of subcutaneous injection. The effective action time of AP2.0 and Vc1.1 is also much longer than that of MVIIA.

Description of drawings

Figure 1. Construction process of pET-32a-AP2.1

The upper left is the linear vector with sticky ends cut by KpnI and EcoRI; the upper right is the single-strand synthesis, annealing, and ligation construction containing the coding sequence of the enterokinase restriction site, the AP2.1 coding region, and the sticky ends of KpnI and EcoRI sequence; the two are connected by T4 ligase to form pET-32a-AP2.1 recombinant vector

Figure 2. Comparison of the analgesic effects of AP2.1, AP2.0, and MVIIA at 1h

1h after subcutaneous injection, the difference between the normal saline group and the MVIIA group was extremely significant (p<0.01), the difference between the Vc1.1 group and the MVIIA group was extremely significant (p<0.01), and the difference between the AP2.1 group and the AP2.0 group was significant (P<0.01). 0.05)

Figure 31h, 4h, 16h moment comparison of the analgesic effect of AP2.1, AP2.0 and MVIIA

In the figure, at all times, the differences in the writhing inhibition rate between all groups and normal saline were extremely significant (p<0.01), and at 1 hour, the differences were significant in the AP2.1, AP2.0 and Vc1.1 groups (p<0.05), the difference between Vc1.1 group and MVIIA group was extremely significant (p<0.01), and the difference between AP2.1 group and AP2.0 group was extremely significant (P<0.01). At 4 hours, except the difference between the AP2.0 group and the Vc1.1 group was not significant, the differences among the other groups were extremely significant (p<0.01). At 16 hours, except for the significant difference between the AP2.1 group and the AP2.0 group (p<0.05), the differences among the other groups were extremely significant (p<0.01)

Detailed ways:

An alpha-conch toxin variant polypeptide compound, the amino acid sequence direction from the N-terminal to the C-terminal is:

AP2.1 GCCADPRDNYDHPEIC*

AP2.0 GCCADPRDNYDHPEIC

Among them, AP2.1 has amidated C-terminus, *—indicates amidation.

(1) Synthesize the gene encoding AP2.1 or AP2.0 and recombine it into the pET-32a(+) expression vector, and construct the genetic engineering bacteria for fermentation

1. According to the codon preference of Escherichia coli, design the nucleotide sequence that can be inserted between the pET-32a (+) vector KpnI and the EcoRI restriction site with the correct reading frame: ggtacc gac gac gac gac aagGGTTGCTGCGCTGACCCGCGTTGCAACTACGACCACCCGGAAATCTGC taa gaattc (order 5' to 3'). Among them, the underlined part is the restriction enzyme cleavage site, the italic part is the enterokinase cleavage site, and the uppercase part is the AP2.1 gene coding region, so that the AP2.1 gene coding region can be inserted into the intestinal tract with the correct reading frame. Between the kinase site and the EcoRI cleavage site.

2. According to the gene sequence designed above, synthesize the following single-stranded DNA (direction 5' to 3'):

S1 CGACG ACGACGACAA GGGTTGCTGC gCTGACCCGC GTTGCA

S2 ACGCGGGTCA GcGCAGCAAC CCTTGTCGTC GTCGTCGGTA C

AS1 ACTA CGACCACCCG GAAATCTGCT AAG

AS2 AATTCTTAGC AGATTTCCGG GTGGTCGTAG TTGCA

3. All of the above single-stranded DNAs were added with a phosphate group at their 5' end with polynucleotide kinase.

4. Anneal the DNA obtained in step 3 according to the following scheme: S1 and S2 are annealed; AS1 and AS2 are annealed, and the annealing reaction procedure is as follows:

94°C for 5 minutes; 3 cycles: 1 minute per cycle, 8°C per minute; 6 cycles: 1 minute per cycle, 4°C per minute; 12 cycles: 1 minute per cycle, 2°C per minute; room temperature 30min.

5. Record the annealed products of S1 and S2 as S, and the annealed products of AS1 and AS2 as AS. Concatenating S with AS results in the sequence shown in the top right of Figure 1.

6. According to the conventional double enzyme digestion technique in molecular cloning, the obtained nucleotide sequence was integrated into the pET-32a(+) vector between KpnI and EcoRI restriction sites. Sequencing verified the correctness of the reading frame and the correctness of the insertion, thereby obtaining the recombinant vector pET-32-AP2.1. The steps above are shown in Figure 1.

7. Transforming the recombinant vectors into Escherichia coli BL21(DE3) respectively to obtain recombinant genetically engineered strains.

(2) Induced expression and purification of expression products, renaturation and fusion protein cleavage

1. The engineered bacteria were grown under the selection pressure of ampicillin, and when they reached the end of logarithmic growth (OD ₆₀₀ =0.8), they were induced by adding 0.1 mM IPTG. After 24 hours of induction at 22°C, the cells were harvested.

2. The cells were enriched by centrifugation at 2000r/min and then ultrasonically disrupted. The inclusion bodies were collected, washed with 2M urea, dissolved in 8M urea and 8mM β-mercaptoethanol, and sealed at room temperature for 12 hours.

