CN102229659A - Rana nigromaculata antibacterial peptide and gene and application thereof - Google Patents

Abstract

The invention relates to a black-spotted frog antibacterial peptide and its gene and application, belonging to the technical field of biomedicine. Black-spotted frog antimicrobial peptide is a single-chain polypeptide encoded by the gene of Chinese amphibian black-spotted frog. Black-spotted frog antimicrobial peptide is a single-chain polypeptide encoded by the gene of Chinese amphibian black-spotted frog. , with an isoelectric point of 9.0, the full sequence of the antimicrobial peptide of the black-spotted frog is: valine-isoleucine-proline-isoleucine-valine-serine-glycine-leucine-leucine-serine - Serine - Leucine - Leucine - Glycine - Lysine. Its coding gene consists of 333 nucleotides, of which the mature part is 142-186 nucleotides. The artificially synthesized antibacterial peptide of the black-spotted frog has strong antibacterial effect, high safety, simple sequence and convenient synthesis, and can be used as a new type of anti-infective drug.

Description

Antimicrobial Peptides and Their Genes and Applications

technical field

The invention provides a Rana nigromaculata antibacterial peptide and its gene and application, belonging to the technical field of biomedicine.

Background technique

In Chinese traditional medicine and ethnic medicine, many amphibians are widely used as medicinal materials, such as Chinese toad ( Bufo gargarizans ), large webbed bell toad ( Bombina maxima ) and black-spotted frog ( Rana nigromaculata ). The skin and viscera of these amphibians have a wide range of pharmacological activities and clinical curative effects. It has been reported that the pharmacological activities include broad-spectrum antibacterial, antitumor, antiviral, analgesic, immune regulation, etc., which have the value of biomedical industry development. At present, there is also a major problem, that is, in traditional Chinese medicine and ethnic medicine, medicinal materials are processed and applied in a holistic manner, and the complexity of its components may cause the active ingredients of the drug to not perform better. Searching for specific active monomeric substances in traditional medicine is one of the important contents of the modernization of traditional Chinese medicine.

Abroad, the search for amphibian skin-specific pharmacologically active monomer compounds has become a hot spot for new drug discovery. For example, maganin isolated from Xenopus laevis has strong antibacterial function. It has completed two phase III clinical trials and achieved good results. Although it has not been approved by the US Food and Drug Administration for marketing, its preclinical Research is still ongoing.

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 few researches on their skin active protein peptides. Black-spotted frog is a kind of traditional Chinese medicine in my country, which is widely distributed in East Asia. Its wide distribution may be related to its strong ability to adapt to the environment. There are few reports on the antimicrobial peptide of black-spotted frog.

The full amino acid sequence of the antimicrobial peptide of the black-spotted frog of the present invention and its coding gene were compared and searched in the protein database and the gene database respectively, and no identical sequence information was found.

Contents of the invention

The object of the present invention is to provide a new antimicrobial peptide of black-spotted frog with strong antibacterial activity and its gene and application based on the prior art.

Black-spotted frog antimicrobial peptide is a single-chain polypeptide encoded by the Chinese amphibian black-spotted frog antibacterial peptide gene, with a molecular weight of 1495.85 Daltons and an isoelectric point of 9.0. The full sequence of the black-spotted frog antibacterial peptide is: valine-isoleucine Acid - Proline - Isoleucine - Valine - Serine - Glycine - Leucine - Leucine - Serine - Serine - Leucine - Leucine - Glycine - Lysine.

