中华生物医学工程杂志
中華生物醫學工程雜誌
중화생물의학공정잡지
CHINESE JOURNAL OF BIOMEDICAL ENGINEERING
2010年
1期
48-51
,共4页
丁宁%肖慧%许立新%余守章
丁寧%肖慧%許立新%餘守章
정저%초혜%허립신%여수장
高迁移率族蛋白质类%细菌噬菌体%肽库%谷胱甘肽转移酶%色谱法,亲和
高遷移率族蛋白質類%細菌噬菌體%肽庫%穀胱甘肽轉移酶%色譜法,親和
고천이솔족단백질류%세균서균체%태고%곡광감태전이매%색보법,친화
High mobility group proteins%Bacteriophages%Peptide library%Glutathione transferase%Chromatography,affinity
目的 构建谷胱甘肽巯基转移酶(GST)标记的人高迁移率族蛋白B1(HMGB1)融合蛋白表达载体并在原核细胞中表达.应用噬菌体展示技术筛选HMGB1的高亲和肽.方法 用RT-PCR方法扩增HMGB1 cDNA,构建于原核表达载体pGEX4T-1并转化大肠杆菌,以异丙基-β-D-硫代半乳糖苷(IPTG)诱导GST-HMGB1蛋白表达;Ni2+-NTA和多粘菌素B亲和层析柱进行纯化重组HMGB1蛋白.以重组HMGB1蛋白为靶分子,进行4轮噬菌体展示环七肽库的筛选,从第4轮洗脱物中随机挑选20个单克隆噬菌体扩增后进行ELISA鉴定,用酶标仪测定450 nm处的吸光度值.对获得的阳性单克隆噬菌体分别进行扩增、纯化,并对DNA测序,以确定插入七肽的氨基酸序列.结果 RT-PCR扩增HMGB1 cDNA大小约648 bp,成功构建了pGEX4T-1-HMGB1重组质粒并纯化了HMGB1蛋白,其相对分子质量为65000.经过4轮筛选后,噬菌体富集了74倍(第4轮与第1轮回收量分别为5.2×108、7.0×106 pfu),随机挑取的20个噬菌体克隆中9个可与HMGB1结合,测序发现其中的6个序列一致,均为DYFVSSV.结论 筛选到1个可与HMGB1高亲和结合的噬菌体展示七肽,其对HMGB1活性的拮抗效应有待进一步阐明.
目的 構建穀胱甘肽巰基轉移酶(GST)標記的人高遷移率族蛋白B1(HMGB1)融閤蛋白錶達載體併在原覈細胞中錶達.應用噬菌體展示技術篩選HMGB1的高親和肽.方法 用RT-PCR方法擴增HMGB1 cDNA,構建于原覈錶達載體pGEX4T-1併轉化大腸桿菌,以異丙基-β-D-硫代半乳糖苷(IPTG)誘導GST-HMGB1蛋白錶達;Ni2+-NTA和多粘菌素B親和層析柱進行純化重組HMGB1蛋白.以重組HMGB1蛋白為靶分子,進行4輪噬菌體展示環七肽庫的篩選,從第4輪洗脫物中隨機挑選20箇單剋隆噬菌體擴增後進行ELISA鑒定,用酶標儀測定450 nm處的吸光度值.對穫得的暘性單剋隆噬菌體分彆進行擴增、純化,併對DNA測序,以確定插入七肽的氨基痠序列.結果 RT-PCR擴增HMGB1 cDNA大小約648 bp,成功構建瞭pGEX4T-1-HMGB1重組質粒併純化瞭HMGB1蛋白,其相對分子質量為65000.經過4輪篩選後,噬菌體富集瞭74倍(第4輪與第1輪迴收量分彆為5.2×108、7.0×106 pfu),隨機挑取的20箇噬菌體剋隆中9箇可與HMGB1結閤,測序髮現其中的6箇序列一緻,均為DYFVSSV.結論 篩選到1箇可與HMGB1高親和結閤的噬菌體展示七肽,其對HMGB1活性的拮抗效應有待進一步闡明.
목적 구건곡광감태구기전이매(GST)표기적인고천이솔족단백B1(HMGB1)융합단백표체재체병재원핵세포중표체.응용서균체전시기술사선HMGB1적고친화태.방법 용RT-PCR방법확증HMGB1 cDNA,구건우원핵표체재체pGEX4T-1병전화대장간균,이이병기-β-D-류대반유당감(IPTG)유도GST-HMGB1단백표체;Ni2+-NTA화다점균소B친화층석주진행순화중조HMGB1단백.이중조HMGB1단백위파분자,진행4륜서균체전시배칠태고적사선,종제4륜세탈물중수궤도선20개단극륭서균체확증후진행ELISA감정,용매표의측정450 nm처적흡광도치.대획득적양성단극륭서균체분별진행확증、순화,병대DNA측서,이학정삽입칠태적안기산서렬.결과 RT-PCR확증HMGB1 cDNA대소약648 bp,성공구건료pGEX4T-1-HMGB1중조질립병순화료HMGB1단백,기상대분자질량위65000.경과4륜사선후,서균체부집료74배(제4륜여제1륜회수량분별위5.2×108、7.0×106 pfu),수궤도취적20개서균체극륭중9개가여HMGB1결합,측서발현기중적6개서렬일치,균위DYFVSSV.결론 사선도1개가여HMGB1고친화결합적서균체전시칠태,기대HMGB1활성적길항효응유대진일보천명.
Objective To construct prokaryotic expression vector of glutathione-S-transferase ( GST) -tagged human high mobility group box 1 protein (HMGB1) and to screen its high affinity binding fused-peptide by phage display technique. Methods Human HMGB1 cDNA gene was amplified by RT-PCR and cloned into vector pGEX4T-1 and then transformed into E. coli. After induction by isopropyl P-D-1-thiogalactopyranoside (IPTG), HMGB1 protein was purified with Ni2+-NTA chromatography and polymyxin B affinity column. Using purified HMGB1 , as target molecule , four rounds of biopanning to a phage display heptapeptides library were carried out. Out of the fourth round eluted product, twenty individual clones were randomly selected and identified by ELISA assay. The absorbance of these clones at 450 nm was determined with a microplate reader. Positive clones were expanded , purified and sequenced to characterize the motifs of fused heptapeptides. Results Recombinant expression plasmid pGEX4T-1-HMGB1 was constructed ( 65 000 by MW) and the purified proteins (648 bp by cDNA) were obtained. Four rounds of biopanning resulted in 74-fold enrichment of the phages (the recovery were 5.2×108、7.0×106 pfu in fourth and first round). Of the twenty phage clones randomly selected, nine were found to bind specifically with HMGB1 proteins. The deduced amino acid sequence analysis showed that six clones displayed a common amino acid sequence (DYFVSSV). Conclusions A heptapeptide is obtained that may bind specifically to HMGB1 protein with high affinity. It remains to be determined whether this heptapeptide may block the effect of HMGB1 or not.
