CAJ | 학술논문

目的克隆表达登革病毒非结构蛋白NS4A,分离纯化与其相互作用的细胞蛋白.方法用特异性引物PCR扩增出FLAG-NS4A-HA基因片段,克隆至真核表达载体pSG5中,将重组质粒通过脂质体法瞬时转染入A549细胞,并通过Western blot检测NS4A融合蛋白的表达情况.利用串联亲和纯化系统(TAP)纯化分离与NS4A相互作用的蛋白,Western blot技术及银染SDS-PAGE验证NS4A蛋白复合物的纯化和分离情况.结果成功构建NS4A蛋白融合表达载体和FLAG、HA串联亲和纯化系统,SDS-PAGE和银染结果显示TAP系统分离得到了NS4A蛋白带和2条可能与NS4A相互作用的蛋白质片段.结论成功分离纯化与NS4A相互作用的细胞蛋白,为进一步研究机体抗登革病毒感染的机制奠定了基础.
목적 극륭표체등혁병독비결구단백NS4A,분리순화여기상호작용적세포단백.방법 용특이성인물PCR확증출FLAG-NS4A-HA기인편단,극륭지진핵표체재체pSG5중,장중조질립통과지질체법순시전염입A549세포,병통과Western blot검측NS4A융합단백적표체정황.이용천련친화순화계통(TAP)순화분리여NS4A상호작용적단백,Western blot기술급은염SDS-PAGE험증NS4A단백복합물적순화화분리정황.결과 성공구건NS4A단백융합표체재체화FLAG、HA천련친화순화계통,SDS-PAGE화은염결과현시TAP계통분리득도료NS4A단백대화2조가능여NS4A상호작용적단백질편단.결론 성공분리순화여NS4A상호작용적세포단백,위진일보연구궤체항등혁병독감염적궤제전정료기출.
Objective To clone and express Dengue virus nonstructural protein 4A (NS4A) gene and express in eukaryotic cells.Then,to isolate and purify and isolate cellular proteins interacted with NS4A.Methods With specific primers,NS4A gene fragment tagged with FLAG and HA (FLAG-NS4A-HA) was amplified by PCR and cloned into an expression vector,pSG5 vector.Recombinant plasmid was transfected into A549 cells by LipofectAMINETM2000.Transient expression of FLAG-NS4A-HA was detected by Western blot.The NS4A interacting proteins were isolated and purified by tandem affinity purification (TAP) system using HA and FLAG antibodies,and then assayed by silver stained SDS-PAGE.Results Dengue virus NS4A gene tagged with FLAG and HA was successfully constructed into pSG5 vector and expressed in A.549 cells.Silver stained SDS-PAGE showed that the expressed NS4A and two potential interacting proteins that interact with NS4A were isolated after TAP purification and SDS-PAGE.Conclusion Cellular proteins that potentially interacted with Dengue virus NS4A were successfully purified and isolated,which provided a basis for further research.