CN1306031C - Novel natural antibacterial peptide Hippocampusin and its preparation and application - Google Patents

Abstract

The present invention relates to a novel natural antibacterial peptide Hippocampusin, a gene hkplp which encodes the protein and application of the protein for preparing medicine used for treating infectious diseases. The present invention uses an RT-PCR method to obtain the novel gene hkplp of the antibacterial peptide through selection from the total RNA of a hippocampus kuda bleeker, and the DNA sequence of the novel gene hkplp is shown in a <400>1 sequence in a sequence table. The amino acid sequence of the protein (Hippocampusin) which is encoded by the gene is shown in a <400>2 sequence in the sequence table. The novel antibacterial peptide of the present invention has the obvious inhibiting effect for Gram <+> bacteria, Gram<-> bacteria, fungi, etc. The novel antibacterial peptide can be used for preparing medicine used for treating infectious diseases.

Description

A kind of new natural antimicrobial peptide Hippocampusin and its preparation method and application

technical field

The present invention relates to a new natural antibacterial peptide Hippocampusin, the gene hkplp encoding the protein, and the application of the protein in the preparation of drugs for treating infectious diseases. .

Background technique

The seahorse (Hippocampus) is a peculiar marine creature, belonging to the subphylum Vertebrate, the class Fish, the order Syngnathidae, and the order Syngnathidae. At present, there are 35 species of seahorses identified in the world. In my country, seahorses mainly inhabit the southeastern waters, and there are 5 common species (Zhang Zhaohui et al., 1998a), namely, H. kelloggi, H. histrix, H. kuda, Zephyr (H. trimaculatus) and Japanese seahorse (H. japonicus).

Hippocampus is a precious traditional Chinese medicinal material, which has the functions of tonifying kidney and strengthening yang, reducing swelling and dissipating stagnation, and calming the nerves. The above five kinds of hippocampus are recorded in Chinese Pharmacopoeia.

At present, domestic research on the medicinal use of hippocampus mainly focuses on species identification, component analysis, pharmacological research and clinical research. In species identification, restriction fragment length polymorphism (RFLP) analysis, DNA sequence analysis (Wu Ping, 1998), scanning electron microscopy and image quantitative analysis (Zhang Zhaohui, 1998), capillary electrophoresis analysis (Zhang Zhaohui, etc.) are generally used. , 1998) and other methods. In terms of component analysis of the hippocampus, it has been identified that the hippocampus contains stearic acid, cholesterol, cholesterol glycol, 2-hydroxy-4-methoxy-acetophenone, cholesterol, acetylcholinesterase, protease and 17 kinds of amino acids, 19 kinds of inorganic elements and so on (Huang Ping, 1982; Wang Qiang, 1998; Jia Yuhai, 1996). The most striking is the pharmacology of the hippocampus. Experiments have proved that ethanol and water extracts of hippocampus can inhibit the calcium influx of rat neurons caused by L-glutamic acid to varying degrees (Zhang Zhaohui et al., 1994), and ethanol extracts can also increase the calcium influx of normal male mice. Sperm count and sperm motility (Zhang Zhaohui et al., 1995), studies by Japanese scholars have shown that the ethanol extract of hippocampus exhibits androgen-like effects; in the study of animal crude drugs based on antioxidant activity, hippocampus showed Inhibition of lipid peroxidation and anti-fatigue effects; in addition, it was also found that the water extract and ethanol extract of the hippocampus can enhance the immune function of the body (Chen Weining et al., 1995), a peptide primary preparation isolated from the hippocampus It can also antagonize the memory-damaging effect of sodium glutamate on young mice (Yan Jiabin et al., 2002).

The magical medicinal effects and unique ornamental value of seahorses have triggered huge demand, and the natural seahorse resources have plummeted, and the breeding industry has emerged as the times require. But this year, the seahorse breeding industry has encountered many difficult problems, one of which is disease control. Due to problems such as drug residues and drug resistance, the application of antibiotics in the aquaculture industry has been strictly restricted. At present, for large-scale seahorse breeding, spraying traditional Chinese medicine has little effect on pathogenic microorganisms in water. Therefore, it is imminent to find alternative drugs to antibiotics. The development of new antimicrobial peptides is not only a research hotspot in the medical field at home and abroad, but also a promising research direction for disease prevention and control in seahorse breeding.

