CN100351271C - Active polypeptide of brown-spotted torrential frog, its gene and application in drug preparation - Google Patents

Abstract

The invention relates to an active polypeptide and its gene and its application in pharmacy, belonging to the field of biomedicine. The active polypeptide is a single-chain polypeptide encoded by the Chinese amphibian brown-spotted frog gene, with a molecular weight of 2664.36 Daltons and an isoelectric point of 9.70. The primary structure of the complete sequence of the polypeptide is: Phe Leu Pro Leu Ala ValSer Leu Ala Ala Asn Phe Leu Pro Lys Leu Phe Cys Lys Ile Thr Lys Lys Cys-AMIDATION. The gene encoding the active polypeptide of the brown-spotted frog consists of 448 nucleotides, wherein the nucleotides 247-318 encode the active polypeptide of the mature brown-spotted frog. The artificially synthesized active polypeptide of the brown-spotted frog has a significant effect of inhibiting the growth of bacteria and fungi, and can be used as a therapeutic drug for preparing pathogenic microorganism infection diseases.

Description

The active polypeptide of the brown-spotted frog and its gene and its application in pharmacy

Technical field:

The invention relates to an active polypeptide of Amolops loloensis and its gene and application in pharmacy, belonging to the field of biomedicine.

Background technique:

Since the invention of antibiotics, humans have made great achievements in the control and treatment of microbial infection diseases. However, with the continuous use of "traditional antibiotics", many new problems continue to arise. At present, microbial resistance has become a major problem in the treatment of clinical microbial infection diseases, so that some pathogenic bacteria have no first-line drugs for clinical treatment. Vancomycin-resistant staphylococci, enterococci and other Gram-negative infectious diseases are currently clinical problems worldwide. Three types of bacteria that mainly cause meningitis have also emerged clinically with strong drug resistance. Penicillin-resistant , chloramphenicol, meningococci, pneumococci, and pneumococci resistant to neocephalosporin antibiotics are also widely present. Therefore, the development of a new class of antibiotics has become a top priority and an international hotspot. Studies have found that peptide antibiotics have broad-spectrum antibacterial activity, and at the same time have incomparable advantages over "traditional antibiotics": such as rapid and broad-spectrum killing of microorganisms (including current clinical drug-resistant bacteria) at the minimum effective concentration; It also has inhibitory effect; it will not induce the production of drug-resistant strains; it is effective for both local infection and systemic infection, and it is expected to become a new generation of antibacterial agents, and its development is currently receiving extensive attention.

Many amphibians are widely used in traditional Chinese medicine and ethnic medicine in China, such as Chinese toad (Bufo gargarizans), large webbed bell toad (Bombina maxima), black-spotted frog (pelophylax nigromaculata), marsh frog (Hylarana guentheri) and marsh frog ( Euphlyctis limnocharis) and so on. The skin and viscera of these amphibians have a wide range of pharmacological activities and clinical efficacy. Pharmacological activities have been reported to include broad-spectrum antimicrobial effects, antitumor effects, analgesia, local anesthesia, immune regulation, and cardiovascular system effects. In foreign countries, the search for specific pharmacologically active monomeric compounds in amphibian skin has become a hot spot in the invention of new drugs. According to domestic and foreign literature reports, different active polypeptides have been isolated from various biological sources, and some have entered clinical treatment. For example, the active polypeptide Magainin obtained from the skin secretion of Xenopus laevis has a broad-spectrum antimicrobial effect and anti-tumor activity. It has been approved as a broad-spectrum antibacterial drug in the United States, and its genetic engineering product has entered Phase III clinical trials; Intrabiotics The active peptide IB-367 produced by the company has been approved for the first phase of clinical trials in the treatment of oral ulcers caused by various bacterial infections in cancer patients; the clinical trial of the active peptide nisin in the treatment of gastric ulcers in cooperation between Applied Microbiology and Astra has also achieved good results Efficacy.

The application of amphibian drugs has a long history in my country, but the research on their active ingredients and pharmacological properties mainly focuses on small organic molecules such as alkaloids, and there are not many studies on their skin active peptides. The brown-spotted turbulent frog (Amolops loloensis) is mainly distributed in Sichuan and Yunnan provinces, and is one of the characteristic resource animals in my country.

