牛支原体武威株fba基因的克

发布时间：2018-05-27 00:13

本文选题：牛支原体 + 醛缩酶　；参考：《农业生物技术学报》2017年02期

【摘要】：牛支原体(Mycoplasma bovis,Mb)是牛(Bos taurus)的一种重要致病性支原体,其感染可引起牛的多种疾病。果糖二磷酸醛缩酶(fructose bisphosphate aldolase,FBA)是参与糖酵解过程中的关键酶,为3-磷酸甘油醛脱氢反应提供底物,并广泛存在于生物界中。为了研究FBA在Mb细胞内的分布,本研究参照Gen Bank中Mb PG45株fba基因序列设计特异性引物,应用PCR扩增获得Mb武威株fba基因并将其克隆至p MD19-T载体,在完成测序及基因优化的基础上,构建原核表达载体p ET-fba并转化大肠杆菌(Escherichia coli)表达菌Transetta(DE3)后经异丙基硫代半乳糖苷(isopropylβ-D-1-thiogalactopyranoside,IPTG)诱导表达,表达产物纯化后免疫新西兰兔(Oryctolagus cuniculus)制备多克隆抗体,并采用间接酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)测定抗体效价。进而应用Western blot对Mb FBA在细胞内的分布进行了初步的定位。结果表明,Mb武威株fba基因编码框全长876 bp(Gen Bank登录号:KX828563),编码292个氨基酸,优化后的基因在大肠杆菌中成功获得表达,重组蛋白r Mb FBA大小约为34 k D,主要以可溶性蛋白的形式存在;ELISA测定制备的多克隆抗体效价为1∶25 600,Western blot分析结果显示,该蛋白具有良好的免疫原性,且在Mb胞浆及膜表面均有分布。上述结果为进一步研究Mb FBA的生物学功能提供了理论依据。
[Abstract]:Mycoplasma bovismb) is an important pathogenic mycoplasma of Bos taurus, and its infection can cause many diseases in cattle. Fructose bisphosphate aldolase (FBA) is a key enzyme involved in glycolysis, which provides a substrate for the dehydrogenation of glyceraldehyde 3-phosphate, and is widely found in the biological world. In order to study the distribution of FBA in Mb cells, a specific primer was designed according to the fba gene sequence of Mb PG45 strain in Gen Bank. The fba gene of Mb Wuwei strain was amplified by PCR and cloned into pMD19-T vector. On the basis of complete sequencing and gene optimization, the prokaryotic expression vector p ET-fba was constructed and transformed into E. coli Escherichia coli expression strain TransettaDE3). The expression was induced by isopropyl 尾 -D-thiogalactopyranoside side ET-fba (isopropyl 尾 -D-thiogalactopyranoside). The polyclonal antibody was prepared by immunizing Oryctolagus cuniculus after purification and the titer of the antibody was determined by indirect enzyme-linked immunosorbent assay (Elisa). Furthermore, the distribution of Mb FBA in cells was preliminarily localized by Western blot. The results showed that the fba gene encoding frame of Mb Wuwei strain was 876 bp(Gen Bank accession number: KX828563G, encoding 292 amino acids. The optimized gene was successfully expressed in E. coli. The r Mb FBA of the recombinant protein was about 34 KD, and the antibody titer of polyclonal antibody determined by Elisa in the form of soluble protein was 1:25. The results of Western blot analysis showed that the protein had good immunogenicity. The distribution of Mb on the cytoplasm and on the surface of the membrane was also observed. These results provide a theoretical basis for further study of the biological function of Mb FBA.
【作者单位】：甘肃农业大学动物医学院;
【基金】：国家自然科学基金(No.31360620) 甘肃省高等学校基本科研业务费项目
【分类号】：S852.62

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