摘要
甲状腺激素受体(thyroid hormone receptor,TR)属于核受体超家族的成员,广泛分布在各种组织中,常与类视黄醇x受体(retinoid X receptor,RXR)组成TRs/RXR杂二聚体的形式,特异结合细胞核内靶基因序列中的甲状腺激素应答元件(thyroid hormone response elements,TREs)。TR存在2种主要的亚型：TRα和TRβ。TRa和TRβ基因的转录本(transcript)因选择性剪接或转录起始位置的不同而产生若干同功体(isoform)。在已报告过的大鼠TRs:TRα1、TRα2、TRα3、TRΔα1、TRΔα2、TRβ1、TRβ2、TRβ3和TRAβ3等9种亚型外,本室在研究甲状腺激素受体过程中又发现了一个新的同功体,被NCBI命名为TRβΔ,其mRNA仅较TRβ1的mRNA多出了一个108bp的片段,TRβΔ蛋白亦与TRβ1具有较高同源性,仅在近氨基端多出了36个氨基酸。TRβΔ蛋白是目前唯一的加长TRs亚型,具有非常重要的科学意义。
目的
为了检测我们新发现的一种甲状腺激素受体TRβΔ新亚型与RXRα、TREs(DR+4)结合的生物活性,我们设计了如下一些实验。
方法
1. pETDuet-1/rRXRα重组表达载体的构建。自本室已经构建好的重组质粒pETDuet-1/rTRβΔ/rRXRα中获取rRXRα的cDNA,并将其插入原核表达载体pETDuet-1, DNA序列分析鉴定。
2.重组质粒在E.coli BL21(DE3)中的诱导表达：重组表达质粒pETDuet-1/rRXRα转化入E.coli BL21(DE3),37℃, IPTG(1mmol/L)诱导表达6小时,SDS-PAGE鉴定表达产物大小和表达量,Western-blot进一步鉴定。重组表达质粒pETDuet-1/rTRβ1与pETDuet-1/rTRβΔ的IPTG诱导表达,并对二者的诱导表达产物进行SDS-PAGE、Western Blot鉴定。
3.免疫共沉淀(Co-immunoprecipitation,Co-IP)鉴定TRβ1/RXRα、TRβΔ/RXRα杂二聚体的形成。分别建立TRβ1-RXRα, TRβΔ-RXRα两种重组蛋白的复合物,分别以兔抗RXRα抗体或小鼠抗His抗体进行免疫共沉淀实验,沉淀物以小鼠抗His抗体或兔抗RXRα抗体进行Western Blot检测。
4.TRβΔ与DNA结合活性鉴定。先将TRβ1、TRβΔ蛋白分别与RXRα蛋白室温下共同孵育,然后运用电泳迁移率改变试验(EMSA)检测杂二聚体的DNA结合能力,同时检测单独的TRβΔ、TRβ1、RXRα蛋白分别与TREs(DR+4)的结合情况。
结果
1.成功构建了pETDuet-1/rRXRα重组表达载体。
2.成功表达了RXRα融合蛋白：重组质粒pETDuet-1/rRXRα转化菌、pETDuet-1/TRβ1转化菌和pETDuet-1/TRβΔ转化菌诱导表达产物的SDS-PAGE均在约55kD附近出现深染条带(RXRα约50kD,TRβ1约52kD,TRβ△约56kD),目的蛋白大小与预期基本一致,且主要存在于包涵体中。RXRα、TRβ1、TRβΔ的表达量分别达到213.8mg/L、251.0mg/L、161.9mg/L培养基,三种重组蛋白占菌体总蛋白的百分比分别为32.2%、30.5%、28.8%。
3.包涵体形式的RXRα、TRβ1、TRβΔ重组蛋白可溶解于高浓度的尿素溶液中,经透析后占上清液的主要部分。
4.免疫共沉淀证实TRp△及TRβ1均能够与RXRα蛋白分别形成TRβΔ/RXRα、TRβ1/RXRα杂二聚体。
5. TRβ1、TRβΔ两种蛋白与RXRα形成杂二聚体后均能够结合TREs (DR+4),而且单独的RXRα、TRβ1、TRβΔ蛋白也分别能够结合TREs (DR+4)。
结论
本文通过构建pETDuet-1/rRXRα重组表达载体成功表达了RXRα蛋白。结果证实甲状腺激素受体TRpΔ同TRβ1一样,能与RXRα形成杂二聚体结合DNA,而且TRβΔ、TRβ1和RXRa三者均具有DNA结合活性。我们的研究结果与Williams报道有所不同,据Williams其文中报道单独的TRβ1、RXRα蛋白均不能够结合TREs (DR+4),只有在TRβ1和RXRα共同孵育形成TRβ1/RXRα杂二聚体之后才能与TREs (DR+4)结合。而本文显示TRβΔ、TRβ1甚至RXRα本身单独也能结合DR+4,但它们是以单体抑或是同二聚体与DNA结合有待于进一步研究。
Thyroid hormone receptors (TRs) belong to the steroid/thyroid hormone receptor superfamily of ligand-dependent transcription factors and are distributed in almost all organs of the body. TRs often modulate transcriptional activity of target genes via heterodimerized with the 9-cis retinoic acid receptor (RXR). On positive thyroid response elements (TREs), RXR favors binding of the TR-RXR complex to DNA and stimulates transcription. TRs are coded by two genes, TRa and TRβ. Because of different transcript start position and alternative splicing, each subtype has several isoforms, including TRal, TRa2, TRa3, TR△α1, TRAa2, TRβ1, TRβ2, TRβ3, TRAβ3. TRPA is the unique extended-pattern TR isoforms which was found by our laboratory as a novel TRβisoform. It's mRNA/cDNA is only 108bps longer than that of TRβ1. The only difference between TRβ△and TRβ1 is an additional 36 amino-acid residues of TRβ△.
