抗对硫磷基因工程四价抗体的制备及其活性研究

发布时间：2018-06-11 21:30

本文选题：对硫磷 + 链霉亲和素　；参考：《中国农业科学院》2010年硕士论文

【摘要】： 本研究通过抗对硫磷单链抗体基因和核心链霉亲和素基因片段的拼接重组,获得抗对硫磷单链抗体-核心链霉亲和素融合基因(scfv-sa),并将该重组基因(scfv-sa)插入到表达载体pET28a(+)中,转化到大肠杆菌BL21(DE3)中进行原核表达,制备出融合蛋白,并对蛋白的生物学活性进行测定。主要研究内容为: 1.查阅链霉亲和素有关知识,分析其主要的功能性成分,检索编码该部分的基因序列,进行合成,其主要功能蛋白定义为核心链霉亲和素或链霉亲和素核心蛋白。 2.设计引物,扩增出抗对硫磷单链抗体的基因,并且用重叠延伸PCR的方法将其和核心链霉亲和素基因拼接,形成新的重组基因。经过酶切和PCR扩增验证其为预期的目的基因,送由公司测序确认。 3.构建重组基因的原核表达载体,优化原核表达中可溶性蛋白的形成条件,诱导重组基因进行原核表达。并回收可溶性蛋白,用Ni+-NTA纯化,SDS-PAGE检测蛋白分子量大小,Western blot鉴定表达产物为特异性的目的蛋白。结果表明,在大肠杆菌BL21(DE3)中该融合基因能表达出分子量约为46kDa的融合蛋白,形成了四价结构域-四价聚合抗体。 4.用ELISA方法测定该融合抗体生物学活性,ELISA测定结果表明该抗体能与对硫磷特异结合,抗体效价在1:1×106以上,亲和常数为4.25×107 L /mol。制备的抗对硫磷四价聚合抗体能与抗原特异结合,与单克隆抗体相比,抗原结合位点显著增加,ELISA检测灵敏度显著提高。
[Abstract]:The fusion protein was prepared by prokaryotic expression and the biological activity of the fusion protein was determined. The main research contents are as follows: 1. Primers were designed to amplify the parathion single-chain antibody (scFv) gene and splice it with core-streptavidin gene by overlapping extension PCR to form a new recombinant gene. After restriction endonuclease digestion and PCR amplification, the gene was confirmed as the intended target gene, and was sent to the company for confirmation by sequencing. 3. 3. The prokaryotic expression vector of the recombinant gene was constructed to optimize the formation conditions of soluble protein in prokaryotic expression and to induce the prokaryotic expression of the recombinant gene. The soluble protein was recovered and its molecular weight was determined by SDS-PAGE. The expressed protein was identified as a specific protein by Western blot. The results showed that the fusion gene could express a fusion protein with a molecular weight of about 46kDa in E. coli BL21DDE3, forming tetravalent domain tetravalent polymerized antibody. The biological activity of the fusion antibody was determined by Elisa. The results showed that the antibody could specifically bind to parathion. The titer of the antibody was above 1:1 脳 106, and the affinity constant was 4.25 脳 107 L / mol. The anti-parathion tetravalent polymerized antibody could specifically bind to the antigen, and compared with the monoclonal antibody, the antigen-binding site significantly increased the sensitivity of Elisa.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R341

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