生物技术通讯
生物技術通訊
생물기술통신
LETTERS IN BIOTECHNOLOGY
2013年
6期
778-781
,共4页
绳纪坡%张宏达%于芳%黄君健%胡宝成
繩紀坡%張宏達%于芳%黃君健%鬍寶成
승기파%장굉체%우방%황군건%호보성
穿膜肽%LacI%LacI前头肽突变体%DNA结合活性
穿膜肽%LacI%LacI前頭肽突變體%DNA結閤活性
천막태%LacI%LacI전두태돌변체%DNA결합활성
cell-penetrating peptides%LacI%LacI headpiece mutant%DNA binding activities
目的:借助穿膜肽TAT高效跨膜的特性和LacI前头肽突变体(LacI HPM)高亲和力结合DNA的特性,构建新型基因转导载体。方法:PCR扩增LacI、LacI基因前头肽序列、前头肽序列突变体、TAT序列的编码基因,构建前头肽序列突变体和TAT的原核表达载体,可溶性表达TAT-LacI HPM融合蛋白并纯化,在缓冲液中氧化获得TAT-LacI HPM二聚体并浓缩,PCR检测二聚体融合蛋白与质粒的体外结合能力。结果:获得了pET-28a(+)-LacI HPM及pET-28a(+)-TAT-LacI HPM表达质粒,表达纯化并获得二聚化融合蛋白,体外结合实验确定TAT-LacI HPM二聚体融合蛋白与检测质粒DNA具有特异的高亲和力结合活性。结论:构建了穿膜肽TAT-LacI HPM,为进一步研究其作为新型DNA转运载体的可行性奠定了基础。
目的:藉助穿膜肽TAT高效跨膜的特性和LacI前頭肽突變體(LacI HPM)高親和力結閤DNA的特性,構建新型基因轉導載體。方法:PCR擴增LacI、LacI基因前頭肽序列、前頭肽序列突變體、TAT序列的編碼基因,構建前頭肽序列突變體和TAT的原覈錶達載體,可溶性錶達TAT-LacI HPM融閤蛋白併純化,在緩遲液中氧化穫得TAT-LacI HPM二聚體併濃縮,PCR檢測二聚體融閤蛋白與質粒的體外結閤能力。結果:穫得瞭pET-28a(+)-LacI HPM及pET-28a(+)-TAT-LacI HPM錶達質粒,錶達純化併穫得二聚化融閤蛋白,體外結閤實驗確定TAT-LacI HPM二聚體融閤蛋白與檢測質粒DNA具有特異的高親和力結閤活性。結論:構建瞭穿膜肽TAT-LacI HPM,為進一步研究其作為新型DNA轉運載體的可行性奠定瞭基礎。
목적:차조천막태TAT고효과막적특성화LacI전두태돌변체(LacI HPM)고친화력결합DNA적특성,구건신형기인전도재체。방법:PCR확증LacI、LacI기인전두태서렬、전두태서렬돌변체、TAT서렬적편마기인,구건전두태서렬돌변체화TAT적원핵표체재체,가용성표체TAT-LacI HPM융합단백병순화,재완충액중양화획득TAT-LacI HPM이취체병농축,PCR검측이취체융합단백여질립적체외결합능력。결과:획득료pET-28a(+)-LacI HPM급pET-28a(+)-TAT-LacI HPM표체질립,표체순화병획득이취화융합단백,체외결합실험학정TAT-LacI HPM이취체융합단백여검측질립DNA구유특이적고친화력결합활성。결론:구건료천막태TAT-LacI HPM,위진일보연구기작위신형DNA전운재체적가행성전정료기출。
Objective: To construct the novel DNA delivery vectors by using cell-penetrating peptides with the ability of high efficient cellular penetration and LacI to specifically bind to its recognizing DNA sequence with high affinity. Methods: The DNA sequence encoding the LacI, LacI HP(headpiece), LacI HPM(headpiece mu-tant) and TAT were firstly amplified by PCR respectively, then the expression plasmids of TAT-LacI HPM were cloned into pET-28a(+) vectors. Fusion proteins expressed in E.coli were purified by Ni-NTA beads, and were di-alyzed against buffer to form the TAT-LacI HPM dimers, then the dimers were concentrated by PEG-8000. DNA-binding activities were examined. Results: The expression plasmids of pET-28a(+)-LacI HPM and pET-28a (+)-TAT-LacI HPM were obtained, after expression and purification, the TAT-LacI HPM dimers were concentrat-ed, and the results showed that TAT-LacI HPM dimers have high affinity with its recognizing DNA sequences. Conclusion: The TAT-LacI HPM fusion proteins may have the potential to serve as a novel DNA delivery vector.