The invention of the natural antimicrobial peptide Hippocampusin is an innovative discovery in the species of hippocampus, which provides a good drug candidate, whether it is for the prevention and treatment of diseases in seahorse breeding, or for infectious diseases in mammals and humans prevention and treatment.

Contents of the invention

The object of the present invention is to provide a new natural antibacterial peptide Hippocampusin and the gene hkplp encoding the protein.

Another object of the present invention is to provide a production method of the protein.

The third object of the present invention is to provide the application of the protein in the preparation of drugs for treating infectious diseases.

The hippocampus antimicrobial peptide gene hkplp isolated from the hippocampus total RNA by RT-PCR in the present invention has a DNA sequence as shown in the <400>1 sequence in the sequence table.

The amino acid sequence of the protein Hippocampusin encoded by the gene of the present invention is shown in the <400>2 sequence in the sequence listing; the isoelectric point of the protein is 12, and the molecular weight is 2719 Daltons.

The protein can be expressed in a secreted form by recombinant yeast, and the expression level can reach 1mg/L.

The protein is sensitive to most common fish and human pathogenic strains, its MIC does not exceed 16 μg/ml, it kills bacteria quickly, and has less hemolysis.

The selected hippocampus of the present invention belongs to Hippocampus kuda Bleeker and is collected from Lufeng, Guangdong.

Construction of whole fish cDNA library of male hippocampus: Firstly, hippocampus tissue was homogenized and total RNA was extracted. Double-stranded cDNA was synthesized after reverse transcription into a single-strand cDNA, and the double-stranded cDNA was connected with the pcDNA3.0 vector to transform E.coli and save each recombinant clone.

The present invention obtains a clone encoding a new hippocampus antimicrobial peptide by performing large-scale random sequence determination on the above recombinant library clone, which is named Hippocampusin (its DNA sequence is shown in the <400>1 sequence in the sequence list). The new gene encodes a mature peptide of 25 amino acids (its amino acid sequence is shown in the <400>2 sequence in the sequence table), with an isoelectric point of 12 and a molecular weight of 2719 Daltons. It has a typical primary structure of antimicrobial peptides, secondary The characteristics of the structure are that it contains more basic amino acids to make the molecule positively charged, and the secondary structure is an amphipathic α-helix.

In the present invention, by designing a pair of primers, the new gene Hippocampusin encoding hippocampal antimicrobial peptide is amplified from the pcDNA3.0 vector by PCR method, cloned into the methanol yeast expression vector pPICZαA, constructed into an expression plasmid and transformed into Pichia pastoris X-33. The expression vector uses _PAOX1 as a promoter, and secretes the recombinant protein into the fermentation supernatant in the form of secreted expression. Through the exploration and optimization of conditions such as culture time, induction time, and methanol concentration, the expression level of the recombinant protein can reach 1mg/L.

The invention also explores and optimizes the purification conditions of the recombinant hippocampus antimicrobial peptide Hippocampusin, and the fermentation supernatant is purified by CMsepharose FF cation exchange column to obtain the mature recombinant hippocampus antibacterial peptide Hippocampusin with a purity of more than 95%.

The recombinant or synthetic hippocampus antimicrobial peptide Hippocampusin obtained in the present invention has biological activity. The recombinant hippocampus antimicrobial peptide Hippocampusin has obvious inhibitory effect on Gram ⁺ bacteria such as Staphylococcus aureus and Gram ^- bacteria such as Escherichia coli K12D31.

The expression plasmid pPICZαA containing the hippocampus antimicrobial peptide mature peptide coding sequence of the present invention is digested with Xho I/Not I to obtain a 75bp fragment, which is the hippocampus antimicrobial peptide Hippocampusin mature peptide coding sequence.