The inventor searched and compared the amino acid structure of the complete sequence of the active polypeptide of the brown-spotted frog of the present invention through protein databases, and found no identical polypeptides. The inventor searched and compared the gene encoding the active polypeptide of the brown-spotted frog of the present invention through a gene database, and found no identical genes.

Invention content:

The purpose of the present invention is to provide a new broad-spectrum anti-microbial (including Gram-negative, positive bacteria, fungi) active polypeptide and its gene and application in pharmacy.

In order to realize the purpose of the present invention, the present invention provides following technical scheme:

Brown dot frog active polypeptide:

Brown-spotted frog active polypeptide is a single-chain polypeptide encoded by the Chinese amphibian brown-spotted frog active polypeptide gene, with a molecular weight of 2664.36 Daltons and an isoelectric point of 9.70. The primary structure of the complete sequence of the polypeptide is: Phe Leu ProLeu Ala Val Ser Leu Ala Ala Asn Phe Leu Pro Lys Leu Phe Cys Lys Ile Thr Lys Lys Cys-AMIDATION.

The cloning of the active polypeptide gene of the brown-spotted frog includes:

Extraction of total RNA from the skin of Rana japonica, purification of mRNA, reverse transcription of mRNA and construction of cDNA library, design of primers, and screening of active polypeptide genes of Rana japonica by PCR. The length of the amplification primer is 25 nucleotides, and its sequence is 5'ATAAGACATCTGATGTGCATTTAGC 3'. The other PCR amplification primer is the 5'PCR Primer in the SMART ^TM cDNA Library Construction Kit of CLONTECH Company, and its sequence is 5'AAGCAGTGGTATCAACGCAGAGT 3 '. The obtained positive single clones were subjected to gene nucleotide sequence determination. The results of gene sequencing show that the gene encoding the active polypeptide of the brown-spotted frog consists of 448 nucleotides, and the sequence from the 5' end to the 3' end is:

taagcagtgg tatcaacgca gagtggccat tacggccggg ggagacctcc actgaagtct 60

tccagctctc tacattctca gcaccaactg aactacccga acccaaagat gttcaccttg 120

aagaaatccc tgttactcct tttcttcctt gggaccatca acttatctct ctgcgagcaa 180

gagagagatg ccgatgagga agaaagaaga gatgatgatg aaatggatgt tgaagtggaa 240

aaacgatttt taccacttgc ggtcagtctg gccgctaatt tcttgcccaa actattttgt 300

aaaataacca aaaaatgttg aaactctgaa aatggaattg gaaatcatct gatgttgaat 360

atcatttagc taaatgcaca tcagatgtct tataaaaaaa taaagatatc tcaaacatca 420

aaaaaaaaaa aaaaaaaaaa aaaaaaaa 448

The 247th-318th nucleotides of the polypeptide coding for the mature activity of the brown-spotted frog are as follows:

Phe Leu Pro Leu Ala Val Ser Leu Ala Ala Asn Phe Leu Pro Lys Leu

1 5 10 15

Phe Cys Lys Ile Thr Lys Lys Cys-AMIDATION.

20

The brown-spotted frog active polypeptide gene is used as an application of genetic engineering to prepare the brown-spotted frog active polypeptide.

The preparation method of the brown-spotted turbulent frog active polypeptide:

The amino acid sequence of the active polypeptide of the frog was deduced according to the gene encoding the active polypeptide of the brown-spotted frog, and its complete sequence was synthesized by an automatic peptide synthesizer. Desalted and purified by HPLC reverse phase C ₁₈ column chromatography. Then its purity was identified by HPLC, the molecular weight was determined by Fast atom bombardment mass spectrometry (FAB-MS), the isoelectric point was determined by isoelectric focusing electrophoresis, and the amino acid sequence structure was determined by an automatic amino acid sequencer.

The brown-spotted frog active polypeptide has a significant effect of inhibiting the growth of bacteria and fungi, and can be used as a therapeutic drug for preparing pathogenic microorganism infection diseases.

