电泳的原理、应用及进展（下）

三、核酸电泳 核酸(包括DNA和RNA)是一类带负电荷的生物大分子,在电场作用下可由负极向正极移动,因此可用电泳的方法进行分离、鉴定和纯化。核酸电泳根据所用凝胶介质的不同可以分两类,即琼脂糖凝胶电泳和聚丙烯酰胺凝胶电泳。琼脂糖电泳几乎都在水平板上进行,操作简单,快速有效;聚丙烯酰胺电泳多在垂直板中进行,主要用于某些特殊需要,如DNA序列分析电泳、寡核苷酸的分离鉴定。根据外加电场的不同,核酸电泳又分为恒定电场的常规电泳和脉冲场电泳。     

Structure Analysis of Streptococcal Protein G Fc Binding Domain

The gene fragment (191 bp) encoding protein G IgG Fc binding domain was isolated by PCR from group G streptococcus (CMCC32138), and a clone containing this gene fragment was found to give fine reactivity to human IgG when expressed in Escherichia coli. The complete nucleotide sequence of the gene fragment was determined. One base pair differs from previously reported protein Gnucleotide sequences, and resultsin an amino acid change (Ala-Thr), but this variation makes no difference in binding to the IgG Fc part by ELISA.The secondary structure of the protein G IgG Fc binding domain has been estimated by circular dichroism and assigned by computer algorithm.It shows a typical α-helix region in this domain.By breaking this α-helix region with recombinant DNA techniques, a 44 peptide, which contained the N-terminal 27 amino acid residues of this domain, was expressed in E. coli and showed no reactivity to IgG.The hydropathicity of this domain was also analyzed and compared with that of protein A relevant     

Engineered Bacterial Fc Receptors

Five new-type Fc receptor molecules were constructed based on streptococcal protein G (SpG) and staphylococcal protein A (SpA). These protein molecules contain one to six Fc binding domains to immunoglobulins which are structurally different from native SpG or SpA. Their expression levels reached 17-30% of the total bacterial proteins after heat induction in E. colt. Immunodiffusion and ELISA results showed that the engineered protein TG (184 amino acid residues) composed of three SpG C3 domain could bind more broadly and efficiently than the native SpG to the IgGs of human, goat, rabbit, etc. , and its optimal pH for binding became wider (pH5-8) compared with the SpG (pH5) ; and the protein TGA (357AA), fused by protein TG and the A, B, C domains of SpA, displayed both the binding pattern of SpG and SpA.     

电泳的原理、应用及进展（上）

一、概述 电泳(Electrophoresis)是一种带电粒子在电场中泳动的现象。早在1808年就已发现这一现象,但直到1937年瑞典的Tiselius建立了“自由电泳法”,成功地将血清蛋白分成5个主要成分,即清蛋白、α_1-、α_2-、β-和γ球蛋白,才使电泳技术在生化分析中得到应用。经过不断地努力,特别是近30年的发展,电泳     

人工构建的新型细菌Fc受体蛋白

本文构建了5个基于链球菌G蛋白(SpG)及金黄色葡萄球菌A蛋白(SpA)的新型Fc受体蛋白,它们分别含有1—6个免疫球蛋白Fc段结合区结构域,且在结构形式上亦不同于天然SpG或SpA。在大肠杆菌中,经热诱导它们的表达量占菌体总蛋白的17—30％。免疫双扩散及酶联实验结果表明,这种人工构建的由SpG C3结构域组成的Protein TG(184 AA)比天然SpG能更广谱、更有效地与人、羊、兔等IgG结合,且最佳结合pH也由SpG的pH5变为pH5—8;而由Protein TG与SpA的A,B,C 3个结构域融合的Protein TGA(357 AA)则兼并了SpG及SpA的结合谱。     

Protein G中Fc段结合区的结构分析

通过对用PCR技术从链球菌染色体中扩增出的Protein G中IgG Fc段结合区基因片段(191bP)的序列分析,我们发现其中有一个碱基与国外文献报道不同,由此导致一个氨基酸发生变化,即该结构域内Ala~(19)→Thr~(19),但这一变化并不影响其与IgG Fc段的结合。同时,我们还制备出一定量的由大肠杆菌表达的该活性多肽纯品,用圆二色谱仪测定了其二级结构组成,并用计算机做了进一步的预测分析,还同Protein A中相关结构域进行了比较,提出了Protein G与IgG Fc段相结合的可能机制。