亚洲带绦虫60S核糖体蛋白L8基因的克隆表达和免疫学分析

目的原核克隆表达亚洲带绦虫成虫60S核糖体蛋白L8基因(TaRPL8),探索其应用前景。方法用RT-PCR方法从亚洲带绦虫成虫cDNA中获取RPL8基因,克隆到原核表达质粒pET-28a(+)中,在大肠埃希菌BL21/DE3中用IPTG诱导表达,表达产物通过SDS-PAGE进行鉴定,用镍离子金属螯合剂亲和层析柱进行纯化,纯化的重组蛋白用蛋白印迹进行免疫学分析。结果 PCR、双酶切及DNA测序结果均表明pET-28a(+)-TaRPL8重组质粒构建成功。SDS-PAGE结果表明目的基因在大肠埃希菌BL21/DE3中获得高效表达,亲和层析获得了高纯度蛋白。重组蛋白可被其免疫的SD大鼠血清识别但不能够被正常大鼠血清识别,表明其具有免疫原性。结论亚洲带绦虫成虫TaRPL8基因可在大肠埃希菌BL21/DE3中获得具有免疫原性的表达,为进一步的功能研究奠定了基础。

Cryo-EM structure of an early precursor of large ribosomal subunit reveals a half-assembled intermediate

Assembly of eukaryotic ribosome is a complicated and dynamic process that involves a series of intermediates.It is unknown how the highly intertwined structure of 60S large ribosomal subunits is established.Here,we report the structure of an early nucleolar pre-60S ribosome determined by cryo-electron microscopy at 3.7 A resolution,revealing a half-assembled subunit.DomainsⅠ,ⅡandⅣof 25S/5.8S rRNA pack tightly into a native-like substructure,but domains Ⅲ,ⅣandⅤare not assembled.The structure contains 12 assembly factors and 19 ribosomal proteins,many of which are required for early processing of large subunit rRNA.The Brx1-Ebp2 complex would interfere with the assembly of domains Ⅳ and Ⅴ.Rpf1,Mak16,Nsa1 and Rrp1 form a cluster that consolidates the joining of domainsⅠandⅡ.Our structure reveals a key intermediate on the path to establishing the global architecture of 60S subunits.

人源MDN1蛋白质的电镜结构研究

目的·利用负染电镜技术分析人源midasin AAA-ATPase 1(MDN1,又称Rea1)蛋白质结构。方法·利用CRISPR/Cas9基因编辑方法在Expi293F细胞内源MDN1蛋白质的氨基端敲入3×FLAG纯化标签,采用ANTI-FLAG^(®)M2 Agarose Affinity Gel亲和层析和甘油密度梯度离心的方法分离纯化目的蛋白质;通过负染色电镜技术和单颗粒(single-particle)重构技术探究人源MDN1蛋白质结构。结果·利用亲和层析及密度梯度离心方法分离纯化获得高纯度、均一性较好的人源MDN1蛋白质样品;采用甲酸铀负染色后利用120 kV电镜初步解析了目的蛋白质MDN1的空间结构。结论·利用单颗粒重构技术搭建了人源MDN1蛋白质的低分辨率的负染模型。