大鼠癌钙调蛋白基因原核表达载体的构建及表达

摘要

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.%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.