PTD-FNK蛋白的原核表达及纯化鉴定
【目的】原核表达Bcl-x L蛋白的突变体PTD-FNK蛋白,为揭示PTD-FNK蛋白的抗凋亡机制及加快其在家畜精液冷冻保存中的应用提供参考依据。【方法】根据FNK蛋白核苷酸序列设计两对突变引物,以p ET-28a（＋）-PTD-Bcl-x L质粒为模板,PCR扩增两段突变序列;回收PCR产物片段进行Dpn I消化,构建重组质粒并转化大肠杆菌DH5α,以IPTG诱导融合蛋白表达,探索IPTG诱导表达的适宜温度,最后以SDS-PAGE电泳和Western bloting对纯化蛋白进行鉴定。【结果】两个突变片段的PCR扩增产物大小分别为462和5616 bp,经Dpn I消化后进行重组反应,再转化...
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| Published in | 南方农业学报 Vol. 47; no. 6; pp. 1019 - 1024 |
|---|---|
| Main Author | |
| Format | Journal Article |
| Language | Chinese |
| Published |
长江大学 动物科学学院，湖北 荆州 434023%广西畜禽品种改良站,南宁,530001
2016
广西大学 动物科学技术学院,南宁,530005%广西大学 动物科学技术学院，南宁 530005 |
| Subjects | |
| Online Access | Get full text |
| ISSN | 2095-1191 |
| DOI | 10.3969/j:issn.2095-1191.2016.06.1019 |
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| Abstract | 【目的】原核表达Bcl-x L蛋白的突变体PTD-FNK蛋白,为揭示PTD-FNK蛋白的抗凋亡机制及加快其在家畜精液冷冻保存中的应用提供参考依据。【方法】根据FNK蛋白核苷酸序列设计两对突变引物,以p ET-28a（＋）-PTD-Bcl-x L质粒为模板,PCR扩增两段突变序列;回收PCR产物片段进行Dpn I消化,构建重组质粒并转化大肠杆菌DH5α,以IPTG诱导融合蛋白表达,探索IPTG诱导表达的适宜温度,最后以SDS-PAGE电泳和Western bloting对纯化蛋白进行鉴定。【结果】两个突变片段的PCR扩增产物大小分别为462和5616 bp,经Dpn I消化后进行重组反应,再转化至大肠杆菌DH5α能成功构建p ET-28a（＋）-PTD-FNK质粒。在30℃下以IPTG诱导7 h可获得可溶性的PTD-FNK蛋白,以NI-NTA Argrose纯化后的融合蛋白经Western blotting鉴定为PTD-FNK蛋白。【结论】通过调控IPTG诱导温度可成功以可溶性形式表达出PTD-FNK蛋白,且诱导时间短,无须复性,有利于蛋白纯化及加快其在家畜精液冷冻保存中的应用。 |
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| AbstractList | 【目的】原核表达Bcl-x L蛋白的突变体PTD-FNK蛋白,为揭示PTD-FNK蛋白的抗凋亡机制及加快其在家畜精液冷冻保存中的应用提供参考依据。【方法】根据FNK蛋白核苷酸序列设计两对突变引物,以p ET-28a（＋）-PTD-Bcl-x L质粒为模板,PCR扩增两段突变序列;回收PCR产物片段进行Dpn I消化,构建重组质粒并转化大肠杆菌DH5α,以IPTG诱导融合蛋白表达,探索IPTG诱导表达的适宜温度,最后以SDS-PAGE电泳和Western bloting对纯化蛋白进行鉴定。【结果】两个突变片段的PCR扩增产物大小分别为462和5616 bp,经Dpn I消化后进行重组反应,再转化至大肠杆菌DH5α能成功构建p ET-28a（＋）-PTD-FNK质粒。在30℃下以IPTG诱导7 h可获得可溶性的PTD-FNK蛋白,以NI-NTA Argrose纯化后的融合蛋白经Western blotting鉴定为PTD-FNK蛋白。【结论】通过调控IPTG诱导温度可成功以可溶性形式表达出PTD-FNK蛋白,且诱导时间短,无须复性,有利于蛋白纯化及加快其在家畜精液冷冻保存中的应用。 TQ936.2; 【目的】原核表达Bcl-xL蛋白的突变体PTD-FNK蛋白，为揭示PTD-FNK蛋白的抗凋亡机制及加快其在家畜精液冷冻保存中的应用提供参考依据。【方法】根据FNK蛋白核苷酸序列设计两对突变引物，以pET-28a（+）-PTD-Bcl-xL质粒为模板，PCR扩增两段突变序列；回收PCR产物片段进行Dpn I消化，构建重组质粒并转化大肠杆菌DH5α，以IPTG诱导融合蛋白表达，探索IPTG诱导表达的适宜温度，最后以SDS-PAGE电泳和Western bloting对纯化蛋白进行鉴定。【结果】两个突变片段的PCR扩增产物大小分别为462和5616 bp，经Dpn I消化后进行重组反应，再转化至大肠杆菌DH5α能成功构建pET-28a（+）-PTD-FNK质粒。在30℃下以IPTG诱导7 h可获得可溶性的PTD-FNK蛋白，以NI-NTA Argrose纯化后的融合蛋白经Western blotting鉴定为PTD-FNK蛋白。【结论】通过调控IPTG诱导温度可成功以可溶性形式表达出PTD-FNK蛋白，且诱导时间短，无须复性，有利于蛋白纯化及加快其在家畜精液冷冻保存中的应用。 |
| Abstract_FL | Objective]The mutant PTD-FNK protein of Bcl-xL protein was expressed in prokaryotic cell , in order to reveal anti-apoptosis mechanism and accelerate its application in livestock semen cryopreservation. [Method]Based on nu-cleotide sequence of FNKT protein, two primers were designed for site-specific mutagenesis, two mutated fragments were amplified by PCR using pET-28a(+)-PTD-Bcl-xL plasmid as template. The extracted PCR products were digested by Dpn l to construct recombinant plasmid, and transformed into Escherichia coli DH5α. Then fusion protein was expressed by IPTG induction, and its optimum induction temperature was studied. Furthermore, the expressed fusion protein was pu-rified by NI-NTA affinity chromatography and identified by SDS-PAGE