The antimicrobial peptide gene of the black-spotted frog was obtained by extracting total RNA from the skin of the black-spotted frog, reverse transcription, designing primers, and screening by PCR. The other amplification primer of PCR is the linker primer added in the process of reverse transcription, its sequence is 5'-TCGACGATTAGGCGGCCGACATGT-3', and the nucleotide sequence of the obtained positive single clone was determined, and the gene sequencing results showed that the gene encoding The gene of the antimicrobial peptide consists of 333 nucleotides, and the sequence from the 5' end to the 3' end is:

atgttcacca tgaagaaatc cctcttactc cttttcttcc ttggaaccat caacttatct 60

ctctgtgagc aagagagaga tgccgatgag gaagaaagaa gagatgatcc atatgaaacg 120

gatactgaag tgcaaaaacg agttatacca attgtgtcag gtctgctctc tagtttgttg 180

gggaagtagc caaaaatttt gaaactttaa aaatggaaaa ggaaatcatc tgatgtgata 240

tatcatttag ctaaatgctc aacagatgtc ttataaaaaa taaataaata tgttgcatgc 300

gaaaaaaaaa aaaaaaaaag aaaaaaaaaa aaa 333

The sequence encoding the mature antimicrobial peptide of black-spotted frog is the 145th-186th nucleotide, and the encoded amino acid sequence is: Vla Ile Pro Ile Vla Ser Gly Leu Leu Ser Ser Ser Leu Leu Gly Lys.

The present invention uses an automatic polypeptide synthesizer to synthesize the complete sequence of the antimicrobial peptide of the black-spotted frog, desalts it through HPLC reversed-phase C18 column chromatography, and uses the HPLC method to identify its purity after purification, and uses matrix-assisted laser analysis ionization time-of-flight mass spectrometry (MALDI-TOF-MS ) to determine its precise molecular weight and determine its amino acid sequence with an automatic amino acid sequencer.

The beneficial effects of the present invention are:

The amino acid structure was deduced from the antimicrobial peptide coding gene of black-spotted frog, and the antibacterial peptide of black-spotted frog synthesized by automatic peptide synthesizer has broad-spectrum antibacterial activity and has no hemolytic effect. The black-spotted frog antibacterial peptide has the characteristics of simple structure, convenient artificial synthesis, strong antibacterial activity, wide antibacterial spectrum and no obvious side effects.

Detailed ways

Further illustrate the present invention with embodiment below, but content of the present invention is not limited thereto.

Embodiment 1: the cloning of antimicrobial peptide gene of black-spotted frog

1. Extraction of total RNA from skin of black-spotted frog:

A. The living black-spotted frog was cleaned with water, put into liquid nitrogen and quick-frozen for 4 hours, the skin tissue was taken, weighed, 300mg of skin tissue was taken, and 10ml of total RNA extraction buffer (Trizol solution, product of Invitrogen, USA) was added to 20ml Homogenize in a glass homogenizer for 10 minutes.

B. Add an equal volume of phenol/chloroform solution, vortex and mix well, place at room temperature for 10 minutes, centrifuge at 12,000 rpm for 10 minutes at 4°C, and absorb the upper aqueous phase liquid.

C. Add 1/2 volume of isopropanol to the supernatant, leave it at room temperature for 10 minutes, centrifuge at 7500g for 10 minutes at 4°C, wash the precipitate once with 75% ethanol, and dry it. The sediment at the bottom of the tube is the skin of the black-spotted frog total RNA.

2. The first strand of cDNA was synthesized by reverse transcription of skin RNA of black-spotted frog

The first strand of cDNA was synthesized by reverse transcription of the skin RNA of the black-spotted frog using the Reverse Transcriptase M-MLV (RNase Hˉ) kit from Japan TAKARA Company.

A. Dissolve 500μg of total RNA in 500μl DEPC water of black-spotted frog skin and set aside.

B. Add 1μl black-spotted frog skin RNA, 1μl reverse transcription primer (5'-TCGACGATTAGGCGGCCGACATGTTTTTTTTTTTTTTCTTT-3') and 4ul DEPC water to a 0.2ml sterile RNase-free centrifuge tube, mix well, incubate at 70°C for 10 minutes, and then quickly incubate Chill on ice for 5 minutes.

