支气管败血波氏杆菌皮肤坏死毒素的重组表达及其生物学特性

Expression and characterization of the dermonecrotic toxin gene of Bordetella bronchiseptica

摘要:

皮肤坏死毒素 (DNT) 是支气管败血波氏杆菌的主要致病因子之一。通过PCR分段扩增和克隆获得了全长4 356 bp的dnt基因，并利用pET-28a/BL21系统对其进行了融合表达。Western blotting检测结果表明，表达产物具有良好的免疫学活性。使用His-band purification kit纯化试剂盒纯化后，得到纯度为93.2％的融合蛋白His6-DNT。在乳鼠皮肤坏死试验中，表达产物His6-DNT和天然DNT均能导致乳鼠皮肤产生坏死性病变。在乳鼠皮肤坏死阻断试验中，兔抗His6-DNT血清能中和天然DNT使其失去对乳鼠的皮肤坏死毒性。试验结果表明，重组蛋白具有天然DNT的生物学毒性和免疫原性，所产生的抗体具有中和活性。文中所获得的重组DNT蛋白具有良好的生物学活性，为DNT的结构和功能研究奠定基础。

Abstract:

Dermonecrotic toxin (DNT) is identified as one of the most important virulence factor of Bordetella bronchiseptica. The complete coding sequence (4 356 bp) of the dnt gene was cloned into the prokaryotic expression vector pET-28a, and expressed in the Eschierichia coli BL21 (DE3) under IPTG (Isopropyl-β-D-thiogalactopyranoside) induction. The recombinant His6-DNT protein showed immunological reactivity in the Western-blot analysis. The recombinant protein was purified from crude lysates of BL21 harboring pET-DNT with the purity of 93.2%. His6-DNT showed the dermonecrotic effects in the infant mouse assay. However, rabbit anti-serum against recombinant DNT protein could neutralize the dermonecrotic effects of native DNT to the infant mice in vivo. These findings suggest that the recombinant DNT protein retained the characteristics and immunogenicity of native DNT. Furthermore, this approach could be used to induce active immunity and serum immunoglobulin for production of a passive therapeutic reagent. In this study, we have shown that the recombinant His6-DNT protein retained the characteristics of native DNT of B. bronchiseptica, which built a good foundation for the further research on the structure and function of DNT.