■母马中的沙门氏菌流产是由肠沙门氏菌亚种肠血清型流产马氏感染引起的,其特征是早产(流产)或无法存活的胎儿出生。虽然所有的马都容易受到感染,这种情况更常见于妊娠母马的临床表现,大多数堕胎记录在年轻女性身上。此外,非特异性临床疾病体征和敏感性差和有效的细菌学诊断方法阻碍了快速可靠的感染诊断。免疫化学方法如酶联免疫吸附测定(ELISA)和免疫层析测定可以促进有效和快速的诊断。然而,它们需要高度特异性和活性的抗原和抗体。本研究旨在制备重组肠沙门氏菌外膜蛋白X(OmpX),并评估其对母马沙门氏菌流产的血清学诊断的适用性。
■从头合成来自肠链球菌抗原的外膜蛋白X,并使用pET28载体在大肠杆菌中表达。在不同条件下培养转化的大肠杆菌细胞以检测重组OmpX(rOmpX)的表达,和rOmpX的纯化和重折叠均使用金属亲和层析进行。重折叠和纯化的rOmpX通过蛋白质印迹进行表征,液相色谱与串联质谱,和ELISA。
■优化后的rOmpX表达式,鉴定出23kDa分子量的蛋白质。使用Mascot程序的氨基酸序列分析表明这些肽是来自肠链球菌的OmpX蛋白。当使用rOmpX对来自流产和接触母马的89份血清样品进行ELISA时,记录了高特异性和诊断效率。
■重组外膜蛋白,与O抗原相比,对流产和接触马的母马的血清表现出更好的诊断特征。
UNASSIGNED: Salmonella abortion in mares is caused by Salmonella enterica subspecies enterica serovar abortus equi infection and is characterized by premature (abortion) or non-viable fetus birth. Although all horses are susceptible to infection, the condition is more often clinically manifested in pregnant mares, with most abortions recorded in young females. In addition, nonspecific clinical disease signs and poorly sensitive and effective bacteriological diagnostic methods hinder rapid and reliable infection diagnoses. Immunochemical methods such as enzyme-linked immunosorbent assay (ELISA) and immunochromatography assays can facilitate effective and rapid diagnoses. However, they require highly specific and active antigens and antibodies. This study aimed to generate a recombinant S. enterica outer membrane protein X (OmpX) and evaluate its suitability for serological diagnosis of Salmonella abortion in mares.
UNASSIGNED: Outer membrane protein X from the S. enterica antigen was synthesized de novo and expressed in Escherichia coli using the pET28 vector. Transformed E. coli cells were cultured under different conditions to detect recombinant OmpX (rOmpX) expression, and rOmpX purification and refolding were both conducted using metal affinity chromatography. Refolded and purified rOmpX was characterized by western blotting, liquid chromatography with tandem mass spectrometry, and ELISA.
UNASSIGNED: After optimized rOmpX expression, a 23 kDa molecular weight protein was identified. Amino acid sequence analysis using Mascot program suggested that these peptides were the OmpX protein from S. enterica. High specificity and diagnostic efficiency were recorded when rOmpX was used in ELISA against 89 serum samples from aborted and contact mares.
UNASSIGNED: Recombinant outer membrane protein, in comparison to the O antigen, demonstrated better diagnostic characteristics against sera from mares who aborted and contact horses.