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 B

acillus BH072

2012.9

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

The Antifungal Peptides Produced by Bacillus BH072

outline

Part 1 Identification and characterization of Bacillus BH072

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

抑菌试验

分析抑菌物质对热、pH、和蛋白酶

的稳定性

用琼脂扩散法，确定纯化抑菌物质的

抑菌谱

提取全细胞DNA作为PCR反应的模板

设计合成扩增16S rDNA的寡核苷酸引

物

PCR扩增芽孢杆菌16S rDNA

结合常规试验结果，确定菌株的种

属

确定鉴定范围

生理生化试验

形态学检测

提交测序结果至NCBI，Blast分析同

源性

Technical route

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

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

Amplification factor: 95 ° C 3 min; 95° C 30 s, 55 °C 60 s, 30 cycles 72 ° C 60s, 72 ° C 5 min,

4 ° C termination reaction.

16S Ribosomal DNA sequence analysis

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

琼脂糖凝胶电泳

PCR 产物 16S rDNA 测序

Blast 分析

结合常规检测 枯草芽胞杆菌

芽胞杆菌 BH072

Part 2 The antifungal activity of Bacillus BH072

Antifungal activity

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

agar well diffusion assay

-0.5

0

0.5

1

1.5

2

2.5

0 10 20 30 40 50 60

吸光度A

抑菌圈直径/cm

时间/h

Antifungal activity graph

Part 3 PCR analysis

Primer design

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

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

Amplification factor: 95 ° C 3 min; 95° C 30 s, 51 °C 60 s, 30 cycles 72 ° C 60s, 72 ° C 5 min,

4 ° C termination reaction.

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])

iturin

tasA

Sequence of the amplified PCR product was determined. Sequences of 675 bp fragments showed elevated similarity (99%) with the gene-encoding iturinA, and only 10 point mutations were observed.

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

Crude extraction stage 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 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

Separation and purificationSeparation and purification

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 ResultsMethods and Results Bacterial strains and growth conditions 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) suppl

emented with 10 g/L glucose, 15 g/L K2HPO4, 5 g/L MgSO4·7H2O. Agrobacterium tumefaciens C58, which is routinely used as an indicator stra

in, was grown on mannitol glutamate agar medium

Antimicrobial activity determination agar well diffusion assay

Bac 14B purification procedure

Methods and ResultsMethods and Results

Centrifuged30min9000*gpH 7

Fraction I

ammonium sulfate saturation

10 mM Tris-HCl pH 9 (buffer A)

supernatant

Precipitation

2.5 × 25 cm Sephadex G-50

column

Fraction II

ElutionMono Q column (2.5 × 20 cm)

attached to a fast proteinliquid chromatography system

Centrifuged30min9000*gpH 7

Elution

Fraction III

Methods and ResultsMethods and Results

Molecular mass determinationmass spectrometryamino acid sequencing

N-Terminal sequence of the purified Bac 14B

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 Effect of pH and temperaturetemperature

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 mo

nth)~ loss activity pH 4-8 retained itsbiological activity, reduced at

pH 9 Triton X-100 , Tween 80~strongly inhibited Completely inactivation after treatment with differ

ent proteolytic enzymes

InhibitoryInhibitory

spectrumspectrum

mode of actionmode of action

Part 5 Discussion

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. 。

Early achievements

Current experiments

Purification of the antifungal peptide1 ammonium sulfate saturation (70%)2 gel chromatograph (seperdex G75)3 HPLC4 mass spectrometry5 amino acid sequencing

Molecular characterization (DNA)1 NCBI→ Bacillus subtilis antifungal peptide gene2 primer design3 PCR amplify aimed sequence4 clone and express5 antifungal assay→if “+”6 sequencing