C. Briefly centrifuge to gather the template RNA/primer denaturing solution at the bottom of the centrifuge tube.

D. Add 2ul 5×M-MLV buffer, 0.5ul dNTP mixture (10 mM each), 0.25ul RNase Inhibitor (40 U/μl), 0.25ul RTase M-MLV (RNase Hˉ) (200 U/μl) and 1ul of DEPC water, after incubation at 42°C for 1 hour, then at 70°C for 15 minutes, then cooling on ice, the obtained cDNA solution can be directly used for the amplification of the antimicrobial peptide coding gene of Rana chinensis.

3. Amplification of the antimicrobial peptide coding gene of the black-spotted frog

A. Preheat the PCR instrument at 95°C.

B. Mix 2 μl cDNA first strand (product in step 2), 75.5 μl deionized water, 10 μl PCR buffer, 8 μl dNTP mixture (2.5uM each), 2 μl 5'PCR primer (5'-ATGTTCACCAAAAGAAATCCAT-3' ), 2 μl 3' PCR primer (5'-TCGACGATTAGGCGGCCGACATGT-3) and 0.5 μl Escherichia coli DNA polymerase were placed in a centrifuge tube for reaction.

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

(1) Pre-denaturation

95°C 5 minutes

(2) 25 cycles:

95℃ for 30 seconds

56℃ for 30 seconds

72℃ for 30 seconds

(3) Post-amplification

72℃ for 5 minutes

D. After the cycle is over, use the DNA product purification kit from TIANGEN to extract and recover the PCR product. The steps are as follows:

(1) Mix the PCR product with an equal volume of membrane-binding buffer 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 12000 rpm for 1 minute and discard the waste in the collection tube.

(2) Add 700 μl of washing solution (containing ethanol) to the centrifugal purification column, centrifuge at 12000 rpm for 1 minute, and discard the waste liquid in the collection tube.

(3) Repeat step (2).

(4) Centrifuge at 12000 rpm for 3 minutes.

(5) Put the centrifugal purification column into a new centrifuge tube.

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

(7) Centrifuge at 12,000 rpm for 1 minute, and the substrate of the tube is the purified PCR product of the antimicrobial peptide coding gene of Rana japonica. the

4. Preparation of Escherichia coli DH5α competent cells:

(1) Pick a single DH5α colony, inoculate it in 3m1 LB medium without ampicillin, and culture it overnight at 37°C. The next day, take the above bacterial solution and inoculate it in 50m1 LB medium at a ratio of 1:100, shake at 37°C 2 hours. Bacterial cultures were harvested when the OD600 value reached 0.35.

(2) Transfer the bacteria to a 50m1 pre-cooled sterile polypropylene tube and place on ice for 10min to cool the culture.

(3) Centrifuge the culture at 4100 rpm for 10 min at 4°C, aspirate the culture medium, and invert the tube for 1 min to drain the residual medium.

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

(5) Centrifuge the resuspension at 4100 rpm for 10 min at 4°C, aspirate the supernatant, and invert the tube for 1 min to drain the residual liquid.

(6) For every 50m1 initial culture, resuspend the cell pellet with 2m1 pre-cooled 0.1mol/L CaCl ₂ solution, aliquot and set aside.

5. Connection and conversion of connection products:

(1) Add 1 μl of Takara pMD19-T simple vector, 4 μl of the PCR product of the antimicrobial peptide encoding gene of Rana spp. and 5 μl of ligase buffer mixture in a microcentrifuge tube.

(2) React at 16°C for 3 hours. the

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

(4) After heating at 42°C for 90 seconds, place in ice for 1 minute.

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

(6) Spread 200 μl on LB medium containing X-Gal, IPTG, and ampicillin, and culture at 37°C for 16 hours to form a single colony.

