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目的 应用基因工程技术表达甲状腺激素反应蛋白 1(TRP 1)。方法 应用RT PCR方法 ,从新生大鼠脑组织RNA中扩增编码TRP 1的cDNA片段 ,构建表达型重组质粒 ,经DNA序列分析确证后 ,在大肠杆菌中表达 ,以Western印迹来初步鉴定是否表达了目的融合蛋白 ,用亲和层析纯化融合目的蛋白 ,并以SDS PAGE电泳测定其相对分子质量。结果 所获特异PCR产物正确地重组入PinpointXa 1表达载体中。Western印迹表明经异丙基硫代半乳糖苷诱导的原核细胞表达产生了目的融合蛋白 ,亲和层析得到了纯度较高、相对分子质量约 2 3 4 0 0的融合目的蛋白。结论 应用原核细胞表达体系成功地表达了TRP 1融合蛋白 ,为进一步研究其在脑发育中的所起的作用以及该蛋白的其它功能提供了可能
Objective To express thyroid hormone reactive protein 1 (TRP 1) by genetic engineering. Methods RT-PCR method was used to amplify the cDNA encoding TRP 1 from the brain tissue of newborn rats. The recombinant plasmid was constructed and confirmed by DNA sequence analysis. The recombinant plasmid was expressed in E. coli and identified by Western blot For the purpose of fusion protein, the fusion protein was purified by affinity chromatography and its relative molecular mass was determined by SDS PAGE electrophoresis. Results The specific PCR products were correctly recombined into PinpointXa 1 expression vector. Western blotting showed that prokaryotic cells induced by isopropylthiogalactoside produced the fusion protein of interest, and the affinity protein obtained the fusion protein of high purity and relative molecular mass of about 2340 by affinity chromatography. Conclusion The successful expression of TRP 1 fusion protein by prokaryotic expression system provided a possibility to further investigate its role in brain development and other functions of this protein