The beneficial effects of the present invention are:

The amino acid structure is deduced from the coding gene of the active polypeptide of the brown-spotted frog, and the synthetic active polypeptide of the brown-spotted frog has a significant effect of inhibiting the growth of bacteria and fungi. The brown-spotted frog active polypeptide has the beneficial characteristics of simple structure, convenient artificial synthesis and wide antibacterial spectrum.

Detailed ways:

Embodiment one:

Cloning of the active polypeptide gene of the brown-spotted turbulent frog:

I. Extraction of total RNA from brown-spotted turbulent frog skin:

A. The living brown-spotted turbulent frog was cleaned with water, put into liquid nitrogen and quick-frozen for 4 hours, got the skin tissue, weighed, got 300mg skin tissue, added 10ml total RNA extraction buffer (Trizol solution, U.S. GIBCOBRL company product), in Homogenize in a 20ml glass homogenizer for 30 minutes.

B. Add an equal volume of phenol/chloroform solution, mix vigorously, leave at room temperature for 10 minutes, centrifuge at 12,000 rpm for 10 minutes at 4°C, and discard the precipitate.

C. Add an equal volume of isopropanol to the supernatant, leave it at room temperature for 10 minutes, centrifuge at 12,000 rpm for 10 minutes at 4°C, wash the precipitate once with 75% ethanol, and dry it in the air.

II. Purification of brown-spotted frog skin mRNA:

To isolate and purify the mRNA from the skin of the brown-spotted frog, the PolyATtract ^(R) mRNA Isolation Systems kit from PROMEGA, USA was used.

A. Dissolve 500 μg of total RNA from brown-spotted frog skin in 500 μl DEPC water, put it in a water bath at 65°C for 10 minutes, add 3 μl of Oligo (dT) probe and 13 μl of 20×SSC solution, mix well, place it at room temperature to cool, weigh For liquid A.

B. Washing of magnetic beads (SA-PMP): Flick and mix the magnetic beads until the magnetic stand absorbs for 30 seconds, discard the supernatant, add 0.5×SSC 0.3ml, let the magnetic stand absorb for 30 seconds, and finally add 0.1ml 0.5 ×SSC suspension, called B solution.

C. Add liquid A to liquid B, let stand at room temperature for 10 minutes, let the magnetic stand absorb for 30 seconds, discard the supernatant, wash 4 times with 0.1×SSC, finally discard the supernatant, add 0.1ml DEPC water to suspend, and put it on the magnetic stand Adsorb for 30 seconds, transfer the supernatant to a new test tube, then add 0.15ml DEPC water to re-suspend, absorb on the magnetic stand for 30 seconds, transfer the supernatant to the above test tube, and the supernatant contains purified brown-spotted frog skin mRNA.

D. Add 1/10 volume of 3M sodium acetate, pH 5.2, equal volume of isopropanol, place at -70°C for 30 minutes, centrifuge at 12000rpm at 4°C for 10 minutes, discard the supernatant, and dissolve the precipitate in 10μl DEPC water.

III. Construction of the cDNA library of the brown-spotted frog skin: the plasmid cDNA library construction kit of Creator ^TM SMART ^TM cDNA Library Construction Kit from CLONTECH Company was used.

A. cDNA first-strand synthesis (mRNA reverse transcription):

1. Add 1 μl brown-spotted frog skin mRNA, 1 μl SMARTIV oligonucleotide, 1 μl CDS III/3’ PCR primer to a 0.5 ml sterile centrifuge tube, and add 2 μl deionized water to make the total volume reach 5 μl.

2. Mix the reagents in the centrifuge tube and centrifuge briefly, and incubate at 72°C for 2 minutes.

3. Incubate the tube on ice for 2 minutes.

4. Add the following reagents to the centrifuge tube: 2.0μl 5×first-strand buffer, 1.0μl 20mM dithiothreitol, 1.0μl 10mM dNTP mixture, 1.0μl PowerScript reverse transcriptase.

