太平洋鳕神经坏死病毒衣壳蛋白(CP)的原核表达及条件优化

为深入研究太平洋鳕Gadus macrocephalus神经坏死病毒(Pacific cod nervous necrosis virus,PCNNV)衣壳蛋白(capsid protein,CP)的功能,将PCNNV cp基因连接到p ET-32a原核表达载体中,构建原核表达重组载体p EVCP,将其转化至大肠杆菌E.coli BL21(DE3)表达系统中进行融合表达,对诱导剂IPTG浓度、诱导时间和温度等诱导表达条件进行优化,采用SDS-PAGE及质谱技术鉴定表达产物。结果表明:本研究中成功构建了重组载体p EVCP,表达的目的蛋白相对分子质量约为55 000;目的蛋白经质谱鉴定,有6个肽段的氨基酸序列与预期一致;对重组菌株p EVCP/BL21的最佳诱导条件为IPTG浓度0.1 mmol/L、最佳诱导时间3 h、温度30℃。本研究结果可为后续冷水鱼类病毒疫苗的研制及病毒快速检测试剂盒的研发奠定基础。

Prokaryotic expression and condition optimization of nervous necrosis virus capsid protein(CP) in Pacific cod Gadus macrocephalus

Capsid protein(CP) gene of nervous necrosis virus in Pacific cod Gadus macrocephalus(PCNNV) was inserted into plasmid pET-32 a to construct recombinant plasmid pEVCP to further study the function of PCNNV CP, and to devise an effective vaccine for PCNNV. The recombinant vector was transformed into Escherichia coli BL21(DE3), and then induced under conditions of different IPTG concentrations, time and temperature. The ex-pressed products were identified by SDS-PAGE and mass spectrum analysis. Results showed that the recombinant plasmid pEVCP was constructed successfully and a protein with molecular weight of 55 000 was identified using SDS-PAGE. 6 peptide segments of recombined CP were sequenced correctly using mass spectrum. The expression level was varied under different induced conditions, with the optimal expression under conditions of 0. 1 mmol/L IPTG, 3 h and 30 ℃. The findings would contribute to exploration of nervous necrosis virus( NNV) vaccine and development of rapid detection kit for NNV.