Brito C F, Fonseca C T, Goes A M, Azevedo V, Simpson A J, Oliveira S C
Department of Biochemistry and Immunology, Belo Horizonte-MG, São Paulo, Brazil.
Parasite Immunol. 2000 Jan;22(1):41-8.
The Schistosoma mansoni gene coding for a 14-kDa fatty acid-binding protein was amplified by PCR and subcloned into the prokaryotic expression vector pMAL-c2. Escherichia coli DH5alpha was transformed with the pMAL-Sm14 construct, and gene expression was induced hr isopropyl-beta-D-thiogalactopyranoside. The resulting recombinant (r) fusion protein was purified by affinity chromatography and confirmed by immunoblot analysis using antimaltose-binding protein or anti-Sm14 antibodies. Additionally, an antibody isotype profile was determined in sera of schistosomiasis patients to rSm14 or soluble adult worm antigen preparation. IgG1 and IgG3 subclass antibodies to rSm14 were predominant in sera of all patients studied whereas low levels of IgM, IgA or IgE were measured. Expression of a S. mansoni gene encoding a vaccine candidate is an important step to better study human immune responses to defined antigens.
通过聚合酶链反应（PCR）扩增编码14 kDa脂肪酸结合蛋白的曼氏血吸虫基因，并将其亚克隆到原核表达载体pMAL-c2中。用pMAL-Sm14构建体转化大肠杆菌DH5α，并用异丙基-β-D-硫代半乳糖苷诱导基因表达。通过亲和层析纯化得到的重组（r）融合蛋白，并用抗麦芽糖结合蛋白或抗Sm14抗体进行免疫印迹分析进行确认。此外，还测定了血吸虫病患者血清中针对rSm14或可溶性成虫虫体抗体制剂的抗体亚型谱。在所有研究患者的血清中，针对rSm14的IgG1和IgG3亚类抗体占主导，而检测到的IgM、IgA或IgE水平较低。曼氏血吸虫编码候选疫苗基因的表达是更好地研究人类对特定抗原免疫反应的重要一步。