5. Mix the reagents in the centrifuge tube and centrifuge briefly, and incubate at 42°C for 1 hour.

6. Place the centrifuge tube on ice to stop first-strand synthesis.

7. Take 2 μl of the synthesized cDNA first strand from the centrifuge tube for use.

B. Amplification of the Second Strand Using the Long-Term Polymerase Chain Reaction (LD-PCR) Method

1. Preheat the PCR instrument at 95°C.

2. Mix 2μl cDNA first strand (mRNA reverse transcription), 80μl deionized water, 10μl 10×Advantage 2PCR buffer, 2μl 50×dNTP mixture, 2μl 5’PCR primer, 2μl CDS III/3’PCR primer and 2μl large intestine Bacillus polymerase centrifuge tube for reaction.

3. Amplify in the PCR instrument according to the following procedures:

①95℃ for 20 seconds

②22 cycles:

95℃ for 5 seconds

68℃ for 6 minutes

4. After the cycle is over, extract the double-strand cDNA synthesized in the centrifuge tube.

C.PCR product is extracted and recovered with the Wizard ^(R) SV Gel and PCR Clean-UpSystem kit of PROMEGA company, and the steps are as follows:

1. Add the double-strand cDNA obtained by PCR into an equal volume of membrane-binding buffer and mix by inversion, then transfer the mixture to a centrifugal purification column, and let it stand at room temperature for 5 minutes to fully bind the DNA to the silica gel membrane. Centrifuge at 16,000g for 1 minute and discard the waste in the collection tube.

2. Add 700 μl of eluent (containing ethanol) to the centrifugal purification column, centrifuge at 16,000 g for 1 minute, and discard the waste liquid in the collection tube.

3. Repeat step 2.

4. Centrifuge at 16,000 g for 5 minutes.

5. Place the spin column into a new centrifuge tube.

6. Add 30 μl of ultrapure water and let stand at room temperature for 5 minutes.

7. Centrifuge at 16,000g for 1 minute, and the solution at the bottom of the tube is the purified double-stranded cDNA.

D. Preparation of Escherichia coli DH5α competent cells:

1. Pick a single DH5α colony, inoculate it in 3ml of LB medium without ampicillin, and culture it overnight at 37°C. The next day, take the above bacterial solution and inoculate it in 50ml of LB culture medium at a ratio of 1:100, shake at 37°C for 2 Hour. Bacterial cultures were harvested when the _OD600 value reached 0.35.

2. Transfer the bacteria to a sterile, single-use, ice-precooled 50ml polypropylene tube, place on ice for 10min, and cool the culture to 0°C.

3. Centrifuge at 4100r/min for 10min at 4°C to recover the cells.

4. Pour off the culture medium and invert the tube for 1 min to drain the last trace of the culture medium.

5. Resuspend each cell pellet in 30 ml of pre-cooled 0.1 mol/L CaCl ₂ -MgCl ₂ solution (80 mmol/L MgCl ₂ , 20 mmol/L CaCl ₂ ) for every 50 ml of initial culture medium.

6. Centrifuge at 4100r/min for 10min at 4°C to recover the cells.

7. Pour off the culture medium and invert the tube for 1 min to allow the last trace of the culture medium to flow out.

8. For every 50ml of initial culture, resuspend each cell pellet with 2ml of ice-cooled 0.1mol/L CaCl ₂ , aliquot and set aside.

E. Digestion, ligation and conversion of ligation products:

1. Add 1 μl of Takara pMD18-T carrier and 4 μl of brown-spotted frog cDNA double-strand solution in a microcentrifuge tube, the total volume is 5 μl.

2. Add 5 [mu]l (equal volume) of ligase buffer mix.

3. React at 16°C for 2 hours.

4. Add the whole amount (10μl) to 100μl DH5α competent cells, and place in ice for 30 minutes.

After heating at 5.42°C for 90 seconds, place in ice for 1 minute.

6. Add 890 μl of LB medium that has been warmed at 37°C, and culture with slow shaking at 37°C for 60 minutes.

7. Take 200 μl and spread it on the LB medium containing X-Gal, IPTG, and Amp and culture it at 37° C. for 16 hours to form a single colony.