3. Bind the above reactants to Ni-NTA or Ni-IDA affinity chromatography column, followed by air-saturated refolding buffer (20mM Tris-HCl; 0.5M arginine; 50mM NaCl; pH8.0) Wash and soak for more than 24 hours.

4. Wash and soak with enterokinase digestion buffer (20 mM Tris-HCl; 2 mM CaCl ₂ ; 50 mM NaCl; pH7.4) for more than 12 hours.

5. Add the enzyme into the column according to the amount of cutting 5 mg of fusion protein per unit of enterokinase, and react at room temperature for 48 hours to carry out sufficient digestion.

6. Elute the cleaved peptide with enterokinase digestion buffer, and the purity can reach more than 95%.

7. Use serine-type carboxypeptidase to catalyze the C-terminal amidation reaction of the target peptide.

(3) The analgesic activity of AP2.1 and AP2.0 by intraperitoneal injection is superior to that of Vc1.1 and MVIIA

The analgesic activity of AP2.1 and AP2.0 was determined by the acetic acid writhing method. ICR mice weigh about 20 g, half male and half male, and randomly divided into 8 mice in each group. Inject 0.5mL/20gbw of the peptide solution at a concentration of 0.1μM intraperitoneally. One hour after administration, 0.2 mL/20 gbw of 0.8% acetic acid was injected intraperitoneally, and the number of writhing times of mice within 15 min was recorded. The average value of each group was taken, and the significance of the difference in writhing times was compared with that of the normal saline control group. The results are shown in Figure 2, the analgesic effects of AP2.1 and AP2.0 were significantly stronger than those of Vc1.1. The analgesic effect of Vc1.1 was significantly stronger than that of MVIIA.

(4) AP2.1 and AP2.0 are superior to Vc1.1 and MVIIA in duration of analgesia after one intraperitoneal injection

Similar to the analgesic drug screening model in Example 3, the analgesic effects of AP2.1, AP2.0, Vc1.1 and MVIIA were compared at 1h, 4h, and 16h respectively, and the normal saline group at 1h was used as a control to measure the writhing inhibition Rate. The results are shown in Figure 3, the analgesic effects of AP2.1 and AP2.0 at 1h and 16h were significantly stronger than those of Vc1.1. The analgesic effect of Vc1.1 was significantly stronger than that of MVIIA at 1h, 4h and 16h. At the same time, the effective action time of AP2.1 and AP2.0 was longer than that of Vc1.1, and the effective action time of AP2.1 was longer than that of AP2.0.

Claims (10)

1. alpha-sea snail toxin variant polypeptide compound is characterized in that, aminoacid sequence direction N holds the C end to be:

AP2.1:GCCADPRCNYDHPEIC* or

AP2.0：GCCADPRCNYDHPEIC

Wherein, AP2.1 has amidated C end, *-expression amidation.

On the basis of polypeptide compound as claimed in claim 1 by taking the compounds that means obtained such as aminoacid deletion, replacement, interpolation and covalency and non-covalent modification alone or in combination.

3. comprise the albumen or the fusion rotein of polypeptide according to claim 1, and on their bases the compound by taking aminoacid deletion, replacement, interpolation and covalency and non-covalent modification means to be obtained alone or in combination.

4. coding is as the gene of polypeptide compound as described in one of claim 1～3.

5. comprise the carrier as gene as described in the claim 4, it is characterized in that, this carrier can generate the described polypeptide compound of one of claim 1～3 in the consistency host cell and when the host is in suitable growth conditions following time.

6. genophore as claimed in claim 5, it is characterized in that, this carrier is with coded polypeptide " GCCSDPRCNYDHPEIC ", hold C end to arrange by direction N, and the nucleotide sequence that designs by the bacillus coli codon preference, according to and the order of sulphur hydrogen reduction albumen synchronous reading frame of (Trx albumen) gene and coding strand 5 ' to 3 ', the enteropeptidase restriction enzyme site that is binned in pET-32a (+) carrier and EcoRI site between constructed recombinant vectors pET-32a-AP2.

7. the host bacterium of claim 4 or 5 described genophores is characterized in that, this genophore is transformed into the constructed genetic engineering bacterium EBAP2.1 of bacillus coli BL21 (DE3).

8. the application of the described genophore host of claim 6 bacterium, it is characterized in that, this genetic engineering bacterium is also concentrated and acquisition bioactive AP2.0 of tool or AP2.1 through abduction delivering Trx-AP2 fusion rotein, broken thalline, metal ion affinity chromatography, renaturation, enteropeptidase cleavage of fusion proteins, wash-out.

9. preparation is characterized in that as the method for polypeptide compound as described in claim 1 or the claim 2, and the technological line by chemosynthesis prepares.

10. application or the application in treatment pain caused by cancer, AIDS slight illness, neurodynia, diabetes ache related and rheumatism cause pain medication such as pain of the described polypeptide compound of one of claim 1～3 in preparation analgesic, nerve injury repair medicine.

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