Identification and molecular analysis of a stress-inducible Hsp70 from Sciaenops ocellatus

Hsp70 proteins are a family of molecular chaperones that are involved in many aspects of protein homeostasis. In this study, an Hsp70 homologue (SoHsp70) was identified from red drum Sciaenops ocellatus and analyzed at molecular level. The open reading frame of SoHsp70 is 1920 bp and intronless, with a 5'-untranslated region (UTR) of 399 bp and a 3'-UTR of 241 bp. The deduced amino acid sequence of SoHsp70 shares 84-92% overall identities with the Hsp70s of a number of fish species. In silico analysis identified in SoHsp70 three conserved Hsp70 domains involved in nucleotide and substrate binding. The coding sequence of SoHsp70 was subcloned into Escherichia coli, from which recombinant SoHsp70 was purified and, upon ATPase assay, found to exhibit apparent ATPase activity. Expressional analysis showed that constitutive expression of SoHsp70 was detectable in heart, liver, spleen, kidney, brain, blood, and gill. Experimental challenges with poly(I:C) and bacterial pathogens of Gram-positive and Gram-negative nature induced SoHsp70 expression in kidney to different levels. Stress-responsive analysis of SoHsp70 expression in primary cultures of red drum hepatocytes showed that acute heat shock treatment elicited a rapid induction of SoHsp70 expression which appeared after 10 min and 30 min of treatment. Exposure of hepatocytes separately to iron, copper, mercury, and hydrogen peroxide significantly unregulated SoHsp70 expression in time-dependent manners. Vaccination of red drum with a Streptococcus iniae bacterin was also found to induce SoHsp70 expression. Furthermore, recombinant SoHsp70 enhanced the immunoprotective effect of a subunit vaccine. Taken together, these results suggest that SoHsp70 is a stress-inducible protein that is likely to play a role in immunity and in coping with environmental and biological stresses. (C) 2010 Elsevier Ltd. All rights reserved.

一株减毒荧光假单胞菌的构建及其交叉免疫保护性研究

铁吸收调节蛋白（Fur）是革兰氏阴性菌中普遍存在的重要的转录调控因子，它调控着不同的生命活动，包括铁的吸收，细胞的新陈代谢，应激反应以及毒力因子的合成。因此，对于许多病原菌来说，Fur在侵染和致病的过程中起到了关键性的作用。本研究从一株分离自患病牙鲆的荧光假单胞菌TSS中克隆得到了fur基因，并且发现TSS Fur能够与fur基因突变的大肠杆菌部分互补。本研究构建了一株TSS fur基因的缺失突变体TFM。研究发现与TSS相比，TFM的生长能力和抵抗宿主血清杀菌能力减弱，外膜蛋白表达异常，在宿主血液和组织中的扩散能力显著降低，并且在以牙鲆为模型的感染实验中发现TFM的毒力较TSS大幅下降。将TFM作为减毒疫苗通过注射、浸泡和口服的途径免疫牙鲆后，发现其对荧光假单胞菌和嗜水气单胞菌皆有很好的保护效应。为了进一步扩展TFM的交叉保护范围，本研究进一步构建了表达哈维氏弧菌抗原AgaV-DegQ的质粒pJAQ，并将其转入TFM中得到TFM/pJAQ。研究发现TFM/pJAQ是一种高效交叉保护疫苗，能同时保护牙鲆抵抗荧光假单胞菌、嗜水气单胞菌以及哈维氏弧菌感染。