8. Wash the colony with 5ml LB liquid medium for each LB plate, add 30% glycerol and freeze it. The constructed cDNA contained approximately 1×10 ⁶ individual clones.

IV. Cloning and Screening of Active Polypeptide Genes of Brown-spotted Frog:

The length of the amplification primer is 25 nucleotides, and its sequence is 5'ATAAGACATCTGATGTGCATTTAGC 3'. The other PCR amplification primer is the 5'PCR Primer in the SMART ^TM cDNA Library Construction Kit of CLONTECH Company, and its sequence is 5'AAGCAGTGGTATCAACGCAGAGT 3 '.

The PCR reaction was carried out under the following conditions: 94°C for 30 seconds, 52°C for 45 seconds and 72°C for 2 minutes and 30 seconds, 35 cycles.

First titrate the constructed bacterial cDNA library, and then dilute it with LB medium containing 100 μg/ml ampicillin to an appropriate bacterial concentration (approximately 5000 bacteria/ml, and 30 bacteria/ml for the first round of screening and the second round of screening respectively) ), plated in an 8×8 matrix on a 96-well culture plate (a total of 64 wells, 100 μl per well), and cultured overnight at 37°C. Bacterial culture solutions were merged in rows and columns, and 16 samples were identified by PCR, and bacterial samples from cross-positive wells entered the second round of screening.

V. Determination and results of the active polypeptide gene sequence of the brown-spotted turbulent frog:

Extract the plasmid DNA and determine the nucleotide sequence by the dideoxy method. The instrument used is the AppliedBiosystems373A automatic nucleotide sequencer in the United States. The sequencing primers are BcaBEST ^TM SequencingPrimer RV-M and BcaBEST ^TM Sequencing Primer M13-47, BcaBEST ^TM SequencingPrimer RV- M sequence: 5'GAGCGGATAACAATTTCACACAGG 3', BcaBEST ^™ Sequencing Primer M13-47: 5'CGCCAGGGTTTTCCCAGTCACGAC 3'. The gene sequencing results from the 5' end to the 3' end sequence are:

taagcagtgg tatcaacgca gagtggccat tacggccggg ggagacctcc actgaagtct 60

tccagctctc tacattctca gcaccaactg aactacccga acccaaagat gttcaccttg 120

aagaaatccc tgttactcct tttcttcctt gggaccatca acttatctct ctgcgagcaa 180

gagagagatg ccgatgagga agaaagaaga gatgatgatg aaatggatgt tgaagtggaa 240

aaacgatttt taccacttgc ggtcagtctg gccgctaatt tcttgcccaa actattttgt 300

aaaataacca aaaaatgttg aaactctgaa aatggaattg gaaatcatct gatgttgaat 360

atcatttagc taaatgcaca tcagatgtct tataaaaaaa taaagatatc tcaaacatca 420

aaaaaaaaaa aaaaaaaaaa aaaaaaaa 448

The nucleotide sequence list of the active polypeptide gene of the brown-spotted frog is as follows: the sequence length is 448 bases, the sequence type: nucleic acid, the number of chains: single-stranded, the topology: linear, the sequence type: cDNA, and the source: brown dot Turbulent Frog Skin.

The 247th-318th nucleotides of the polypeptide coding for the mature activity of the brown-spotted frog are as follows:

Phe Leu Pro Leu Ala Val Ser Leu Ala Ala Asn Phe Leu Pro Lys Leu

1 5 10 15

Phe Cys Lys Ile Thr Lys Lys Cys-AMIDATION.

20

The brown-spotted frog active polypeptide gene is used as an application of genetic engineering to prepare the brown-spotted frog active polypeptide.

Embodiment two:

Preparation of brown-spotted frog active polypeptide:

I. The preparation method of the active polypeptide of Rana variegata: infer the amino acid sequence of the active polypeptide of Rana variegata according to the gene encoding the active polypeptide of Rana variegata, and synthesize its full sequence with an automatic polypeptide synthesizer. Desalted and purified by HPLC reverse phase C ₁₈ column chromatography.

