抗芽殖酵母端粒G4-DNA结构的单克隆抗体和基因工程单链抗体片段的制备和定性研究

大多数真核生物端粒3'末端由富含鸟苷酸的重复序列组成,并可以在体外形成四链G4- DNA结构。为了解这种结构是否在体内存在,本文中我们以芽殖酵母作为研究对象,将G4-DNA作为抗原免疫BALB／c小鼠,制备抗G4-DNA的单克隆抗体,结果显示该抗体能够特异性识别富含鸟苷酸重复序列DNA。为了提高抗体的特异性,我们通过基因工程制备抗体:利用RT-PCR的方法,得到抗体重链和轻链可变区的基因,然后克隆到载体pET22b中得到表达质粒pET22b-scFv,转入大肠杆菌进行表达。在细胞周质中我们检测并纯化到了目的基因的表达产物。另外,我们还利用该基因序列进行了初步的结构分析。基因工程抗体在大肠杆菌中的成功表达及结构分析为今后利用该抗体进行定点突变来研制高特异性和亲和力的抗G4-DNA抗体奠定了基础。

GENERATION AND CHARACTERIZATION OF THE MONOCLONAL ANTIBODY AND scFv AGAINST YEAST TELOMERIC GUANINE-QUADRUPLEX DNA

Most eukaryotic telomeres contain many tandem repeats of G-rich sequence. In budding yeast,Sacchromyces cerevisiae,the G-rich sequence can form parallel-stranded quadruplex comformation (G4-DNA) in vitro. Whether this structure exsits in vivo is unknown. To address this question,we generated the antibodies against the G4-DNA of the S.cerevisiae by immunizing the BALB/c mouse with in vitro synthesized G4-DNA oligonucleotides. The antibodies recognize G4-DNA,as well as G-rich DNA sequence in vitro. In order to improve the affinity to G4-DNA substrate, we cloned VH and VL genes of the antibodies and constructed the single-chain antibody fragment (scFv) with a peptide linker. The recombinated scFv was successfully expressed in E.coli and purified by the affinity chromotography. Based on the sequence of the scFv,we proposed the structure of the antibody by computer-remodeling. The engineered antibodies will be used to detect the existence of the G4-DNA structure in vivo.