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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。
Mycoplasma bovis (Mb) is an important pathogenic mycoplasma of Bos taurus, and its infection can cause many diseases of cattle. Fructose bisphosphate aldolase (FBA), a key enzyme involved in glycolysis, supplies substrates for dehydrogenation of glyceraldehyde-3-phosphate and is widely found in the organism. In order to study the distribution of FBA in Mb cells, specific primers were designed according to the fba gene sequence of Mb PG45 in Gen Bank. The fba gene of Mb Wuwei strain was obtained by PCR amplification and cloned into pMD19-T vector. Sequencing, and gene optimization, the prokaryotic expression vector p ET-fba was constructed and transformed into Transetta (DE3) of Escherichia coli. The recombinant plasmid was induced by isopropylβ-D-1-thiogalactopyranoside ), And polyclonal antibody was prepared by immunization of New Zealand rabbit (Oryctolagus cuniculus) after purification. The antibody titer was determined by enzyme linked immunosorbent assay (ELISA). Furthermore, the localization of Mb FBA in the cell was preliminarily determined by Western blot. The results showed that the full-length coding sequence of fba gene in Mb Wuwei strain was 876 bp (GenBank accession number: KX828563), encoding 292 amino acids. The optimized gene was successfully expressed in E. coli. D, mainly in the form of soluble protein. The titer of polyclonal antibody prepared by ELISA was 1:25 600. The result of Western blot showed that the protein had good immunogenicity, distributed. These results provide a theoretical basis for further study on the biological function of Mb FBA.