CAJ | 학술논문

应用PCR方法从感染香蕉束顶病毒的香蕉植株幼嫩假茎和叶片总DNA中克隆NSP (Nuclear shuttle protein)基因的编码区,并通过Gateway技术定向重组到原核表达载体pDEST~(TM)-17中的6xHis标签下游,经菌落PCR和测序鉴定结果表明,成功构建了原核表达载体pDEST~(TM)-17-NSP.阳性克隆转化Ecoli BL21(DE3),经IFFG诱导表达、SDS-PAGE分析和western blot检测,结果显示,融合蛋白以包涵体的形式稳定表达,分子量约为20 ku,与预计值相符.通过诱导条件的优化,确定了最佳的融合蛋白表达条件为25℃、0.1 mmol/L IPTG条件下诱导4h.从而为制备NSP多克隆抗体和进一步研究NSP的功能奠定了基础.
응용PCR방법종감염향초속정병독적향초식주유눈가경화협편총DNA중극륭NSP (Nuclear shuttle protein)기인적편마구,병통과Gateway기술정향중조도원핵표체재체pDEST~(TM)-17중적6xHis표첨하유,경균락PCR화측서감정결과표명,성공구건료원핵표체재체pDEST~(TM)-17-NSP.양성극륭전화Ecoli BL21(DE3),경IFFG유도표체、SDS-PAGE분석화western blot검측,결과현시,융합단백이포함체적형식은정표체,분자량약위20 ku,여예계치상부.통과유도조건적우화,학정료최가적융합단백표체조건위25℃、0.1 mmol/L IPTG조건하유도4h.종이위제비NSP다극륭항체화진일보연구NSP적공능전정료기출.
The opening reading frame of NSP gene of banana bunchy top virus was cloned by polymerase chain reaction (PCR) using total DNA isolated from tender pseudostem and leaf of banana plant infected with the virus as a template, and directionally cloned to the downstream of 6 His tag by the Gateway technology. The construction of prokaryotic expressive vector, pDESTTM-17-NSP have been identified by the colony PCR and sequencing, and transformed into E.coli BL21(DE3) . Transformants were cultured and induced by IPTG, and then analyzed by SDS-PAGE and Western blot. The results showed that the fusion protein of approximately 20 ku was expressed steadily as an inclusion body and accorded with the predicted size. The expression of NSP gene was optimized under the conditions that the transformants were induced with 0.1 mmo1/1 IPTG at 25℃for 4 hours. This result might provide a foundation for preparation of the polyclonal antibody against NSP and further functional study of NSP.