II. The molecular weight is determined by fast atom bombardment mass spectrometry (FAB-MS), with glycerol: m-nitrobenzyl alcohol: dimethyl sulfoxide (1: 1: 1, V: V: V, volume ratio) As the substrate, Cs ⁺ as the bombardment particles, the current is 1μA, and the emission voltage is 25Kv.

III. The purity of the purified brown-spotted frog active polypeptide was identified by high-performance liquid chromatography (HPLC), the molecular weight was determined by fast atom bombardment mass spectrometry, the isoelectric point was determined by isoelectric focusing electrophoresis, and the amino acid sequence structure was determined by an automatic amino acid sequencer.

Brown-spotted frog active polypeptide is a single-chain polypeptide encoded by the Chinese amphibian brown-spotted frog active polypeptide gene, with a molecular weight of 2664.36 Daltons and an isoelectric point of 9.70. The primary structure of the complete amino acid sequence of the polypeptide is: phenylalanine -Leucine-Proline-Leucine-Alanine-Valine-Serine-Leucine-Alanine-Alanine-Asparagine-Phenylalanine--Leucine-Pro Amino Acid-Lysine-Isoleucine-Threonine-Lysine-Lysine-Amidated Cysteine (Phe Leu Pro Leu Ala Val Ser Leu Ala Ala Ala Asn Phe Leu Pro Lys LeuPhe Cys Lys Ile Thr Lys Lys Cys-AMIDATION).

Embodiment three:

The effect of the active polypeptide of the brown-spotted frog on inhibiting the growth of bacteria:

The antibacterial activity was detected by the cup and saucer method, and the medium was ordinary agar medium. Inject 20ml of heated and melted medium into the plate as the bottom layer, spread it evenly in the bottom of the plate, after solidification, take another appropriate amount of medium and heat and melt, add 5ml of bacterial suspension to each plate, shake well, Spread it evenly on the bottom layer as a bacterial layer. After cooling, put 6 sterilized stainless steel cups evenly in the plate at equal distances. Add 0.1ml of the test compound solution at a concentration of 0.1-0.3mg/ml to the first steel cup, add the sample solution to the other steel cups by doubling dilution method, incubate at 37°C, and observe the size of the inhibition zone. The bacteriostatic zone above 10mm was regarded as the minimum inhibitory concentration (minimal inhibitory concentration, MIC). The bacterial strains were obtained from the First Affiliated Hospital of Kunming Medical College. The test was repeated four times and the average value was taken. The results are shown in Table 1.

Table 1, the effect of the active polypeptide of the brown-spotted frog on inhibiting the growth of bacteria: strain Minimal inhibitory concentration (ug/ml) Brown-spotted frog active polypeptide Escherichia coli ATCC25922 (Escherichia coli ATCC25922) Staphylococcus aureus ATCC2592 (Staphylococcus auneus ATCC2592) Pseudomonas aeruginosa CMCCB10104 (Pseudomonas aeruginosa CMCCB10104) 4.51810.6

Klebsiella pneumoniae Bacillus megaterium Bacillus subtilis 17.315.23.2

It can be seen from Table 1 that the synthesized active polypeptide of the brown-spotted frog has a significant effect of inhibiting the growth of bacteria.

The effect of the active polypeptide of the brown-spotted frog on inhibiting the growth of fungi:

The antifungal activity was detected by the cup and saucer method, and the medium was modified Sabousand medium. Inject 20ml of heated and melted medium into the plate as the bottom layer, and spread it evenly on the bottom of the plate. After solidification, take another appropriate amount of medium and heat it to dissolve. Add 5ml of bacterial suspension to each plate, shake well, and make it Spread evenly on the bottom layer as a bacterial layer. After cooling, put 5 sterilized stainless steel cups at equal distances in the plate. Add 0.1ml of the test compound solution at a concentration of 0.3mg/ml to the first steel cup, add the sample solution to the other steel cups by doubling dilution method, incubate at 37°C, and measure the size of the inhibition zone after 24h-48h. The minimum inhibitory concentration (MIC) was defined as the zone of inhibition above 10 mm. The bacterial strains come from the Institute of Microbiology, Yunnan University. This experiment was performed in three parallels, and the geometric mean value was taken. The results are shown in Table 2.

