Transcript Antifungal peptide抗真菌肽

Antifungal peptide抗真菌肽
A bacterial strain isolated from a honey sample
showed antifungal activity against mold.
What is this strain?
The essence of this antifungal material?
？
芽胞杆菌BH072产生的抗真菌肽研究
The Antifungal Peptides Produced by
Bacillus BH072
2012.9
The Antifungal Peptides Produced by Bacillus BH072
outline
①
Identification and characterization of Bacillus BH072
②
The antifungal activity of Bacillus BH072
③
PCR analysis
④
Purification and characterization of this peptide
⑤
Discussion
Part 1 Identification and characterization
of Bacillus BH072
Technical route
选择适当的培养
基，培养芽孢杆菌
离心去沉淀获得培
养物的上清液
形态学检测
提取全细胞DNA作
为PCR反应的模板
抑菌试验
生理生化试验
设计合成扩增16S
rDNA的寡核苷酸引
物
用琼脂扩散法，确
定纯化抑菌物质的
抑菌谱
确定鉴定范围
PCR扩增芽孢杆菌
16S rDNA
分析抑菌物质对
热、pH、和蛋白酶
的稳定性
结合常规试验结
果，确定菌株的种
属
提交测序结果至
NCBI，Blast分析同
源性
Morphological，Biochemical and Physiological tests
注：+表示实验结果为阳性；﹣表示实验结果为阴性
pH
temperature
16S Ribosomal DNA sequence analysis
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8F
（5’-AGAGTTTGATCATGGCTCAG-3’）
1492R（5’-ACGGTTACCTTGTTACGACTT-3’）
The PCR amplification system :25µ L system,
0.2 µ L Taq enzyme ( 0.5 U/mL )
2.5 µ L 10× Buffer
1.8 µ L Mg2+
1 µ L dNTPs Mixture
1 µ L template DNA
0.5 µ L forward primer ( 10 µ mol/L )
0.5 µ L reverse primer ( 10 µ mol/L )
17.5µ L ddH2O
16S Ribosomal DNA sequence analysis
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Amplification factor:
95 ° C 3 min;
95° C 30 s,
55 °C 60 s,
30 cycles
72 ° C 60s,
72 ° C 5 min,
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4 ° C termination reaction.
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The 16S rDNA sequence of the amplified PCR product was determined.
Sequences of 1120 bp fragments showed similarity (minimum identity
98%) with the Bacillus subtilis.
Results
细菌DNA的制备
PCR引物的设计
PCR扩增
结合常规检测
枯草芽胞杆菌
琼脂糖凝胶电泳
PCR产物16S
rDNA测序
Blast分析
芽胞杆菌BH072
Part 2 The antifungal activity of
Bacillus BH072
 Antifungal
activity
agar well diffusion assay
编号
指示菌
抑制作用
A
黑曲霉
B
黄瓜炭疽菌
﹣
C
酵母菌
﹣
D
金黄色葡萄球菌
E
腐霉
++
F
青霉
﹣
G
灰霉
+
H
大肠杆菌
﹣
++
﹣
注：++表示抑菌作用显著；+表示抑菌作用一般；﹣表示无抑菌作用
2.5
2
Antifungal activity graph
1.5
吸光度A
1
抑菌圈直径/cm
0.5
0
0
-0.5
10
20
30
时间/h
40
50
60
Part 3
PCR analysis
Primer design
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iturinA gene
上游引物：5’- atgaaaatttacggagtatatatg - 3’
下游引物：5’- ttataacagctcttcatacgtt - 3’
扩增长度：675 bp
tasA gene
上游引物：5’- atgggtatgaaaaagaaattaag - 3’
下游引物：5’- ttagtttttatcctcactgtga - 3’
扩增长度：786 bp
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The PCR amplification system :25µ L system,
0.2 µ L Taq enzyme ( 0.5 U/mL )
2.5 µ L 10× Buffer
1.8 µ L Mg2+
1 µ L dNTPs Mixture
1 µ L template DNA
0.5 µ L forward primer ( 10 µ mol/L )
0.5 µ L reverse primer ( 10 µ mol/L )
17.5µ L ddH2O
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Amplification factor:
95 ° C 3 min;
95° C 30 s,
51 °C 60 s,
30 cycles
72 ° C 60s,
72 ° C 5 min,
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4 ° C termination reaction.
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The amplified sequences were purified,
connected to the pUCm-T carrier, transformated,
cultivated, extracted of plasmid, and then
sequenced in both directions by Sangon Biotech
Co., Ltd. (Shanghai, China). Sequencing data
were collected. The BLAST algorithm was used
to retrieve for homologous sequences in
GenBank (National Center for Biotechnology
Information [http://www.ncbi.nlm. nih.gov])
tasA
iturin
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Sequence of the amplified PCR product was
determined. Sequences of 675 bp fragments
showed elevated similarity (99%) with the geneencoding iturinA, and only 10 point mutations
were observed.
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Sequences of 786 bp fragments showed
elevated similarity (99%) with the gene-encoding
tasA, and only 10 point mutations were observed.