6. Cloning, screening and identification of antimicrobial peptide gene of black-spotted frog:

Pick the above-mentioned single colony in the LB medium containing ampicillin, and culture it with slow shaking at 37°C for 4 hours, and perform PCR amplification. After plasmid extraction of the positive clones confirmed by PCR, the nucleotide sequence was determined with the Applied Biosystems 3730A automatic nucleotide sequence analyzer in the United States. The sequencing primer is BcaBESTTM Sequencing Primer M13-47 (5' CGCCAGGGTTTTTCCCAGTCACGAC 3'), and the gene sequencing results from the 5' end to the 3' end sequence are:

atgttcacca tgaagaaatc cctcttactc cttttcttcc ttggaaccat caacttatct 60

ctctgtgagc aagagagaga tgccgatgag gaagaaagaa gagatgatcc atatgaaacg 120

gatactgaag tgcaaaaacg agttatacca attgtgtcag gtctgctctc tagtttgttg 180

gggaagtagc caaaaatttt gaaactttaa aaatggaaaa ggaaatcatc tgatgtgata 240

tatcatttag ctaaatgctc aacagatgtc ttataaaaaa taaataaata tgttgcatgc 300

gaaaaaaaaa aaaaaaaaag aaaaaaaaaa aaa 333

The nucleotide sequence length of the black-spotted frog antimicrobial peptide gene is 333 bases, sequence type: nucleic acid; chain number: single-stranded; topology: linear; sequence type: cDNA; source: black-spotted frog skin.

The sequence encoding the mature antimicrobial peptide of black-spotted frog is 142-186 nucleotides, and its amino acid sequence is: Vla Ile Pro Ile Vla Ser Gly Leu Leu Ser Ser Ser Leu Leu Gly Lys.

 

Example 2 Preparation and Pharmacological Experiment of Antimicrobial Peptide of Black-spotted Rana

1. Preparation of antimicrobial peptides from black-spotted frog:

(1) Preparation method of the antimicrobial peptide of the black-spotted frog: deduce the amino acid sequence of the antibacterial peptide of the black-spotted frog according to the gene encoding the antibacterial peptide of the black-spotted frog, synthesize the whole sequence with an automatic peptide synthesizer, and perform HPLC reversed-phase C18 column chromatography Desalting and purification.

(2) The purity of the purified black-spotted frog antimicrobial peptide was identified by high-performance liquid chromatography (HPLC), and its precise molecular weight was determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), and finally determined by an automatic amino acid sequencer Amino acid sequence structure for confirmation.

Black-spotted frog antimicrobial peptide is a single-chain polypeptide encoded by the Chinese amphibian black-spotted frog gene, with a molecular weight of 1495.85 Daltons and an isoelectric point of 9.0. The full sequence of the black-spotted frog antibacterial peptide is: valine-isoleucine - Proline - Isoleucine - Valine - Serine - Glycine - Leucine - Leucine - Serine - Serine - Leucine - Leucine - Glycine - Lysine.

the

2. Pharmacological experiment of antimicrobial peptide of black-spotted frog:

(1) Detection of antibacterial activity

The minimum inhibitory concentration (minimal inhibitory concentration, MIC) was detected by double dilution method, and the tested strains were Staphylococcus aureus, Escherichia coli and Candida albicans.

The three strains were inoculated on LB solid medium and cultured upside down in a 37°C incubator. After the colony grows, pick a single colony with an inoculation loop and transfer it to LB liquid medium, and shake it in a 37°C incubator until the logarithmic growth phase. The OD600 of the bacterial solution was detected on an ultraviolet spectrophotometer, and the bacterial solution was diluted to 2×10 ⁵ CFU/ml with liquid LB medium according to 1 OD600=1×10 ⁹ CFU/ml. Add 100 μl of LB liquid medium to each well of a sterile 96-well plate, and then add 100 μl of antimicrobial peptide samples diluted to a certain concentration to the first well and filter and sterilize through a 0.22 μm microporous membrane, mix well Take 100 μl and add it to the second well, serially doubly dilute, aspirate 100 μl from the tenth well and discard, at this point the two-fold concentration gradient sample is prepared. The concentrations in each well were 320 ug/ml, 160 ug/ml, 80 ug/ml, 40 ug/ml, 20 ug/ml, 10 ug/ml, 5 ug/ml, 2.5 ug/ml, 1.25 ug/ml and 0.625ug/ml. A negative control and a positive control were set up at the same time.

