鸡β-防御素5在大肠杆菌中的串联表达及生物学活性检测  

Tandem Expression and Biological Activity Detection of Chickenβ-defensin 5 in Escherichia coli

作　　者：温钰蓉 尹姣姣 田文霞[1] 马海利[1] 高荣琨 张鼎[1] 李桢[1] 乔梦丽 王颖[1] 宁官保[1] WEN Yurong;YIN Jiaojiao;TIAN Wenxia;MA Haili;GAO Rongkun;ZHANG Ding;LI Zhen;QIAO Mengli;WANG Ying;NING Guanbao(College of Veterinary Medicine,Shanxi Agricultural University,Taigu 030801,China)

机构地区：[1]山西农业大学动物医学学院,山西太谷030801

出　　处：《山西农业科学》2021年第6期772-776,共5页Journal of Shanxi Agricultural Sciences

基　　金：山西省重点研发计划项目(201703D221024-5;201703D211001)。

摘　　要：为提高蛋白表达量,研究选用串联表达技术对鸡β-防御素5(Av BD-5,也称Gal-5)重组蛋白进行表达及其生物学活性检测。按照NCBI上鸡β-防御素5的c DNA编码区序列,优化其基因序列后串联得到2×Gal-5,将2×Gal-5与p ET-28a双酶切后连接为重组表达载体p ET-28a-2×Gal-5,之后将p ET-28a-2×Gal-5转化到大肠杆菌(BL21)中诱导表达蛋白;重组蛋白表达成功后对其进行纯化以及体外抗菌活性的测定。结果表明,重组蛋白2×Gal-5在BL21中成功表达,分子质量约为12 ku,主要以包涵体形式存在;纯化后的重组蛋白对大肠杆菌有明显的抗菌活性,对沙门氏菌、金黄色葡萄球菌和链球菌抗菌活性较弱。试验成功构建可串联表达Gal-5且具有生物学活性的大肠杆菌原核表达载体。To improve the protein expression level,the tandem expression technology was used in this study,and the recombinant protein ofβ-Defensin 5(AvBD-5,or Gal-5)was expressed and its biological activity was detected.According to NCBI,the sequence of the coding region of Gal-5 cDNA was optimized and 2×Gal-5 would be obtained.After digestion with Gal-5 and pET-28a,the recombinant expression vector pET-28a-2×Gal-5 was constructed,then pET-28a-2×Gal-5 was transformed into Escherichia coli(BL21)to induce the expression of Gal-5 protein.After successful expression,the recombinant protein was purified and its antibacterial activity was determined in vitro.The results showed that the recombinant protein 2×Gal-5 was successfully expressed in BL21,it was about 12 ku and mainly in the form of inclusion bodies.The purified recombinant protein had obvious antibacterial activity against Escherichia coli,but weak antibacterial activity against Salmonella,Staphylococcus aureus and Streptococcus.The prokaryotic expression vector of Gal-5 with biological activity was constructed successfully.

关 键 词：鸡Gal-5 串联表达技术 大肠杆菌 表达载体 抗菌活性 

分 类 号：Q78[生物学—分子生物学]