CAJ | 학술논문

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。
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.