The replication method of the expression plasmid of the present invention: with reference to the Sambrook (Sambrook, et al.1989, Molecular cloing.Cold Spring Harbor Laboratory Press.USA) method, by CaCl ₂ methods transform the plasmid in the E.coli.DH5α bacterial strain, use ampicillin-containing LB medium with penicillin (100 μg/mL) was used to transform the bacteria, and the plasmid was extracted by alkaline method.

Description of drawings

Fig. 1 is the comparison result of hippocampus natural antimicrobial peptide Hippocampusin of the present invention and several other homologous antimicrobial peptides, wherein "*" means the same; ":" means the difference is small; "." means the difference is obvious; blank space means completely different .

Figure 2 is the result of electrophoresis of total RNA in the hippocampus.

Fig. 3 is the RT-PCR electrophoresis result of the hippocampus antimicrobial peptide hkplp gene. 1: PCR target band; 2: 100bp DNAmarker.

Fig. 4 is an enzyme digestion identification diagram of the expression plasmid pPICZαA-hkplp containing the gene hkplp. 1: 100bp DNA marker; 2: pPICZαA-hkplp was digested with Xho I/Not I double enzymes.

Fig. 5 is a schematic diagram of the α-Helix conformation that Hippocampusin can form parents predicted by Helical Wheel software.

Figure 6 is the result of the inhibition zone experiment of Hippocampusin on Staphylococcus aureus (1 is a negative control, 2 and 3 are 25 μg and 50 μg Hippocampusin respectively).

Fig. 7 is the SDS-PAGE electrophoresis picture (silver staining) of the purified antimicrobial peptide hippocampusin Tricine-Tris. M: peptide marker; 1: purified antimicrobial peptide hippocampusin.

Fig. 8 is a construction diagram of the recombinant expression plasmid pPICZαA-hkplp containing the gene hkplp.

Table 1 shows the antibacterial spectrum, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of hippocampus antimicrobial peptide Hippocampusin.

Detailed ways

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

Example 1: Extraction and RT-PCR amplification of hippocampal total RNA

Extraction of total RNA and synthesis of cDNA: The hippocampal tissue was homogenized in Trizol, and the total RNA of the hippocampus was extracted by one-step thermal phenol method, and the protein was removed by extraction. The total RNA concentration obtained was 1.04 μg/μl, and its A ₂₆₀ /A ₂₈₀ = 1.89. Two clear bands of 18s and 28s were detected by 1% agarose electrophoresis, and the ratio was >1 (see Figure 2), indicating the integrity of the total RNA good. Take 1ug of total RNA and use SMART III olignuclotide (5'-AAGCAGTGGTATCAACGCAGAGTGGCCATTATGGCCGGG-3') and CDSIII/3'PCR primers (5'-ATTCTAGAGGCCGAGGCGGCCGACATG-d(T) ₃₀ N-1N-3') to synthesize the first strand by reverse transcription to obtain 10 μl cDNA first-strand product. 2 μl of the first-strand product was amplified with 5'PCR primer (5'-AAGCAGTGGTATCAACGCAGAGT-3') and CDS III/3'PCR primer (5'-ATTCTAGAGGCCGAGGCGGCCGACATGd(T) ₃₀ N-1N-3') for second-strand amplification. After digesting and purifying the second chain with proteinase K, it was cut with Sfi I and ligated into the pcDNA3.0 library vector that had been treated with the same Sfi I digestion. The ESTs of two antimicrobial peptide genes Hippocampusin were obtained by random sequencing, and the ESTs were found to be full-length by software analysis.

