Sheikholvaezin Ali, Blomberg Fredrik, Ohrmalm Christina, Sjösten Anna, Blomberg Jonas
Section of Clinical Microbiology, Department of Medical Sciences, Uppsala University, Sweden.
Protein Expr Purif. 2011 Dec;80(2):176-84. doi: 10.1016/j.pep.2011.08.007. Epub 2011 Aug 19.
Diagnosis of infectious diseases often requires demonstration of antibodies to the microbe (serology). A large set of antigens, covering viruses, bacteria, fungi and parasites may be needed. Recombinant proteins have a prime role in serological tests. Suspension arrays offer high throughput for simultaneous measurement of many different antibodies. We here describe a rational process for preparation, purification and coupling to beads of recombinant proteins prepared in Escherichia coli derivate Origami B, to be used in a serological Luminex suspension array. All six Gag and Env proteins (p10, p12, p15, p30, gp70 and p15E), from the xenotropic murine leukemia virus-related virus (XMRV), were prepared, allowing the creation of a multiepitope XMRV antibody assay. The procedure is generic and allows production of protein antigens ready for serological testing in a few working days. Instability and aggregation problems were circumvented by expression of viral proteins fused to a carrier protein (thioredoxin A; TrxA), purification via inclusion body formation, urea solubilization, His tag affinity chromatography and direct covalent coupling to microspheres without removal of the elution buffer. The yield of one preparation (2-10mg fusion protein per 100ml culture) was enough for 20-100 coupling reactions, sufficing for tests of many tens of thousands of sera. False serological positivity due to antibodies binding to TrxA and to traces of E. coli proteins remaining in the preparation could be reduced by preabsorption of sera with free TrxA and E. coli extract. The recombinant antigens were evaluated using anti-XMRV antibodies. Although hybrid proteins expressed in E. coli in this way will not have the entire tertiary structure and posttranslational modifications of the native proteins, they contain a large subset of the epitopes associated with them. The described strategy is simple, quick, efficient and cheap. It should be applicable for suspension array serology in general.
传染病的诊断通常需要证明针对微生物的抗体（血清学检测）。可能需要一大组涵盖病毒、细菌、真菌和寄生虫的抗原。重组抗原。重组蛋白在血清学检测中起着重要作用。悬浮阵列可实现高通量，用于同时测量多种不同的抗体。我们在此描述了一个合理的流程，用于制备、纯化在大肠杆菌衍生菌株Origami B中制备的重组蛋白，并将其偶联到磁珠上，以用于血清学Luminex悬浮阵列。制备了来自嗜异性小鼠白血病病毒相关病毒（XMRV）的所有六种Gag和Env蛋白（p10、p12、p15、p30、gp70和p15E），从而能够创建一种多表位XMRV抗体检测方法。该方法具有通用性，能够在几个工作日内生产出可供血清学检测的蛋白质抗原。通过表达与载体蛋白（硫氧还蛋白A；TrxA）融合的病毒蛋白、经由包涵体形成进行纯化、尿素溶解、His标签亲和层析以及直接共价偶联到微球而无需去除洗脱缓冲液，解决了不稳定性和聚集问题。一次制备的产量（每100ml培养物产生2 - 10mg融合蛋白）足以进行20 - 100次偶联反应，足够检测数万份血清。通过用游离TrxA和大肠杆菌提取物对血清进行预吸收，可以减少由于抗体与TrxA以及制备物中残留的痕量大肠杆菌蛋白结合而导致的假血清学阳性。使用抗XMRV抗体对重组抗原进行了评估。尽管以这种方式在大肠杆菌中表达的杂合蛋白不会具有天然蛋白的完整三级结构和翻译后修饰，但它们包含与之相关的大部分表位。所描述的策略简单、快速、高效且成本低廉。它一般应适用于悬浮阵列血清学检测。
