北京生物医学工程
北京生物醫學工程
북경생물의학공정
Beijing Biomedical Engineering
2015年
5期
458-461,508
,共5页
倪斌%张玲%李坚%张金喜%周健%杨可威%张君智%戴一鸣%夏雅琴%鲁燕华%刘俊平%李建峰
倪斌%張玲%李堅%張金喜%週健%楊可威%張君智%戴一鳴%夏雅琴%魯燕華%劉俊平%李建峰
예빈%장령%리견%장금희%주건%양가위%장군지%대일명%하아금%로연화%류준평%리건봉
α-突触核蛋白%猫%帕金森病%克隆%原核表达%纯化
α-突觸覈蛋白%貓%帕金森病%剋隆%原覈錶達%純化
α-돌촉핵단백%묘%파금삼병%극륭%원핵표체%순화
α-synuclein%cat%Parkinson disease%cloning%prokaryotic expression%purification
目的：对猫突触核蛋白（α?synuclein ）进行克隆、表达及纯化，并探讨其生物信息学特征。方法在Genebank中α?synuclein基因的保守区域内设计引物，从猫脑cDNA文库中PCR扩增得到猫的α?synuclein基因，再将此基因双酶切后克隆到pET28a原核表达载体中，构建重组质粒，测序正确的重组质粒转化BL21（ DE3）大肠杆菌，采用IPTG诱导表达。然后对猫α?synuclein氨基酸的同源性和疏水性进行分析。结果实验成功从猫脑cDNA文库中扩增出α?synuclein基因，基因全长381个碱基，编码126个氨基酸。获得的全长基因成功克隆进入pET28a，最后转染大肠杆菌BL21（ DE3），获得可溶性表达的α?synuclein蛋白质，蛋白质分子量为13?12kD，与预期分子量一致。生物信息学分析显示猫α?synuclein蛋白与人源及鼠源α?synuclein氨基酸具有很高的同源性，分别为87?35％和83?15％，但是与鼠和人的氨基酸序列比较，猫α?synuclein氨基酸缺失41～54位氨基酸。蛋白质结构预测显示猫α?synuclein具有很好的疏水性，有助于诱导表达时形成可溶性蛋白，这一结果在本研究中得到证实。结论本研究首次克隆了猫α?synuclein基因，并在大肠杆菌中实施了可溶性表达，为后期研究α?synuclein的进化、蛋白晶体结构、生物学功能和帕金森动物模型的构建奠定了一定的基础。
目的：對貓突觸覈蛋白（α?synuclein ）進行剋隆、錶達及純化，併探討其生物信息學特徵。方法在Genebank中α?synuclein基因的保守區域內設計引物，從貓腦cDNA文庫中PCR擴增得到貓的α?synuclein基因，再將此基因雙酶切後剋隆到pET28a原覈錶達載體中，構建重組質粒，測序正確的重組質粒轉化BL21（ DE3）大腸桿菌，採用IPTG誘導錶達。然後對貓α?synuclein氨基痠的同源性和疏水性進行分析。結果實驗成功從貓腦cDNA文庫中擴增齣α?synuclein基因，基因全長381箇堿基，編碼126箇氨基痠。穫得的全長基因成功剋隆進入pET28a，最後轉染大腸桿菌BL21（ DE3），穫得可溶性錶達的α?synuclein蛋白質，蛋白質分子量為13?12kD，與預期分子量一緻。生物信息學分析顯示貓α?synuclein蛋白與人源及鼠源α?synuclein氨基痠具有很高的同源性，分彆為87?35％和83?15％，但是與鼠和人的氨基痠序列比較，貓α?synuclein氨基痠缺失41～54位氨基痠。蛋白質結構預測顯示貓α?synuclein具有很好的疏水性，有助于誘導錶達時形成可溶性蛋白，這一結果在本研究中得到證實。結論本研究首次剋隆瞭貓α?synuclein基因，併在大腸桿菌中實施瞭可溶性錶達，為後期研究α?synuclein的進化、蛋白晶體結構、生物學功能和帕金森動物模型的構建奠定瞭一定的基礎。
목적：대묘돌촉핵단백（α?synuclein ）진행극륭、표체급순화，병탐토기생물신식학특정。방법재Genebank중α?synuclein기인적보수구역내설계인물，종묘뇌cDNA문고중PCR확증득도묘적α?synuclein기인，재장차기인쌍매절후극륭도pET28a원핵표체재체중，구건중조질립，측서정학적중조질립전화BL21（ DE3）대장간균，채용IPTG유도표체。연후대묘α?synuclein안기산적동원성화소수성진행분석。결과실험성공종묘뇌cDNA문고중확증출α?synuclein기인，기인전장381개감기，편마126개안기산。획득적전장기인성공극륭진입pET28a，최후전염대장간균BL21（ DE3），획득가용성표체적α?synuclein단백질，단백질분자량위13?12kD，여예기분자량일치。생물신식학분석현시묘α?synuclein단백여인원급서원α?synuclein안기산구유흔고적동원성，분별위87?35％화83?15％，단시여서화인적안기산서렬비교，묘α?synuclein안기산결실41～54위안기산。단백질결구예측현시묘α?synuclein구유흔호적소수성，유조우유도표체시형성가용성단백，저일결과재본연구중득도증실。결론본연구수차극륭료묘α?synuclein기인，병재대장간균중실시료가용성표체，위후기연구α?synuclein적진화、단백정체결구、생물학공능화파금삼동물모형적구건전정료일정적기출。
Objective To study cloning, expression, purification and characterization analysis of catα?synuclein protein? Methods According to the conserved regions ofα?synuclein gene, primers were designed and cat α?synuclein gene was amplified by PCR from cat brain cDNA? The gene was cloned into pET28a and pET28a carrying the coding DNA sequence of catα?synuclein gene was transformed into E?coli BL21( DE3) to express cat α?synuclein protein by IPTG induction? And the homology and hydrophobicity of cat α?synuclein amino acid were analyzed? Results There was a 381 bp cat α?synuclein gene amplified from the cat brain cDNA and cloned into pET28a? The transfected E? coli BL21 ( DE3) expressed soluble cat α?synuclein protein? The molecular weight of cat α?synuclein protein was consistent with the expected molecular weight ( 13?12kD ) ? Bioinformatics analysis showed that cat, murine and humanα?synuclein amino acids had high homology, yet the 41-54 amino acids in cat α?synuclein protein were missing? Protein structure prediction showed that cat α?synuclein had good hydrophobicity, contributing to the formation of soluble protein expression? Conclusions This study successfully cloned cat α?synuclein gene and implemented a soluble expression in E? coli, which provided a basis for the further studies of the evolution of α?synuclein, protein crystal structure, biological function and Parkinson animal models.
