东南大学学报(医学版)
東南大學學報(醫學版)
동남대학학보(의학판)
JOURNAL OF SOUTHEAST UNIVERSITY(MEDICAL SCIENCE EDITION)
2013年
4期
408-412
,共5页
陈洋%何泽%张永臣%万青%赵虎子%赵蕾%沈传陆
陳洋%何澤%張永臣%萬青%趙虎子%趙蕾%瀋傳陸
진양%하택%장영신%만청%조호자%조뢰%침전륙
MLC2%原核表达%GST融合蛋白%蛋白纯化
MLC2%原覈錶達%GST融閤蛋白%蛋白純化
MLC2%원핵표체%GST융합단백%단백순화
myosin%prokaryotic expression%GST fusion protein%protein purification
目的:构建pGEX-MLC2重组质粒,在大肠杆菌中表达并纯化谷胱甘肽硫转移酶( GST )-MLC2融合蛋白,以用于MLC2抗体的制备。方法:从人乳腺癌MCF-7细胞中提取总RNA,通过RT-PCR获得人MLC2 cDNA全长开放读码框架(open reading frame, ORF),并将其重组于原核蛋白表达质粒pGEX-6P-1中,经限制性内切酶和DNA测序鉴定,将该重组质粒转化大肠杆菌E.coli BL21,用异丙基β-D-硫代半乳糖苷( IPTG)诱导表达 GST-MIC2融合蛋白后,进行 Western blotting 分析,鉴定 GST-MLC2蛋白;通过 Glutathione Sepharose 4B亲和层析柱纯化融合蛋白,并用SDS-PAGE电泳鉴定该融合蛋白的纯度。结果:人乳腺癌MCF-7细胞MLC2的RT-PCR产物为535 bp,符合预期;重组质粒pGEX-MLC2经BamHI和EcoRI双酶切出现特征性的525 bp片段;DNA测序证实MLC2全长ORF序列正确无误。 IPTG诱导表达的GST-MLC2融合蛋白分子质量约为46.6 kDa,纯化后其蛋白纯度约95%,经Western blotting 分析表明诱导表达的蛋白是GST-MLC2融合蛋白。结论:人MLC2蛋白原核表达质粒的构建、GST-MLC2融合蛋白的表达和纯化,为MLC2抗体的制备及MLC2功能的深入研究奠定了基础。
目的:構建pGEX-MLC2重組質粒,在大腸桿菌中錶達併純化穀胱甘肽硫轉移酶( GST )-MLC2融閤蛋白,以用于MLC2抗體的製備。方法:從人乳腺癌MCF-7細胞中提取總RNA,通過RT-PCR穫得人MLC2 cDNA全長開放讀碼框架(open reading frame, ORF),併將其重組于原覈蛋白錶達質粒pGEX-6P-1中,經限製性內切酶和DNA測序鑒定,將該重組質粒轉化大腸桿菌E.coli BL21,用異丙基β-D-硫代半乳糖苷( IPTG)誘導錶達 GST-MIC2融閤蛋白後,進行 Western blotting 分析,鑒定 GST-MLC2蛋白;通過 Glutathione Sepharose 4B親和層析柱純化融閤蛋白,併用SDS-PAGE電泳鑒定該融閤蛋白的純度。結果:人乳腺癌MCF-7細胞MLC2的RT-PCR產物為535 bp,符閤預期;重組質粒pGEX-MLC2經BamHI和EcoRI雙酶切齣現特徵性的525 bp片段;DNA測序證實MLC2全長ORF序列正確無誤。 IPTG誘導錶達的GST-MLC2融閤蛋白分子質量約為46.6 kDa,純化後其蛋白純度約95%,經Western blotting 分析錶明誘導錶達的蛋白是GST-MLC2融閤蛋白。結論:人MLC2蛋白原覈錶達質粒的構建、GST-MLC2融閤蛋白的錶達和純化,為MLC2抗體的製備及MLC2功能的深入研究奠定瞭基礎。
목적:구건pGEX-MLC2중조질립,재대장간균중표체병순화곡광감태류전이매( GST )-MLC2융합단백,이용우MLC2항체적제비。방법:종인유선암MCF-7세포중제취총RNA,통과RT-PCR획득인MLC2 cDNA전장개방독마광가(open reading frame, ORF),병장기중조우원핵단백표체질립pGEX-6P-1중,경한제성내절매화DNA측서감정,장해중조질립전화대장간균E.coli BL21,용이병기β-D-류대반유당감( IPTG)유도표체 GST-MIC2융합단백후,진행 Western blotting 분석,감정 GST-MLC2단백;통과 Glutathione Sepharose 4B친화층석주순화융합단백,병용SDS-PAGE전영감정해융합단백적순도。결과:인유선암MCF-7세포MLC2적RT-PCR산물위535 bp,부합예기;중조질립pGEX-MLC2경BamHI화EcoRI쌍매절출현특정성적525 bp편단;DNA측서증실MLC2전장ORF서렬정학무오。 IPTG유도표체적GST-MLC2융합단백분자질량약위46.6 kDa,순화후기단백순도약95%,경Western blotting 분석표명유도표체적단백시GST-MLC2융합단백。결론:인MLC2단백원핵표체질립적구건、GST-MLC2융합단백적표체화순화,위MLC2항체적제비급MLC2공능적심입연구전정료기출。
Objective: To construct pGEX-MLC2 recombinant plasmid and to express and purify GST-MLC2 fusion protein .The fusion protein was prepared for MLC 2 polyclonal antibody .Methods: The total RNA was extracted from human breast cancer cells MCF-7, the full-length open reading frame ( ORF) of MLC2 cDNA was amplified by RT-PCR and then was cloned into the expression vector pGEX-6P-1.After identification by restriction enzyme digestion analysis and DNA sequencing , the recombinant clone was transformed into the competent cells E. coli BL21.GST-MLC2 fusion protein was induced expression by IPTG and was identified by Western blotting;Then the fusion protein was purified by Glutathione Sepharose 4 B affinity chromatography and the purity was identified by SDA-PAGE electrophoresis .Results: The RT-PCR product of MLC2 was 535 bp in line with expectations; The recombinant plasmid of pGEX-MLC2 was identified containing a distinctive 525 bp fragment by BamH I and EcoR I double digestion;DNA sequencing confirmed that the MLC 2 full-length ORF sequence was correct .The molecular weight of GST-MLC2 fusion protein by IPTG induced expression was about 46.6 kDa and the purity of GST-MLC2 fusion protein was about 95%after purification .Western blotting analysis proved that the protein was GST-MLC2 fusion protein .Conclusion:The construction of prokaryotic expression plasmid for human MLC 2 protein and the expression and purification of GST-MLC2 fusion protein lay the foundations for the preparation of MLC 2 antibody and the further study on the function of MLC 2.