医学分子生物学杂志
醫學分子生物學雜誌
의학분자생물학잡지
FOREIGN MEDICAL SCIENCES
2010年
2期
131-135
,共5页
许成燕%刘友生%段光杰%朱江%徐小明
許成燕%劉友生%段光傑%硃江%徐小明
허성연%류우생%단광걸%주강%서소명
内毒素结合肽%定点突变%原核表达%纯化
內毒素結閤肽%定點突變%原覈錶達%純化
내독소결합태%정점돌변%원핵표체%순화
endotoxin binding peptide%site-directed mutation%prokaryotic expression,protein purification
目的 构建新型人内毒素结合肽(a new endotoxin binding peptide consisting of 25 amino acid residues,EBP_(25))及其突变体(mutant of EBP_(25),mEBP_(25))的原核表达重组质粒,并在大肠埃希菌中诱导表达.方法 采用PCR法,扩增EBP_(25)基因,构建pET-30-EBP_(25)融合表达载体并转化E.coli DH5α扩增.重组质粒经酶切和测序鉴定后,应用快速定点突变法将EBP_(25)第2位缬氨酸和第5位谷氨酰胺所对应碱基均替换成赖氨酸所对应的碱基,突变后重组质粒再经测序鉴定后,将二者转化至E.coli BL_(21)(DE_3)PlysS后经IPTG诱导表达,表达产物采用Western印迹进行鉴定后,用His-Tag亲和层析对融合蛋白进行纯化. 结果 两次测序结果 显示人EBP_(25)和mEBP_(25)重组序列和理论设计序列完全一致后,经IPTG诱导表达获得目的融合蛋白,通过SDS-PAGE电泳、Western 印迹证实蛋白表达的特异性,并对蛋白进行纯化,获得EBP_(25)和mEBP_(25)融合蛋白. 结论 构建、表达纯化了EBP_(25)和mEBP_(25)融合蛋白,为进一步研究其中和内毒素/脂多糖活性奠定了基础.
目的 構建新型人內毒素結閤肽(a new endotoxin binding peptide consisting of 25 amino acid residues,EBP_(25))及其突變體(mutant of EBP_(25),mEBP_(25))的原覈錶達重組質粒,併在大腸埃希菌中誘導錶達.方法 採用PCR法,擴增EBP_(25)基因,構建pET-30-EBP_(25)融閤錶達載體併轉化E.coli DH5α擴增.重組質粒經酶切和測序鑒定後,應用快速定點突變法將EBP_(25)第2位纈氨痠和第5位穀氨酰胺所對應堿基均替換成賴氨痠所對應的堿基,突變後重組質粒再經測序鑒定後,將二者轉化至E.coli BL_(21)(DE_3)PlysS後經IPTG誘導錶達,錶達產物採用Western印跡進行鑒定後,用His-Tag親和層析對融閤蛋白進行純化. 結果 兩次測序結果 顯示人EBP_(25)和mEBP_(25)重組序列和理論設計序列完全一緻後,經IPTG誘導錶達穫得目的融閤蛋白,通過SDS-PAGE電泳、Western 印跡證實蛋白錶達的特異性,併對蛋白進行純化,穫得EBP_(25)和mEBP_(25)融閤蛋白. 結論 構建、錶達純化瞭EBP_(25)和mEBP_(25)融閤蛋白,為進一步研究其中和內毒素/脂多糖活性奠定瞭基礎.
목적 구건신형인내독소결합태(a new endotoxin binding peptide consisting of 25 amino acid residues,EBP_(25))급기돌변체(mutant of EBP_(25),mEBP_(25))적원핵표체중조질립,병재대장애희균중유도표체.방법 채용PCR법,확증EBP_(25)기인,구건pET-30-EBP_(25)융합표체재체병전화E.coli DH5α확증.중조질립경매절화측서감정후,응용쾌속정점돌변법장EBP_(25)제2위힐안산화제5위곡안선알소대응감기균체환성뢰안산소대응적감기,돌변후중조질립재경측서감정후,장이자전화지E.coli BL_(21)(DE_3)PlysS후경IPTG유도표체,표체산물채용Western인적진행감정후,용His-Tag친화층석대융합단백진행순화. 결과 량차측서결과 현시인EBP_(25)화mEBP_(25)중조서렬화이론설계서렬완전일치후,경IPTG유도표체획득목적융합단백,통과SDS-PAGE전영、Western 인적증실단백표체적특이성,병대단백진행순화,획득EBP_(25)화mEBP_(25)융합단백. 결론 구건、표체순화료EBP_(25)화mEBP_(25)융합단백,위진일보연구기중화내독소/지다당활성전정료기출.
Objective To construct prokaryotic expression vectors expressing a new human endotoxin binding peptide consisting of 25 amino acid residues and its mutant and purify their fusion proteins.Methods The EBP_(25) gene was amplified by PCR and inserted into the prokaryotic expression vector pET-30. The recombinant plasmid pET-30-EBP_(25) was transformed into E.coli. DH5a and identified by DNA sequencing .Then the bases corresponding to the second valine and the fifth glutamine were replaced respectively with the bases of lysine by use of Quikchange site-directed mutation. The recombinant mutant plasmid pET-30-mEBP_(25) was again identified by DNA sequencing. Both EBP_(25) and mEBP_(25) were transfected into E.coli.BL_(21)(DE_3)PlysS and expressed under IPTG induction. Expressed EBP_(25) and mEBP_(25) proteins were analyzed by SDS-PAGE and western blotting and purified with His-Tag affinity chromatography.Results Both EBP_(25) and mEBP_(25) recombinants were confirmed by DNA sequencing. Highly expressed and purified EBP_(25) and mEBP_(25) protein were obtained,and their specificities were verified by SDS-PAGE and western blot.Conclusion The preparation of EBP_(25) and mEBP_(25) fusion proteins may be useful in studying biological function of neutralizing endotoxin/lipopolysaccaride.