Embodiment 2: Analysis of hippocampal antimicrobial peptide gene sequence

Blast homology analysis showed that the ORF of the new hippocampal antimicrobial peptide gene was 165 bp in length, encoding a precursor protein of 55 amino acid residues, including a signal peptide of 22 amino acids at the N-terminus and a Pro-region region of 8 amino acids at the C-terminus and the mature peptide of the middle 25 amino acids. The final Gly residue of the mature peptide is the amino donor for the amidation of the carboxy-terminal Arg. The compositional pattern of the precursor protein and amidation at the C-terminus are typical characteristics of the primary structure of antimicrobial peptides. The predicted isoelectric point of the mature peptide is 12 and its molecular weight is 2719 Daltons. The mature peptide, because it contains 7 basic amino acids, makes the molecule positively charged, hydrophobic amino acids account for 48%, and its secondary structure is an amphipathic α-helix (see Figure 5 for the predicted results). Structure.

Example 3: Construction of hippocampal recombinant antimicrobial peptide yeast expression plasmid

A pair of primers were synthesized according to the sequences at both ends of the hkplp gene, the upstream primer contained the Xho I cleavage site, and the downstream primer contained the Not I cleavage site.

Upstream primer (B1): CCTTAGGGCCCCTCGAGaaaaga TTT TTG GGA CTC ATT TTT CAC;

Downstream primer (B2): GCCGAGCTCTGCAGCGGCCGCtca GCC GCG ATT CCT GTG GAT CAA

Using the pcDNA3.0 plasmid containing the hkplp gene as a template and using B1 and B2 as primers for PCR amplification, a specifically amplified single band was obtained, and the product size was about 122 bp. The PCR amplified product was digested with Xho I/Not I and cloned into the methanolic yeast expression vector pPICZαA to obtain the recombinant expression vector pPICZαA-hkplp (the construction process is shown in Figure 8). The expression vector was identified by Xho I/Not I double enzyme digestion (see Figure 4 for the enzyme digestion results). The exogenous gene in the expression vector pPICZαA-hkplp was identified correctly by sequencing.

Example 4: Expression of hippocampal recombinant antimicrobial peptides

Transform pPICZαA-hkplp into Pichia pastoris strain X-33 by electroporation to obtain recombinant yeast genetically engineered bacteria. After the expression of the recombinant yeast strain was induced by methanol, the fermentation supernatant was analyzed by Tricine/Tris SDS-PAGE electrophoresis (silver staining). The results showed that the induced bacterial cells had obvious specific expression product bands, and its molecular weight was consistent with the predicted theoretical value of 2.8kD.

After exploring conditions such as medium pH value, methanol induction concentration, and induction time, the expression conditions of medium pH 6.0, methanol concentration 1%, and induction time 6 days were determined. The final expression level was 1mg protein/L in BMMY medium (1% yeast extract, 2% peptone, 100mM potassium phosphate, pH6.0, 1.34% YNB, 4×10 ^-5 % biotin, 0.5% methanol).

Embodiment 5: Purification of hippocampal recombinant antimicrobial peptide

The fermentation supernatant containing hippocampus recombinant antimicrobial peptide obtained in Example 4 was replaced with equilibration buffer (20mM PBS, pH6.3) by Sephadex G-25 column, and then loaded on CM sepharose FF cation exchange column. Collect the flow-through peak and wash the column with the aforementioned equilibration buffer until the plateau. Then, use solution A (20mM PBS, pH 7.0) and solution B (20mM PBS containing 1M NaCl, pH 7.0) for linear gradient elution, collect each elution peak, and use Tricine/Tris SDS-PAGE electrophoresis (Silver stain) analysis. The results showed that the target protein was eluted in 20mM PBS (pH7.0) containing 400mM NaCl. Finally, the recombinant hippocampus antimicrobial peptide Hippocampusin with a purity of more than 95% can be obtained, and the yield is about 100 μg/L medium (see FIG. 7 ).

Embodiment 6: hemolysis test

Leakage of hemoglobin was used to assess the magnitude of hemolysis. Wash human erythrocytes with 10mM Tris-HCl (containing 154mM NaCl) buffer at pH 7.6, resuspend erythrocytes with the aforementioned buffer containing antimicrobial peptides, incubate for 0.5 hours and then centrifuge to remove complete erythrocytes, take supernatant, and dilute Measure A ₅₄₀ . When the concentration of Hippocampusin was 4 μg/ml, no more than 5% hemolysis occurred. Compared with many other published antimicrobial peptides, it is less hemolytic, indicating that hippocampal antimicrobial peptides are less toxic to normal cells.

