摘要
【目的】对哈维氏弧菌(Vibrio harveyi)ZJ0603的PspF基因进行克隆与原核表达分析,优化表达条件。【方法】克隆哈维氏弧菌菌株ZJ0603的FspF基因,对其编码蛋白进行理化性质、信号肽、亚细胞定位、二级结构及三级结构分析,构建表达载体pET28a-PspF,经BamHI和XhoI双酶切和测序鉴定正确后转入表达菌株大肠杆菌BL21,对表达重组菌株进行表达条件优化及Western Blot鉴定。【结果与结论】PspF基因的开放阅读框(ORF)全长为1179bp,编码336个氨基酸,分子质量为38.04ku,理论等电点5.27,不稳定系数41.97,总平均亲水性-0.382,PspF蛋白整体表现为亲水性蛋白。成功构建含pET28a-PspF的表达菌株,其最佳诱导条件为37℃下异丙基-β-D-硫代半乳糖苷(IPTG)0.2mmol/L诱导6h,其表达蛋白为38ku。Western Blot结果表明PspF重组蛋白成功获得,蛋白同源性高,具有作为抗弧菌病疫苗的潜力。
【Objective】The PspF gene of Vibrio harveyi ZJ0603 was cloned and expressed in prokaryotic, and the expression conditions were optimized.【Method】The FspF gene in V.harveyi strain ZJ0603 was cloned, and its physical and chemical properties, signal peptide, subcellular localization, secondary structure and tertiary structure were analyzed.The expression vector pET28 a and the PCR product of the amplified FspF gene were digested with BamHI and XhoI.The pET28 a-PspF expression construct was produced after ligation of the digested vector and insert with T4 DNA ligase.The recombinant plasmid was transformed into E.coli BL21(DE3), and the expression conditions of the recombinant strain were optimized and identified by Western Blot.【Result and conclusion】The open reading frame of PspF gene was 1179 bp in length, encoding 336 amino acids, the relative molecular weight and the theoretical isoelectric point was 38.04 ku and 5.27 respectively.The instability coefficient of the deduced protein were 41.97, and the total average hydrophilicity was-0.382.The optimal induction conditions for the expression of pET28 a-PspF were adding 0.2 mmol/L IPTG at 37 ℃ for 6 h, and the expressed protein was 38 ku in size.Western Blot results showed that PspF recombinant protein was successfully obtained.The protein has high homology and potential as an anti-Vibrio vaccine.
作者
马少鸿
黄郁葱
简纪常
蔡双虎
MA Shao-hong;HUANG Yu-chong;JIAN Ji-chang;CAI Shuang-hu(Fisheries College, Guangdong Ocean University/Guangdong Provincial Key Laboratory of PathogenicBiology and Epidemiology for Aquatic Economic Animals, Zhanjiang 524088, China)
出处
《广东海洋大学学报》
CAS
2019年第5期1-7,共7页
Journal of Guangdong Ocean University
基金
广东省科技计划(2016A020209010)
广东省自然科学基金(2017A0303030975)
关键词
哈维氏弧菌
PspF基因
原核表达
表达优化
Vibrio harveyi
PspF gene
prokaryotic expression
expression optimization
