PBD1基因的克隆及其在大肠杆菌中的表达  

Cloning of PBD1 Gene and Its Expression in Escherichia coli

作　　者：郭奎[1,2] 杨兴武 张鸿鑫[1] 赵宇 郑慧华 杨明凡 陈红英[1,3] GUO Kui1,2 YANG Xing-Wu1 ZHANG Hong-Xin1 ZHAO Yu1 ZHENG Hui-Hua1 YANG Ming-Fan1,3 CHEN Hong-Ying1,3(1 College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou 450002, China; 2 Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences, Harbin 150069, China; 3 Zhengzhou Major Pig Disease Prevention and Control Laboratory, Henan Agricultural University, Zhengzhou 450002, Chin)

机构地区：[1]河南农业大学牧医工程学院,郑州450002 [2]中国农业科学院哈尔滨兽医研究所,哈尔滨150069 [3]河南农业大学郑州市猪重大疫病防控重点实验室,郑州450002

出　　处：《农业生物技术学报》2018年第4期635-641,共7页Journal of Agricultural Biotechnology

基　　金：河南省产学研合作计划项目(No.152107000003);河南省基础与前沿技术研究计划项目(No.142300410156)

摘　　要：猪(Sus scrofa)β防御素1(porcineβ-defensin 1,PBD1)是一种含有3对二硫键的阳离子活性肽,具有很强的抗菌活性,因此,在养猪业可能成为一种候选抗菌药剂。本研究根据Gen Bank中发表的PBD1全基因序列(登录号:NM213838)设计并合成2对特异性引物(P1/P2和P3/P4),应用引物P1/P2和逆转录聚合酶链反应(reverse transcription polymerase chain reaction,RT-PCR)技术,从猪舌组织样品总RNA中扩增包含PBD1全基因的片段(310 bp),并克隆到pMD18-T载体,命名为p MD18-T-PBD1Q。用引物P3/P4,将编码成熟蛋白的PBD1基因亚克隆到原核表达载体p ET28a-SUMO中,再转化入大肠杆菌(Escherichia coli)Rosetta(DE3),获得重组菌株,并优化表达融合蛋白的异丙基-β-D-硫代半乳糖甘(isopropy-β-Dthiogalactoside,IPTG)浓度、诱导时间以及诱导温度。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate-polyacrylamide gel electrophoresis,SDS-PAGE)及Western blot显示,重组菌破碎后上清中出现1条约18.3 kD的条带,在20℃、0.7 mmol/L IPTG诱导6 h,融合蛋白SUMO-PBD1表达量最高,且以可溶性形式存在;经小分子泛素样修饰蛋白(small ubiquitin-like modifier,SUMO)蛋白酶切割,获得了重组PBD1蛋白(约4.3 kD)。该研究结果为进一步研发具有广谱抗菌作用的微生态制剂提供技术支持。Porcine（Sus scrofa） β-defensin 1（PBD1） is a cationic antimicrobial peptide with 3 pairs of disulfide bonds, and has very strong antibacterial activity, and thus could be a good candidate as a bactericidal agent for pigs. Two pairs of specific primers（P1/P2 and P3/P4） were designed and synthesized according to the nucleotide sequence of the full porcine β-defensin 1（PBD1） gene published in Gen Bank（No.NM213838）. The fragment containing the full PBD1 gene was amplified by P1/P2 and reverse transcription polymerase chain reaction（RT-PCR） from total RNA extracted from porcine tongue tissue samples, PCRproduct was cloned into p MD18-T vector for sequencing. The result revealed that the nucleotide sequence of PBD1 gene was consisted of 310 bp in length. The PBD1 gene of mature protein was amplified using primers P3/P4. A prokaryotic plasmid of p ET28 a-SUMO-PBD1 was obtained by subcloning the encoding region of the mature peptide of PBD1 gene into p ET28 a-SUMO vector and transformed Escherichia coli Rosetta（DE3）competent cell. The expressed products were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis（SDS-PAGE） and Western-blot test after optimizing the various expression conditions such as concentration of isopropy-β-D-thiogalactoside（IPTG） and its cultured time and temperature. Fusion protein SUMO-PBD1 was presented as a form of soluble and its expression was highest when it was induced by addition of IPTG to a final concentration of 0.7 mmol/L for 6 h at 20 ℃. The recombinant PBD1（about 4.3 k D） was successfully obtained by small ubiquitin-like modifier（SUMO） protease digestion and purified. This study will lay a foundation for further study of the antimicrobial activity of defensins.

关 键 词：猪β防御素1 克隆 表达 纯化 小分子泛素样修饰蛋白(SUMO)蛋白酶 

分 类 号：S852.4[农业科学—基础兽医学] Q816[农业科学—兽医学]