Franke S, Gunzer F, Wieler L H, Baljer G, Karch H
Institut für Hygiene und Mikrobiologie, Universität Würzburg, Germany.
Vet Microbiol. 1995 Jan;43(1):41-52. doi: 10.1016/0378-1135(94)00071-4.
We constructed and purified recombinant B-subunits of the SLT-IIv as well as tested their usefulness in an immunoblot assay. The slt-IIvB gene amplified by PCR was ligated into the fusion vector pGEX-2T, and expressed in E. coli K 12 laboratory strains. Deletion of the signal sequence was necessary for optimal expression. High quantities of the fusion protein could be purified by affinity chromatography and subsequently used as antigen for immunoblot analysis with serum samples from diseased pigs and healthy controls. IgG antibodies against SLT-IIv were detected in the sera of 11 of 52 (21.15%) healthy pigs. By contrast, only in 1 of 28 (3.57%) serum samples of pigs with edema disease caused by SLT-IIv-producing E. coli we could demonstrate SLT-IIv-specific antibodies. During an outbreak of edema disease, sera from 10 pigs were taken at 4, 20, and 40 days after disease onset to investigate the immune response elicited by SLT-IIv. Immunoblot analysis with the recombinant SLT-IIv fusion protein revealed that the number of IgG-positive serum samples increased within this period of 40 days from one on day 4, to seven on day 20, to ten on day 40; the number of IgM-positive samples also increased from one after 4 days to eight after 20 days. Forty days after disease onset, IgM reactivity was no longer detectable. Since all animals seroconverted in the follow-up sera, the antigenicity of SLT-IIv during infection of pigs seems to differ from that of SLT-II in human hemolytic uremic syndrome where only a minority of patients are known to mount an immune response. The recombinant SLT-IIvB described here may be a possible candidate for vaccination trials.
我们构建并纯化了志贺样毒素II变异体（SLT-IIv）的重组B亚基，并在免疫印迹分析中测试了它们的效用。通过PCR扩增的slt-IIvB基因被连接到融合载体pGEX-2T中，并在大肠杆菌K12实验室菌株中表达。删除信号序列对于最佳表达是必要的。大量的融合蛋白可以通过亲和层析纯化，随后用作抗原，用于对患病猪和健康对照的血清样本进行免疫印迹分析。在52头健康猪中，有11头（21.15%）的血清中检测到了抗SLT-IIv的IgG抗体。相比之下，在由产SLT-IIv的大肠杆菌引起水肿病的猪的28份血清样本中，只有1份（3.57%）检测到了SLT-IIv特异性抗体。在水肿病暴发期间，在发病后4天、20天和40天采集了10头猪的血清，以研究SLT-IIv引发的免疫反应。用重组SLT-IIv融合蛋白进行免疫印迹分析表明，在这40天内，IgG阳性血清样本的数量从第4天的1份增加到第20天的7份，再到第40天的10份；IgM阳性样本的数量也从4天后的1份增加到20天后的8份。发病40天后，不再能检测到IgM反应性。由于所有动物在后续血清中都发生了血清转化，因此猪感染期间SLT-IIv的抗原性似乎与人类溶血尿毒综合征中SLT-II的抗原性不同，在人类溶血尿毒综合征中，只有少数患者会产生免疫反应。这里描述的重组SLT-IIvB可能是疫苗试验的一个潜在候选物。
