目的利用大肠杆菌原核表达系统优化表达纯化肠道病毒71型(EV71)VP3结构蛋白,为后续单克隆抗体制备及检测试剂盒研发提供前期基础。方法采用PCR方法扩增EV71病毒VP3基因,将其插入表达载体pET28a(+),构建pET28a-VP3重组质粒,转化大肠杆菌B...目的利用大肠杆菌原核表达系统优化表达纯化肠道病毒71型(EV71)VP3结构蛋白,为后续单克隆抗体制备及检测试剂盒研发提供前期基础。方法采用PCR方法扩增EV71病毒VP3基因,将其插入表达载体pET28a(+),构建pET28a-VP3重组质粒,转化大肠杆菌BL21(DE3)菌株,分别在25℃、37℃下经IPTG诱导表达,重组表达的蛋白产物经凝胶电泳初步分析,比较不同温度诱导表达的蛋白产物。结果成功构建pET28a-VP3重组质粒,不同温度下诱导表达的蛋白产物在30.5 k Da左右位置均出现目的条带;37℃下诱导表达的蛋白超声破碎并离心后,目的蛋白基本位于沉淀中,而25℃诱导表达的蛋白产物有少量目的蛋白溶解于上清液中。结论在25℃或37℃下均能利用大肠杆菌原核表达系统有效表达EV71病毒VP3蛋白;37℃诱导时蛋白可融性表达低,目的蛋白获取效率较高。展开更多
Objective:To clone the virB12 gene in pET28a expression vector for production of recombinant protein to be used as antigenic component for future serological test development.Methods: Brucella melitensis(B.melitensis)...Objective:To clone the virB12 gene in pET28a expression vector for production of recombinant protein to be used as antigenic component for future serological test development.Methods: Brucella melitensis(B.melitensis) 16M strain was cultured and bacterial DNA was extracted by Bioneer AccuPrep~ Genomic DNA Extraction Kit.Oligonucleotide primer pair was designed based on Brucella virB12 gene sequence with BamHI and HindIII restriction site at 5’ end of the forward and reverse primers,respectively.DNA amplification was performed using PrimSTAR~ HS DNA polymerase and the PCK product was purified by DNA AccuPrepGel Purification Kit.Purified DNA was cloned into pJET1.2 cloning vector.VirB12 gene fragment was excised from pJET1.2 asing BamHI/HindIII and subsequendy subcloned into pET28a(+).Results:Brucella virB12 gene was successfully cloned in pJET1.2 and then in pET28a(+) plasmids.PCR and restriction enzyme digestion confirms the procedure.Conclusion:We cloned and expressed the Brucella virB12 gene which could be used as antigenic component for specific serological assay development.展开更多
文摘目的利用大肠杆菌原核表达系统优化表达纯化肠道病毒71型(EV71)VP3结构蛋白,为后续单克隆抗体制备及检测试剂盒研发提供前期基础。方法采用PCR方法扩增EV71病毒VP3基因,将其插入表达载体pET28a(+),构建pET28a-VP3重组质粒,转化大肠杆菌BL21(DE3)菌株,分别在25℃、37℃下经IPTG诱导表达,重组表达的蛋白产物经凝胶电泳初步分析,比较不同温度诱导表达的蛋白产物。结果成功构建pET28a-VP3重组质粒,不同温度下诱导表达的蛋白产物在30.5 k Da左右位置均出现目的条带;37℃下诱导表达的蛋白超声破碎并离心后,目的蛋白基本位于沉淀中,而25℃诱导表达的蛋白产物有少量目的蛋白溶解于上清液中。结论在25℃或37℃下均能利用大肠杆菌原核表达系统有效表达EV71病毒VP3蛋白;37℃诱导时蛋白可融性表达低,目的蛋白获取效率较高。
基金supported by grant from Tehran University of Medical Sciences
文摘Objective:To clone the virB12 gene in pET28a expression vector for production of recombinant protein to be used as antigenic component for future serological test development.Methods: Brucella melitensis(B.melitensis) 16M strain was cultured and bacterial DNA was extracted by Bioneer AccuPrep~ Genomic DNA Extraction Kit.Oligonucleotide primer pair was designed based on Brucella virB12 gene sequence with BamHI and HindIII restriction site at 5’ end of the forward and reverse primers,respectively.DNA amplification was performed using PrimSTAR~ HS DNA polymerase and the PCK product was purified by DNA AccuPrepGel Purification Kit.Purified DNA was cloned into pJET1.2 cloning vector.VirB12 gene fragment was excised from pJET1.2 asing BamHI/HindIII and subsequendy subcloned into pET28a(+).Results:Brucella virB12 gene was successfully cloned in pJET1.2 and then in pET28a(+) plasmids.PCR and restriction enzyme digestion confirms the procedure.Conclusion:We cloned and expressed the Brucella virB12 gene which could be used as antigenic component for specific serological assay development.