Table 2. The effect of the active polypeptide of the brown-spotted frog on inhibiting the growth of fungi: strain Minimal inhibitory concentration (ug/ml) Brown-spotted frog active polypeptide Candida albicans ATCC2002 (Candia albicans ATCC2002) Aspergillus flavus IFFI4015 (Aspergillus flavus sp ATCC2592) Penicillium IFFI2002 (Penicillium sp CMCCB10104) Mucor pusillus (Sacharomyces cervisiae) 18.5720.512.310.7

It can be seen from Table 2 that the synthesized active polypeptide of the brown-spotted frog has a significant effect of inhibiting the growth of fungi.

                                                                                                                                                                                                                                              

SEQUENCE LISTING

<110> Nanjing Agricultural University

<120> Brown-spotted frog active polypeptide and its gene and its application in pharmaceuticals

<130>1

<160>2

<170>PatentIn version 3.3

<210>1

<211>448

<212>DNA

<213>Amolops loloensis

<400>1

taagcagtgg tatcaacgca gagtggccat tacggccggg ggagacctcc actgaagtct 60

tccagctctc tacattctca gcaccaactg aactacccga acccaaagat gttcaccttg 120

aagaaatccc tgttactcct tttcttcctt gggaccatca acttatctct ctgcgagcaa 180

gagagagatg ccgatgagga agaaagaaga gatgatgatg aaatggatgt tgaagtggaa 240

aaacgatttt taccacttgc ggtcagtctg gccgctaatt tcttgcccaa actattttgt 300

aaaataacca aaaaatgttg aaactctgaa aatggaattg gaaatcatct gatgttgaat 360

atcatttagc taaatgcaca tcagatgtct tataaaaaaa taaagatatc tcaaacatca 420

aaaaaaaaaa aaaaaaaaaa aaaaaaaa 448

<210>2

<211>24

<212>PRT

<213>Amolops loloensis

<220>

<221> AMIDATION

<222>(24)

<400>2

Phe Leu Pro Leu Ala Val Ser Leu Ala Ala Asn Phe Leu Pro Lys Leu

1 5 10 15

Phe Cys Lys Ile Thr Lys Lys Cys-AMIDATION

20

Claims (3)

1. Brown-spotted frog active polypeptide, characterized in that the active polypeptide is a single-chain polypeptide encoded by the Chinese amphibian brown-spotted frog active polypeptide gene, with a molecular weight of 2664.36 Daltons and an isoelectric point of 9.70. The complete sequence of the polypeptide is: Phe Leu Pro Leu Ala Val Ser Leu Ala Ala Asn Phe Leu Pro Lys Leu Phe Cys LysIle Thr Lys Lys Cys-AMIDATION. 2. The active polypeptide gene of brown-spotted turbulent frog is characterized in that: the cDNA consists of 448 nucleotides, and its sequence from the 5' end to the 3' end is: taagcagtgg tatcaacgca gagtggccat tacggccggg ggagacctcc actgaagtct 60 tccagctctc tacattctca gcaccaactg aactacccga acccaaagat gttcaccttg 120 aagaaatccc tgttactcct tttcttcctt gggaccatca acttatctct ctgcgagcaa 180 gagagagatg ccgatgagga agaaagaaga gatgatgatg aaatggatgt tgaagtggaa 240 aaacgatttt taccacttgc ggtcagtctg gccgctaatt tcttgcccaa actattttgt 300 aaaataacca aaaaatgttg aaactctgaa aatggaattg gaaatcatct gatgttgaat 360 atcatttagc taaatgcaca tcagatgtct tataaaaaaa taaagatatc tcaaacatca 420 aaaaaaaaaaaaaaaaaaaaaaaaaaa 448. 3. The application of the active polypeptide of the brown-spotted frog according to claim 1, characterized in that the active polypeptide of the brown-spotted frog is used in the preparation of therapeutic drugs for diseases caused by pathogenic microorganisms.