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1 微生物学通报 Microbiology China Sep. 20, 2016, 43(9): DOI: /j.microbiol.china 研究报告 重组鹅 β- 防御素 12 蛋白的原核表达及生物学特性 * 王秋玲徐倩倩张婷婷赵文钧陈玉秋齐甜铭马得莹 ( ) 摘要 : 目的 研究重组鹅 β- 防御素 12 蛋白的原核表达并探究其生物学特性 方法 采用 His 标签蛋白原核表达系统, 将鹅防御素 12 (AvBD12) 基因亚克隆到表达载体 pproex-hta 上, 构建重组表达质粒 将重组表达质粒转化到大肠杆菌 Rosseta 感受态中, 用 IPTG 进行诱导表达, 并对该重组蛋白进行纯化 进一步采用菌落计数法测定其体外抗菌活性和盐离子稳定性 结果 经 Tricine-SDS-PAGE 电泳分析, 诱导表达的鹅 AvBD12 重组蛋白分子量约为 12 kd, 大部分以包涵体形式存在 该重组蛋白对大肠杆菌 鸡白痢沙门氏菌 金黄色葡萄球菌 四联球菌 枯草芽孢杆菌均具有抗菌活性, 高浓度盐离子显著抑制重组蛋白的抗菌活性 此外, 该重组蛋白对鸡红细胞没有溶血活性 结论 该重组蛋白具有广谱抗菌活性, 高浓度盐离子显著降低其抗菌活性, 且该重组蛋白不具有溶解鸡红细胞的活性 关键词 : 鹅 β- 防御素 12, 重组蛋白, 抗菌活性 Recombinant expression and biological characteristics of goose β-defensin 12 WANG Qiu-Ling XU Qian-Qian ZHANG Ting-Ting ZHAO Wen-Jun CHEN Yu-Qiu QI Tian-Ming MA De-Ying * (College of Animal Science and Technology, Northeast Agricultural University, Harbin, Heilongjiang , China) Abstract: [Objective] The study was to express and characterize the fusion protein of goose β-defensin (AvBD) 12 in Escherichia coli. [Methods] The cdna of goose AvBD12 was cloned into EcoR I and Xho I sites of pproex-hta vector. The recombinant expression plasmid was translated into E. coli Rosseta and the bacteria were induced by isopropyl β-d-1-thiogalactopyranoside (IPTG). The recombinant protein was purified. Furthermore, antibacterial activity and salt ionic stability of the recombinant protein were determined by colony-counting assays. [Results] It was demonstrated by Tricine-SDS-PAGE that the recombinant protein (molecular weight, 12 kd) was produced as Foundation item: Specialized Research Fund for the Doctoral Program of Higher Education (No ); Specialized Research Fund for the Science and Technological Innovation Talent of Harbin (No. 2013RFXXJ019); Research Program for Applied Technology of Heilongjiang Province (No. PC13S02) *Corresponding author: Tel: ; madeying@neau.edu.cn Received: November 09, 2015; Accepted: March 03, 2016; Published online ( March 18, 2016 基金项目 : (No ) (No. 2013RFXXJ019) (No. PC13S02) * 通讯作者 :Tel madeying@neau.edu.cn 收稿日期 : 接受日期 : 优先数字出版日期 (

2 1974 微生物学通报 Microbiol. China 2016, Vol.43, No.9 insoluble bodies in the cells. The recombinant protein showed extensive antibacterial activity against bacteria, including E. coli, Micrococcus tetragenus, Salmonella pullorum, Bacillus subtilis, and Staphylococcus aureus. At high NaCl concentrations, the antibacterial activity of goose AvBD12 decreased significantly. In addition, goose AvBD12 showed little hemolytic activity. [Conclusion] Recombinant goose AvBD12 exhibited extensive antibacterial activity. High salt concentration significantly decreased its antibacterial activity. In addition, AvBD12 showed little hemolytic activity. Keywords: Goose β-defensin 12, Recombinant protein, Antibacterial activity β- (Avian β-defensin AvBD) [1] β- [2-3] β- β- [4-6] 11 β- (AvBD1 7 AvBD9 10 AvBD12 AvBD16) AvBD bp 65 GST AvBD1 AvBD3 AvBD5 AvBD6 AvBD10 GST β- His AvBD12 AvBD12 β- 1 材料与方法 1.1 材料 菌株及质粒 : pmd18-t-goose AvBD12 [7] pmd18-t-simple Vector TaKaRa pproex-hta Escherichia coli Rosetta Bacillus subtilis (ATCC 9193) Staphyloccocus aureus (ATCC 29213) Micrococcus tetragenus (ATCC 2835) Salmonella pullorum (C79-11-S11) Escherichia coli (BL21) 主要试剂和仪器 :ExTaq DNA EcoR I Xho I T4 DNA Marker -β-d- (IPTG) ( ) OMEGA Novagen Forma Scientific (ULTB86-3 ) REVCD (DK-8D ) - (DYY-6C ) (Bio imaging system) Gene Genius (CV18) (J2-21M ) Beckman Pharmacia 1.2 方法 原核表达重组质粒的构建 重组蛋白的表达及纯化 : pproex-hta pmd18-t-avbd12