The minimum inhibitory concentration was the concentration where no microbial growth was observed with the naked eye.

(2) Detection of hemolytic activity

For human venous blood collection, mix the human venous blood and Alfred's solution at a ratio of 1:1 and place in a centrifuge tube, centrifuge at 1000 rpm for 5 min, and wash with normal saline until the supernatant is no longer red. The washed erythrocytes were diluted with physiological saline to form a suspension with a concentration of 10 ⁷ -10 ⁸ /ml. The above-mentioned diluted erythrocyte suspension was incubated with samples of different concentrations dissolved in normal saline for 30 min at 37°C, and then centrifuged at 1000 rpm for 5 min, and the supernatant was measured for absorbance at 540 nm. Normal saline was used as the negative control, and Triton X-100 was used as the positive control. The concentrations in each well were 500ug/ml, 400ug/ml, 300ug/ml, 200ug/ml, 100ug/ml and 50ug/ml. Calculate the hemolysis rate of the sample based on the measured absorbance value.

From the results of the above antibacterial and hemolytic activity experiments, it was shown that the antimicrobial peptides of the black-spotted frog had good antibacterial activity against the strains, and the minimum inhibitory concentrations (MIC) against Staphylococcus aureus, Escherichia coli and Candida albicans were respectively It is 5ug/ml, 2.5 ug/ml and 5ug/ml, and there is still no obvious hemolytic activity at the concentration of 500 ug/ml. This shows that the antimicrobial peptide of the black-spotted frog has a strong antibacterial effect, and its safety is high, so it can be used as a new type of antibacterial drug.

 

Claims (4)

1. the Rana nigromaculata antibacterial peptide is a kind of single chain polypeptide of Chinese amphibian animal Rana nigromaculata genes encoding, it is characterized in that molecular weight 1495.85 dalton, iso-electric point 9.0, the polypeptide amino acid total order is classified as: Xie Ansuan-Isoleucine-proline(Pro)-Isoleucine-Xie Ansuan-Serine-glycine-leucine-leucine-Serine-Serine-leucine-leucine-glycine-Methionin.

2. the gene nucleotide series of the described Rana nigromaculata antibacterial peptide of claim 1, it is characterized in that: cDNA is made up of 333 Nucleotide, and it from 5 ' end to 3 ' terminal sequence is:

atgttcacca?tgaagaaatc?cctcttactc?cttttcttcc?ttggaaccat?caacttatct 60

ctctgtgagc?aagagagaga?tgccgatgag?gaagaaagaa?gagatgatcc?atatgaaacg 120

gatactgaag?tgcaaaaacg?agttatacca?attgtgtcag?gtctgctctc?tagtttgttg 180

gggaagtagc?caaaaatttt?gaaactttaa?aaatggaaaa?ggaaatcatc?tgatgtgata 240

tatcatttag?ctaaatgctc?aacagatgtc?ttataaaaaa?taaataaata?tgttgcatgc 300

gaaaaaaaaa?aaaaaaaaag?aaaaaaaaaa?aaa 333。

3. Rana nigromaculata antibacterial peptide gene nucleotide sequence according to claim 2, it is characterized in that, 186 nucleotide coding Rana nigromaculatas of the 142nd – antibacterial peptide mature sequence, its aminoacid sequence is: Vla Ile Pro Ile Vla Ser Gly Leu Leu Ser Ser Leu Leu Gly Lys.

4. the application of the described Rana nigromaculata antibacterial peptide of claim 1 is characterized in that as the novel anti-infection medicine.