Embodiment 7: Antibacterial activity experiment of recombinant hippocampus antimicrobial peptide Hippocampusin

The biological activity of the recombinant hippocampus antimicrobial peptide Hippocampusin was analyzed with reference to the method of Wu and Hancock to analyze its minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against common sensitive and pathogenic strains (see Table 1 for data). Simultaneously, the inhibition zone experiment (see Figure 6) and the time-dependence (figure omitted) analysis of the bactericidal activity were also carried out. The results show that Hippocampusin is sensitive to most common fish and human pathogenic strains, and its MIC does not exceed 16 μg/ml, and it can kill more than 98% of Staphylococcus aureus within 20 minutes, indicating that Hippocampusin can be effective, Antibacterial rapidly.

Table 1. MIC ₅₀ and MBC of HKPLP strain MIC50(μg/ml) MBC (μg/ml) Staphylococcus aureus <4 8 Escherichia coli K12D31 <16 16 Morganella morganii <4 >16 Bacillus subtilis <16 32 Vibrio mermaid <16 >32 Candida albicans 32 ND Pseudomonas aeruginosa >128 ND Vibrio anguillarum >128 ND Saccharomyces cerevisiae AH109 >128 ND

ND: not detected.

Sequence Listing

<110> Sun Yat-Sen University

<120> A new natural antimicrobial peptide Hippocampusin and its production method and application

<160>2

<210>1

<211>75

<212>DNA

<213> Hippocampus kuda Bleeker

<220>

<221>mat peptide

<222>(1)...(75)

<400>1

ttt ttg gga ctc att ttt cac ggc ctg gtt cac gcc gga aag ctg atc 48

Phe Leu Gly Leu Ile Phe His Gly Leu Val His Ala Gly Lys Leu Ile

1 5 10 15

cac ggg ttg atc cac agg aat cgc ggc 75

His Gly Leu Ile His Arg Asn Arg Gly

20 25

<210>2

<211>25

<212>PRT

<213> Hippocampus kuda Bleeker

<220>

<221>mat peptide

<222>(1)...(25)

<400>2

Phe Leu Gly Leu Ile Phe His Gly Leu Val His Ala Gly Lys Leu Ile

1 5 10 15

His Gly Leu Ile His Arg Asn Arg Gly

20 25

Claims (4)

1. separate the hippocampus antibacterial peptide gene hkplp that obtains from the total RNA of big hippocampus, its dna sequence dna is as follows: ttt ttg ggactc att ttt cac ggc ctg gtt cac gcc gga aag ctg atc cac ggg ttg atc cac agg aat cgc ggc.

2. the hippocampus antibacterial peptide Hippocampusin of the described genes encoding of claim 1, its aminoacid sequence is: Phe Leu GlyLeu Ile Phe His Gly Leu Val HisAla Gly Lys Leu Ile His Gly Leu Ile His Arg Asn Arg Gly.

3. the production method of the described hippocampus antibacterial peptide of claim 2 Hippocampusin, carry out according to the following steps:

(1) the described gene hkplp of claim 1 is cloned on the methanol yeast expression vector pPICZ α A, obtains recombinant expression vector pPICZ α A-hkplp;

(2) pPICZ α A-hkplp electric shock is transformed Pichi strain X-33, obtain recombination yeast gene engineering fungus;

(3) at the substratum of pH6.0, be 1% methyl alcohol with concentration, above-mentioned recombination yeast gene engineering fungus was carried out abduction delivering 6 days;

(4) fermented supernatant fluid can obtain purity at the reorganization of the maturation more than 95% hippocampus antibacterial peptide Hippocampusin through CM sepharose FF cationic exchange column purification.

4. the application of the described hippocampus antibacterial peptide of claim 2 in preparation treatment infectious disease medicament.

Images