CAJ | 학술논문

目的在原核表达载体中构建癌钙调蛋白(OM)基因,并对构建的原核表达质粒进行表达和鉴定.方法取SD大鼠6只,提取腹腔巨噬细胞并活化,提取总RNA,设计引物对OM基因进行RT-PCR扩增.通过中间载体pUC57进行目的基因克隆,将阳性克隆质粒酶切,与原核表达载体pET-22b(+)连接,转化入大肠杆菌感受态细胞DH5α,菌检PCR筛选阳性克隆后小量提取质粒,送样行核苷酸序列分析鉴定.成功构建的原核表达质粒,在大肠杆菌BL21中经异丙基-β-D-硫代半乳糖苷(IPTG)诱导重组体的表达,并用Western blot鉴定表达产物.结果琼脂糖凝胶电泳获得目的基因OM,大小为350 bp左右,与预期的目的基因大小一致.构建的重组质粒pET-22b(+)/OM菌检PCR获得长度为500 bp左右的阳性克隆,与预期大小一致.抽提质粒经核苷酸序列分析后表明,克隆的片段与OM基因序列一致.在IPTG的诱导下,重组体表达出分子量约11.7 kD的表达产物,Western blot结果证实其为目的蛋白.结论实验成功构建了OM基因原核表达载体及其表达,为该蛋白促进视神经损伤后再生的研究奠定了基础.
목적 재원핵표체재체중구건암개조단백(OM)기인,병대구건적원핵표체질립진행표체화감정.방법 취SD대서6지,제취복강거서세포병활화,제취총RNA,설계인물대OM기인진행RT-PCR확증.통과중간재체pUC57진행목적 기인극륭,장양성극륭질립매절,여원핵표체재체pET-22b(+)련접,전화입대장간균감수태세포DH5α,균검PCR사선양성극륭후소량제취질립,송양행핵감산서렬분석감정.성공구건적원핵표체질립,재대장간균BL21중경이병기-β-D-류대반유당감(IPTG)유도중조체적표체,병용Western blot감정표체산물.결과 경지당응효전영획득목적 기인OM,대소위350 bp좌우,여예기적목적 기인대소일치.구건적중조질립pET-22b(+)/OM균검PCR획득장도위500 bp좌우적양성극륭,여예기대소일치.추제질립경핵감산서렬분석후표명,극륭적편단여OM기인서렬일치.재IPTG적유도하,중조체표체출분자량약11.7 kD적표체산물,Western blot결과증실기위목적 단백.결론 실험성공구건료OM기인원핵표체재체급기표체,위해단백촉진시신경손상후재생적연구전정료기출.
Objective To construct and express the prokaryotic recombinant vectors for rat oncomodulin gene, and to identify their expression. Methods Peritoneal macrophages were extracted from six selected SD rats and activated. Total RNA was extracted from activated rat macrophages,and design primers to obtain whole length of oncomodulin gene by RT-PCR. The oncomodulin gene was cloned into pUC57 vector. Then the gene was inserted into pET-22b(+) expression vector and and the inserting plasmid was transformed into Escherichia coli. host DH5α. The positive clone was characterized by PCR examination of bacterium liquid pET-22b(+)/OM and DNA sequence analysis.Then the recombinant plasmids were transformed into E. coli BL21 and the expressions of recombinant plasmids were induced by adding isopropylthiogalactoside (IPTG). The expression product was identified by Western blot assay. Results The target DNA sequence of oncomodulin was obtained by RT-PCR which was about 350 bp length, exactly in accordance with prospective. The positive clones about 500 bp length were obtained by PCR examination of bacterial colony pET-22b(+)/OM plasmid, which were consistent with the expected size. The result of DNA sequence analysis for recombinant plasmids extraction revealed that the gene encoding oncomodulin was coloned into vector pET-22b(+)accurately. The protein bands with the molecular weight of about 11.7 kD were induced by IPTG in the recombinant plasmids. Western blot assay revealed that the protein was OM. Conclusion The prokaryotic recombinant vectors for rat oncomodulin gene is successfully constructed and expressed. It might provide a foundation for further study on the functional test of oncomodulin that can promote regeneration of damaged optic nerve.