Objective:
In order to detect the novel thyroid hormone receptor isoform-TRPA could bind with RXRa as forming a heterodimer, and TRβ△/RXRa heterodimer has biological activity of binding target DNA, some experiments are disigned as following.
Methods:
1. Construction of recombinant expression vector pETDuet-1/rRXRa. RXRa target gene was obtained from recombinant plasmid pETDuet-1/rTRβ△/rRXRa,and then inserted into prokaryotic expression vector pETDuet-1, and tested by DNA sequencing.
2. The recombinant plasmids were transformed into E.coliBL21(DE3) and expressed.The recombinant plasmid pETDuet-1/rRXRαwas transformed into E.coli BL21(DE3) and induced with IPTG(1mmol/L) at 37℃for 6h. It's amount and molecular weight of the recombinant protein were tested by SDS-PAGE and Western blot. At the same time we also completed induced expression, SDS-PAGE and Western blot of pETDuet-1/rTRβ1 and pETDuet-1/rTRβ△.
3. Heterodimers of TRβ1/RXRa and TRβ△/RXRa were detected by Co-immunoprecipitation. Complexes of TRβ1-RXRa and TRβ△-RXRa were co-immunoprecipitated with Rabbit polyclonal to Retinoid X Receptor alpha or anti-His antibody.The immunocomplexes were detected by Western blot with anti-His or Retinoid X Receptor alpha antibody to identify the formation of heterodimers.
4. Biological activity assay of TRβ△. First TRβ1 and TRβ△were incubated separately with RXRa at room temperature,then their biological activity of binding target DNA was confirmed by electrophoretic mobility shift assay (EMSA).
Results:
1. We constructed recombinant expression vector pETDuet-1/rRXRa.
2. We expressed recombinant RXRa protein. RXRa,TRβ1 and TRβ△fusion proteins were expressed in Ecoli. Fusion proteins were analyzed by SDS-PAGE and Western blot.The result showed that fusion proteins were stained near 55kD (RXRa 50kD,TR(3156kD and TRβ△5kKD) and fusion proteins were mainly in inclusion bodies. The yield of RXRa,TRβ1 and TRβ△reached 213.8mg/L、251.0mg/L.161.9mg/L medium and accounted for 32.2%、30.5%、28.8% of total protein in E.coli BL21(DE3).
3. Inclusion body of RXRa, TRβ1 and TRβ△dissolved in a high concentration of urea solution and accounted for the major part of the supernatant.
4. Co-immunoprecipitation confirmed that recombinant protein TRβ△, like TRβ1, could bind with RXRa as forming a heterodimer.
5. Complexes of RXRa with TRβ1 or TRβ△resulted in binding of TRβ1/RXRa and TRβ△/RXRa heterodimers, could bind DNA when incubated with the element. RXRa, TRβ1 and TRβ△could also bind DNA when incubated alone with the element.
Conclusion:
In this study we constructed recombinant expression vector pETDuet-1/rRXRαand expressed fusion protein RXRa. Our result shows that TRβ△, like TRβ1, could bind with RXRa as forming a heterodimer and complexes of TRβ△/RXRa and TRβ1/RXRa could bind with DNA. Interestingly, TRβ△,TRβ1 and RXRa also have the biological activity of binding target DNA when incubated alone with the element. Our results are different from Williams's report. Williams reported that RXRa and TRβ1 failed to bind DNA when incubated alone with the element, and coincubation of RXR with TRβ1 resulted in binding of TRβ1-RXR heterodimers. Therefore, it is need to study the DNA-binding of TRβ△,TRβ1,and even RXRa alone furthermore.
引文
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