Marcipar Iván S, Olivares María Laura, Robles Lucía, Dekanty Andrés, Marcipar Alberto, Silber Ariel M
Instituto de Tecnología Biológica (INTEBIO), Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral, Santa Fe, Argentina.
Protein Expr Purif. 2004 Mar;34(1):1-7. doi: 10.1016/j.pep.2003.11.022.
In the present work, we have determined the effect of expression vectors and their corresponding host bacteria on the antigenic performance of Trypanosoma cruzi P2beta (TcP2beta) full-length recombinant protein. The gene encoding the TcP2beta ribosomal protein was cloned in pMAL-c2 and pET-32a vectors that allow the expression of high levels of soluble fusion proteins. A panel of 32 positive and 32 negative sera was assayed with the purified proteins expressed using pMal-c2 (TcP2beta-MBP) and pET-32a (TcP2beta-TRX) vectors and with MBP and TRX purified from pMAL-c2 and pET-32a vectors, respectively. The antigenic behavior of each TcP2beta recombinant protein differed in the diagnostic performance in terms of DI(+) (93.7 for TcP2beta-MBP vs 100% for TcP2beta-TRX), in DI(-) (90.5 for TcP2beta-MBP vs 100% for TcP2beta-TRX) and in cross-reaction with negative sera. To determine if the higher reactivity of expressed pMAL-c2 protein was due to folding during protein expression or to a steric effect related to the protein adsorption at the titration plate, the reactivity of sera against soluble proteins was assessed by ELISA inhibition assays. As each soluble protein preserved its level of reactivity, we concluded that differences in reactivity were due to intrinsic characteristics of the proteins and not to differences in patterns of adsorption to the plates.
在本研究中，我们确定了表达载体及其相应宿主细菌对克氏锥虫P2β（TcP2β）全长重组蛋白抗原性能的影响。编码TcP2β核糖体蛋白的基因被克隆到pMAL-c2和pET-32a载体中，这两种载体能够表达高水平的可溶性融合蛋白。使用pMal-c2（TcP2β-MBP）和pET-32a（TcP2β-TRX）载体表达的纯化蛋白，以及分别从pMAL-c2和pET-32a载体中纯化的MBP和TRX，对一组32份阳性血清和32份阴性血清进行了检测。每种TcP2β重组蛋白的抗原行为在诊断性能方面存在差异，在DI(+)方面（TcP2β-MBP为93.7%，TcP2β-TRX为100%），在DI(-)方面（TcP2β-MBP为90.5%，TcP2β-TRX为100%），以及与阴性血清的交叉反应方面。为了确定表达的pMAL-c2蛋白的较高反应性是由于蛋白质表达过程中的折叠，还是与蛋白质在滴定板上的吸附有关的空间效应，通过ELISA抑制试验评估了血清对可溶性蛋白的反应性。由于每种可溶性蛋白都保持了其反应水平，我们得出结论，反应性的差异是由于蛋白质的内在特性，而不是与平板吸附模式的差异有关。
