PNAi相关基因RDE-4表达、蛋白纯化及结晶
发布时间：2018-08-20 17:56
【摘要】：根据已报道的RDE-4基因序列,设计一对引物,从含C.elegance的cDNA文库中,通过PCR扩增得到大小为1158nt的RDE-4基因,并把它重组到细菌表达载体pET-21b(+)中,该载体C-端具有6个组氨酸标记多肽。酶切鉴定及序列测定表明,重组表达质粒pET-21b-RDE-4连接区域符合设计要求,具有正确的开放阅读框架,插入片段含有385个氨基酸的完整编码区,其核苷酸序列与报道的RDE-4基因的同源性为100%。重组表达载体导入宿主菌E.coli BL21(DE3),用终浓度为3mmol/L IPTG诱导,37℃培养6小时后,RDE-4基因的表达量达到最高,每毫升菌液含目的蛋白的量约为2mg。通过大量培养,利用亲和纯化和FPLC纯化技术将RDE-4基因蛋白纯化、浓缩,并用PEG等条件养晶,进行条件筛选。全长基因蛋白晶形不好,进而用胰蛋白酶对RDE-4基因蛋白限制性酶切,通过NI-ATK亲和柱层析,判断目的蛋白的稳定结构域在N-端。根据以上分析结果,重新设计N-端引物,亚克隆RDE-4(1-280Aa,315Aa)基因片段,并将它们插入GEX-6P-1原核载体,该载体N-端带有GST融合标签,构建pGEX-6P-1-RDE-4(1-280Aa,315Aa)表达载体。重组表达载体导入E.coli BL21(DE3),用IPTG诱导高效表达。通过GST-column亲和层析,纯化RDE-4(1-280Aa)基因片段蛋白,并用PPase在柱上对其进行限制性酶切,一般过夜后洗脱、10KD孔径的超滤管浓缩至1mL,注射器被动注入FPLC上样孔,通过凝胶分子筛进一步纯化,获得高浓度和纯度蛋白,浓度为50mg/mL。用悬吊液滴蒸汽扩散法和40%PEG-600,0.1MCHES,pH6.0[Cryoll screen kit,Enedd Biostructure]加入stock Options~(TM) pH screen(Hampton Research)蛋白质养晶条进行养晶,一星期,晶体成规则八面体,通过X-Ray衍射,收到晶体电子密度图及相关数据。本论文研究为进一步研究RNAi的作用机制奠定基础,为研究在此过程中相关蛋白之间的相互作用提供结构依据。
[Abstract]:According to the reported RDE-4 gene sequence, a pair of primers were designed. The RDE-4 gene of 1158nt was amplified by PCR from the cDNA library containing C.elegance. The RDE-4 gene was recombined into the bacterial expression vector pET-21b (), which had six histidine labeled peptides at the C-terminal. The restriction endonuclease analysis and sequencing showed that the pET-21b-RDE-4 ligation region of the recombinant plasmid met the design requirements and had a correct open reading frame. The inserted fragment contained a complete coding region of 385 amino acids. Its nucleotide sequence is 100% homology with the reported RDE-4 gene. The recombinant expression vector was introduced into the host strain E.coli BL21 (DE3). After 6 hours of incubation at 37 鈩，
本文编号：2194514
[Abstract]:According to the reported RDE-4 gene sequence, a pair of primers were designed. The RDE-4 gene of 1158nt was amplified by PCR from the cDNA library containing C.elegance. The RDE-4 gene was recombined into the bacterial expression vector pET-21b (), which had six histidine labeled peptides at the C-terminal. The restriction endonuclease analysis and sequencing showed that the pET-21b-RDE-4 ligation region of the recombinant plasmid met the design requirements and had a correct open reading frame. The inserted fragment contained a complete coding region of 385 amino acids. Its nucleotide sequence is 100% homology with the reported RDE-4 gene. The recombinant expression vector was introduced into the host strain E.coli BL21 (DE3). After 6 hours of incubation at 37 鈩，
本文编号：2194514
本文链接：https://www.wllwen.com/yixuelunwen/binglixuelunwen/2194514.html
最近更新
教材专著
