摘要
目的在酿酒酵母中分泌表达牛乳铁蛋白肽(bovine lactoferricin,LfcinB),并检测其活性。方法根据LfcinB的氨基酸序列,按照酿酒酵母密码子偏好性设计LfcinB基因序列,人工合成LfcinB基因,扩增后插入质粒pYES2-α中,构建重组表达质粒pYES2-α-LfcinB,转化至酿酒酵母INVSc1中,半乳糖诱导表达。采用抗生素微生物效价测定法中的管碟法一剂量法检测表达的LfcinB对大肠埃希菌DH5α和枯草杆菌的抑菌活性。对重组酵母菌的诱导时间、诱导剂浓度和诱导温度进行优化,并检测重组LfcinB的稳定性。结果重组表达质粒pYES2-α-LfcinB经双酶切和测序证明构建正确;重组酵母菌诱导表达产物可见相对分子质量分别为5 164和4 355的特异蛋白条带,每升工程菌发酵液中目的蛋白含量约为2.878 5 mg;表达的重组LfcinB对大肠埃希菌DH5α和枯草杆菌均有明显的抑菌活性;在诱导温度为26℃、半乳糖浓度为3%的条件下诱导96 h时,LfcinB的抑菌活性最高;重组LfcinB具有热稳定性,且二硫键的存在对其抑菌活性无影响。结论已成功在酿酒酵母中分泌表达了具有活性的LfcinB,为进一步研究LfcinB的生物功能及其应用奠定了基础。
Objective To achieve secretory expression of LfcinB in Saccharomyces cescerevisiae and determine its activity.Methods The DNA sequence of LfcinB was designed based on the codon bias of S.cescerevisiae,then synthesized chemically and inserted into vector pYES2-α.The constructed recombinant plasmid pYES2-α-LfcinB was transformed to S.cescerevisiae INVSc1 cells and induced with galactose.The inhibiting activities of expressed LfcinB to E.coli DH5α and Bacillus subtilis were determined by a single dose cylinder-plate method.The concentration of inducer as well as time and temperature for induction were optimized,and stability of recombinant LfcinB was determined.Results Restriction analysis and sequencing proved that recombinant plasmid pYES2-α-LfcinB was constructed correctly.The expressed product showed specific protein bands with relative molecular masses of 5 164 and 4 355 on SDS-PAGE profile.The target protein content in each liter of fermentation liquid was about 2.878 5 mg.The expressed LfcinB showed significantly inhibiting activities to E.coli DH5α and B.subtilis.The inhibiting activities reached the peak values after induction of recombinant S.cerevisiae with 3% galactose at 26 ℃ for 96 h.Recombinant LfcinB showed heat stability,of which the activity was not influenced in presence of disulfide bond.Conclusion Biologically active LfcinB was successfully expressed in S.cerevisiae in secretory form,which laid a foundation of further study on biological function and application of LfcinB.
出处
《中国生物制品学杂志》
CAS
CSCD
2014年第9期1132-1137,共6页
Chinese Journal of Biologicals
基金
安徽省自然科学基金(1408085MC50)
安徽省高校自然科学自然科学研究重点项目(KJ2010A025)
关键词
牛乳铁蛋白肽
酿酒酵母
分泌表达
抗菌活性
Bovine lactoferricin； Saccharomyces cescerevisiae； Secretory expression； Antibacterial activity