and Western bloting. [Result]The PCR products of two site-specific mutagenesis were 462 and 6093 bp in length. After restriction enzyme digestion , recombination and transformation, pET-28a(+)-PTD-FNK plasmid was successfully constructed. The fusion protein was well expressed by inducing its gene with IPTG at 30 ℃ for 7 h. SDS-PAGE and Western bloting results showed the purified protein was PTD-FNK protein. [Conclusion]The soluble PTD-FNK fusion protein is expressed successfully by regulating induction temperature. Furthermore, it has a lots of advantages, such as shorter induction time, without refolding, easier protein purification, accelerating practical application of PTD-FNK protein in livestock semen cryopreservation. |
| Author | 王晓晔 石博妹 王英群 刘德玉 李铭 李芳芳 李珣 胡传活 |
| AuthorAffiliation | 广西大学动物科学技术学院,南宁530005 长江大学动物科学学院,湖北荆州434023 广西畜禽品种改良站,南宁530001 |
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| Author_FL | LIU De-yu WANG Xiao-ye SHI Bo-mei HU Chuan-huo WANG Ying-qun LI Ming LI Xun LI Fang-fang |
| Author_FL_xml | – sequence: 1 fullname: WANG Xiao-ye – sequence: 2 fullname: SHI Bo-mei – sequence: 3 fullname: WANG Ying-qun – sequence: 4 fullname: LIU De-yu – sequence: 5 fullname: LI Ming – sequence: 6 fullname: LI Fang-fang – sequence: 7 fullname: LI Xun – sequence: 8 fullname: HU Chuan-huo |
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| Copyright | Copyright © Wanfang Data Co. Ltd. All Rights Reserved. |
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| Keywords | prurification and identification site-specific mutagenesis 原核表达 点突变 PTD-FNK protein prokaryotic expression 纯化鉴定 PTD-FNK蛋白 |
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| Notes | WANG Xiao-ye1, SHI Bo-mei1,2, WANG Ying-qun3, LIU De-yu3, LIMing3, LI Fang-fang3, LI Xun1, HU Chuan-huo1（1College of Animal Science and Technology, Guangxi University, Nanning 530005, China; 2College of Animal Science, Yangtze University, Jingzhou, Hubei 434023, China; 3Guangxi Work Station of Livestock ＆ Poultry Breed Improvement, Nanning 530001, China） 45-1381/S PTD-FNK protein; site-specific mutagenesis; prokaryotic expression; prurification and identification [Objective]The mutant PTD-FNK protein of Bcl-xL protein was expressed in prokaryotic cell, in order to reveal anti-apoptosis mechanism and accelerate its application in livestock semen cryopreservation. [ Method ] Based on nu- cleotide sequence of FNKT protein, two primers were designed for site-specific mutagenesis, two mutated fragments were amplified by PCR using pET-28a（＋）-PTD-Bcl-xL plasmid as template. The extracted PCR products were digested by Dpn 1 to construct recombinant plasmid, and transformed into Escherichia coli DHSα. Then fusion protein |
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| PublicationTitle | 南方农业学报 |
| PublicationTitleAlternate | Journal of Southern Agriculture |
| PublicationYear | 2016 |
| Publisher | 长江大学 动物科学学院，湖北 荆州 434023%广西畜禽品种改良站,南宁,530001 广西大学 动物科学技术学院,南宁,530005%广西大学 动物科学技术学院，南宁 530005 |
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| Title | PTD-FNK蛋白的原核表达及纯化鉴定 |
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