Part 4 Purification and characterization
of this peptide
Separation and purification
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Crude extraction stage
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ammonium sulphate precipitation
硫酸铵沉淀法。该法是细菌素粗提阶段最常用的一种有效方法,其优点在于
成本低,操作简便, 条件温和, 不改变蛋白质活性。
organic solvent precipitation
有机溶剂沉淀法。该法的优点在于有机溶剂不会残留在细菌素中,容易蒸发
除去。有机溶剂沉淀法比硫酸铵沉淀法容易使细菌素变性,所以很少使用。
pH absorption
在细菌素粗提阶段, 如果对细菌素的性质不了解时, 可以首先采用硫酸铵分级
沉淀法, 如果细菌素具有吸附性,可采用吸附法提取细菌素。也可根据实验要
求,采用几种方法相结合对细菌素进行粗提。
Purification stage
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gel chromatography
是利用蛋白质分子量大小的差异来得到目的蛋白的一种方法。
ion-exchange chromatography
根据蛋白质所带电荷的性质与离子交换剂的结合力大小的差别而进行分离
的一种层析方法。大多细菌素带正电荷,在细菌素纯化时常常选择阳离子交
换剂。
HPLC
纯度达到99 %以上
New discovery：
 Bac 14B is the first antimicrobial protein to show a spectrum of
action that is particularly inhibitory against Agrobacterium spp.
Strains.
 It also revealed that this bacteriocin contained a unique
sequence, namely M-L-K-A-N-L-Q-N-P-L-N-A, suggesting the
identification of a novel compound.
Methods and Results
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Bacterial strains and growth conditions
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Bac 14B from B. subtilis 14B strain
 pH7.2 and 37°C
 LB (peptone, 10 g/L; yeast extract, 5 g/L; NaCl, 5 g/L; pH 7.4)
supplemented with 10 g/L glucose, 15 g/L K2HPO4, 5 g/L
MgSO4·7H2O.
Agrobacterium tumefaciens C58, which is routinely used as an indicator
strain, was grown on mannitol glutamate agar medium
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Antimicrobial activity determination
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agar well diffusion assay
Methods and Results
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Bac 14B purification procedure
Centrifuged
30min
9000*g
pH 7
supernatant
Centrifuged
30min
9000*g
pH 7
ammonium
sulfate saturation
Precipitation
2.5 × 25 cm
Sephadex G-50
column
Fraction I
Elution
Fraction II
Elution
Mono Q column (2.5 × 20 cm)
attached to a fast protein
liquid chromatography system
10 mM Tris-HCl
pH 9 (buffer A)
Fraction III
Methods and Results
Molecular mass determination
mass spectrometry
amino acid sequencing
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N-Terminal sequence of the purified Bac 14B
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N-terminal sequencing of the blotted purified Bac 14B led to
the identification of 12 residues, M-L-K-A-N-L-Q-N-P-L-N-A.
This sequence was submitted to the GenBank nonredundant
nucleotide database for comparison with protein sequences
using the BLASTP and tBlastn search programs. It was
further submitted to the Swiss-Prot database for comparison
using BLASTP search software. No similar sequence was
found in either database, indicating that Bac 14B has unique
N-terminal amino acids.
Effect of pH and
temperature
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100°C for 2 h
90°C for 45 min~loss 20% activity
120°C for 20 min~complete loss activity
Low temperature storage (−20 and 4°C for 1
month)~ loss activity
pH 4-8 retained itsbiological activity, reduced at
pH 9
Triton X-100 , Tween 80~strongly inhibited
Completely inactivation after treatment with
different proteolytic enzymes
Inhibitory
spectrum
mode of action
Part 5 Discussion
Early achievements
A bacterial strain isolated from a honey sample showed antifungal
activity against mold. Based on the morphological, biochemical and
physiological tests, the strain was like Bacillus sp. The 16S rDNA
sequence of the Bacillus sp. was amplified by PCR and sequenced. By
Blast assay, the strain was identified to be Bacillus subtilis.
 The antibacterial spectrum against different microorganisms was
detected by agar diffusion method, the results indicated that it had
antibacterial effects against Aspergillus, Pythium, Botrytis cinerea, but
not E. coli, Staphylococcus aureus and Saccharomyces cerevisiae.
 The Bacillus subtilis culture was centrifuged and the supernatant was
used to conduct antibacterial test. The antibacterial substances were
found in the supernatant. The antibacterial substances could
remain inhibitory effect at pH between 5 and 9, and at temperature
between 40 and 100℃. The antibacterial activity was found lost after
proteinase K treatment. It seemed to be a proteinous material and had a
stable physical and chemical properties.。
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Current experiments
Purification of the antifungal peptide
1 ammonium sulfate saturation (70%)
2 gel chromatograph (seperdex G75)
3 HPLC
4 mass spectrometry
5 amino acid sequencing
Molecular characterization (DNA)
1 NCBI→ Bacillus subtilis antifungal peptide gene
2 primer design
3 PCR amplify aimed sequence
4 clone and express
5 antifungal assay→if “+”
6 sequencing