3 : β AvBD12 P1/P2 EcoR I Xho I P1 P2 5 P1 5 -GAATTCATGGAC AGCTGTAACCACG-3 P2 5 -CTCGAGTCAGGTC TTGGTGGGAGTTG-3 pmd18-t- AvBD12 PCR PCR ExTaq TM DNA (2 ) 12.5 μl ( 200 mg/l) 0.5 μl (10 μmol/l) 1 μl 25 μl 94 C 5 min 94 C 30 s 58 C 30 s 72 C 30 s C 10 min EcoR I Xho I pproex-hta pproex-avbd12 PCR PCR 3 μl EcoR I Xho I 0.5 μl 10 H Buffer 1 μl 10 μl 37 C 2 h 50 µl 1:100 5 ml Amp + LB [6] 37 C 250 r/min h OD ml 100 mmol/l IPTG 0.6 mmol/l h 1 ml 4 C r/min 5 min 1/10 1 SDS 5 min 2 min Tricine-SDS-PAGE - Novagen 1 ml 100 μl PBS (ph 7.4) Tricine-SDS- PAGE 鹅 AvBD12 重组蛋白抗菌活性测定 : AvBD12 AvBD ml EP ph 7.4 PBS mg/l 250 μl CFU/mL 10 μl EP PBS 3 37 C 4 h 100 μl LB 37 C 3 Excel 2003 GraphPad Prism 5 (%)= / 不同盐离子浓度对鹅 AvBD12 重组蛋白抗菌活性的影响 : (G ) (G + ) PBS mmol/l 1.5 ml EP 250 mg/l 250 μl NaCl 10 μl 37 C 4 h 鹅 AvBD12 重组蛋白对鸡红细胞的溶血活性测定 : PBS (ph 7.4) 2% 3% PBS mg/l 20 μl PBS 0.2% Triton μl 3 37 C 1 h r/min

4 1976 微生物学通报 Microbiol. China 2016, Vol.43, No.9 10 min OD (%)=(OD OD PBS )/(OD Triton OD PBS ) 统计分析 : SAS 结果与分析 2.1 重组表达质粒 pproex-goose AvBD12 的鉴定 PCR (147 bp) 1 pproex-hta 2.2 鹅 AvBD12 重组蛋白的原核表达与纯化 Tricine-SDS-PAGE 12 kd 35% h 6 h Novagen AvBD12 Tricine-SDS-PAGE 12 kd ( 2) mg/l 图 1 重组质粒 pproex-goose-avbd12 的双酶切及 PCR 产物的鉴定 Figure 1 Identification of pproex-goose-avbd12 recombinant plasmid digested and PCR product F pproex HTa-AvBD12 E pproex HTa-AvBD12 PCR M 100 bp Ladder DNA marker. Note: F: Product of plasmid pproex HTa-AvBD12 restriction enzyme digestion; E: Plasmid pproex HTa-AvBD12 PCR product; M: 100 bp Ladder DNA marker. 图 2 鹅 AvBD12 重组蛋白的表达与纯化 Figure 2 Expression and purification of recombinant goose AvBD12 protein IPTG h 7 M Marker. Note: 1: Supertant; 2: Inclusion body; 3: Total protein of Rosseta containing goose AvBD12 without IPTG induction; 4 6: Total protein of Rosseta containing goose AvBD12 on 2, 4, 6 h, after induction with IPTG; 7: Purified protein; M: Protein marker. 2.3 鹅 AvBD12 重组蛋白的抗菌活性 AvBD12 AvBD mg/l AvBD12 ( 3) 2.4 不同 NaCl 浓度对抗菌活性的影响 4 ( ) ( ) AvBD mg/l NaCl 150 mmol/l (P<0.01) 2.5 对鸡红细胞的溶血活性 5 ( mg/l) AvBD12 PBS (P>0.05) AvBD12

5 : β 图 3 鹅 AvBD12 重组蛋白的抗菌活性 Figure 3 Antimicrobial activity of recombinant goose AvBD12 protein A B C D E. Note: A: E. coli; B: S. pullorum; C: M. tetragenus; D: B. subtilis; E: S. aureus. 图 4 NaCl 浓度对鹅 AvBD12 重组蛋白抗菌活性的影响 Figure 4 Effect of different NaCl concentration on the antimicrobial activity of recombinant goose AvBD12 protein 图 5 鹅 AvBD12 重组蛋白的溶血活性 Figure 5 Hemolysis activity of recombinant goose AvBD12 protein

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