甲状腺激素反应蛋白1的原核生物表达

Prokaryotic expression of thyroid hormone-response protein-1 gene

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 。

Objective To e xp ress thyroid hormone-response protein-1 (TRP-1) by gene engeneering technique . Methods The gene encoding TRP-1 of RNA from neonatal rat brain tissue was amplified by RT-PCR, and was subcloned into Pinpo int Xa-1 T vector to construct recombinant expression plasmid. After confirming the sequence and open reading frame by DNA sequencing, the expression of target protein in E. coli was induced by isopropyl β-D-thiogalacto pyranoside (IPTG ). The fusion protein was detected by Western blotting. Using avidin resin, the biotinylated fusion protein was purified under non-denaturing condition by affi nity chromatograph and its molecutar weight was measured by SDS-PAGE. Results The specific PCR products were correctly inserte d into restriction sites of pinpoint Xa-1 expression vector. The result of West ern blotting indicated that the fusion protein was expressed as expected, the fu sion protein was purified by affinity chromatograph, and SDS-PAGE showed that t he molecular weight of the TRP-1 fusion protein was 23400 as ca lculated according to its amino sequence. Conclusion Using prokaryocyte expression system, TRP-1 fusion protein is expressed, thus, it is possible to further investigate the function of this protein and its role in brain development and other tissues.