中南民族大学学报（自然科学版）
中南民族大學學報（自然科學版）
중남민족대학학보（자연과학판）
JOURNAL OF SOUTH-CENTRAL UNIVERSITY FOR NATIONALITIES(NATURAL SCIENCE EDITION)
2013年
2期
36-40
,共5页
钾离子通道阻断剂%Im58%原核表达%蛋白纯化
鉀離子通道阻斷劑%Im58%原覈錶達%蛋白純化
갑리자통도조단제%Im58%원핵표체%단백순화
potassium channel blocker%Im58%prokaryotic expression%protein purification
为表达钾离子通道阻断剂Im58,设计了4条搭桥 PCR 引物,通过 overlap PCR 的方法扩增出蝎毒肽 Im58 DNA全长片段,选用表达载体pGEX-4T-1,将测序正确的克隆转化至大肠杆菌 E. coli/Rosetta(DE3)中.通过检测不同诱导时间和IPTG浓度下融合蛋白的表达量,筛选出最优表达条件.表达蛋白经 IPTG 诱导后,融合蛋白再经GSH亲和层析柱纯化,将洗脱液经超滤浓缩脱盐、纯化后得GST-Im58融合蛋白,蛋白用小肠激酶酶切后通过 RP-HPLC C18柱分离获得色谱纯的蝎毒肽.蝎毒肽通过 Tricine 系统的 SDS-PAGE 蛋白质电泳鉴定重组多肽的分子量大小和纯度,并由 MALDI-TOF-MS 来精确测定重组多肽的分子量.结果表明：Im58阳性克隆子证明成功构建了Im58原核表达载体,Tricine 系统的 SDS-PAGE 蛋白质电泳和MALDI-TOF-MS鉴定Im58多肽表达纯化成功.
為錶達鉀離子通道阻斷劑Im58,設計瞭4條搭橋 PCR 引物,通過 overlap PCR 的方法擴增齣蝎毒肽 Im58 DNA全長片段,選用錶達載體pGEX-4T-1,將測序正確的剋隆轉化至大腸桿菌 E. coli/Rosetta(DE3)中.通過檢測不同誘導時間和IPTG濃度下融閤蛋白的錶達量,篩選齣最優錶達條件.錶達蛋白經 IPTG 誘導後,融閤蛋白再經GSH親和層析柱純化,將洗脫液經超濾濃縮脫鹽、純化後得GST-Im58融閤蛋白,蛋白用小腸激酶酶切後通過 RP-HPLC C18柱分離穫得色譜純的蝎毒肽.蝎毒肽通過 Tricine 繫統的 SDS-PAGE 蛋白質電泳鑒定重組多肽的分子量大小和純度,併由 MALDI-TOF-MS 來精確測定重組多肽的分子量.結果錶明：Im58暘性剋隆子證明成功構建瞭Im58原覈錶達載體,Tricine 繫統的 SDS-PAGE 蛋白質電泳和MALDI-TOF-MS鑒定Im58多肽錶達純化成功.
위표체갑리자통도조단제Im58,설계료4조탑교 PCR 인물,통과 overlap PCR 적방법확증출갈독태 Im58 DNA전장편단,선용표체재체pGEX-4T-1,장측서정학적극륭전화지대장간균 E. coli/Rosetta(DE3)중.통과검측불동유도시간화IPTG농도하융합단백적표체량,사선출최우표체조건.표체단백경 IPTG 유도후,융합단백재경GSH친화층석주순화,장세탈액경초려농축탈염、순화후득GST-Im58융합단백,단백용소장격매매절후통과 RP-HPLC C18주분리획득색보순적갈독태.갈독태통과 Tricine 계통적 SDS-PAGE 단백질전영감정중조다태적분자량대소화순도,병유 MALDI-TOF-MS 래정학측정중조다태적분자량.결과표명：Im58양성극륭자증명성공구건료Im58원핵표체재체,Tricine 계통적 SDS-PAGE 단백질전영화MALDI-TOF-MS감정Im58다태표체순화성공.
To express potassium channel blocker Im58, full length DNA sequences of Im58 was amplified by overlap PCR with four designed primers and the cloning with the right sequence was transformed to E. coli/Rosetta ( DE3 ) by vector pGEX-4T-1. Under different inducing time and IPTG concentrations, the expressing quantities of fusion protein were investigated to optimize the inducing conditions. Then the recombinant GSH-fusion proteins were purified by GSH affinity chromatography, treated with enterokinase and purified by C18 column of RP-HPLC. The sample of Im58 purifying peptides was obtained to identify the molecular weight and purity by SDS-PAGE Gel of Tricine system. Accurate molecular weight was also identified by MALDI-TOF-MS. The results showed that the prokaryotic expression plasmid of Im58 was successfully constructed by positive clone. Moreover, the expression, purification and identification of Im58 were confirmed by SDS-PAGE Gel of Tricine system and MALDI-TOF-MS.
