CAJ | 학술논문

杆状病毒Bac-to-Bac表达系统由于操作简便等优点,被广泛应用于外源蛋白的表达.利用新构建的家蚕核型多角体病毒Bac-to-Bac系统可溶性表达一个大分子质量的融合蛋白—piwi相关基因编码蛋白PRG-1.为了提高目的蛋白的表达量,对重组病毒感染剂量及产物收获时间进行优化,并利用GST亲和层析纯化融合蛋白.结果表明在家蚕卵巢培养细胞BmN中表达的重组GST-PRG-1是可溶性蛋白；最佳的感染条件是病毒粒子对BmN细胞的感染复数(MOI)=1,并在感染后96 h收获细胞.试验结果表明,新构建的家蚕核型多角体病毒Bac-to-Bac表达系统可用于大分子蛋白的可溶性表达.
간상병독Bac-to-Bac표체계통유우조작간편등우점,피엄범응용우외원단백적표체.이용신구건적가잠핵형다각체병독Bac-to-Bac계통가용성표체일개대분자질량적융합단백—piwi상관기인편마단백PRG-1.위료제고목적단백적표체량,대중조병독감염제량급산물수획시간진행우화,병이용GST친화층석순화융합단백.결과표명재가잠란소배양세포BmN중표체적중조GST-PRG-1시가용성단백；최가적감염조건시병독입자대BmN세포적감염복수(MOI)=1,병재감염후96 h수획세포.시험결과표명,신구건적가잠핵형다각체병독Bac-to-Bac표체계통가용우대분자단백적가용성표체.
The baculovirus expression system Bac-to-Bac has been widely used in expression of exogenous proteins owing to its convenience in operation and other advantages.In this study,a fusion protein of high molecular mass containing PRG-1,encoded by piwi-related gene,was expressed using the newly constructed Bombyx mori nucleopolyhedrovirus (BmNPV) Bac-to-Bac system.To achieve better expression level,the dose of infection and cells' harvest time were optimized,and the fusion protein was purified by GST affinity chromatography.Our results indicated that the recombinant GST-PRG-1 expressed in BmN cells was in soluble form.The optimized expression condition was to infect the cells by 1 multiplicity of infection (MOI) and to harvest cells at 96 h post infection.The obtained data show that the newly constructed BmNPV Bac-to-Bac expression system can be used to express protein of hiah molecular mass in soluble form.