Roche P W, Winter N, Triccas J A, Feng C G, Britton W J
Centenary Institute of Cancer Medicine and Cell Biology, Newtown, Australia.
Clin Exp Immunol. 1996 Feb;103(2):226-32. doi: 10.1046/j.1365-2249.1996.d01-613.x.
Proteins secreted across the cell wall of mycobacteria are important antigens recognized early in the host response to mycobacterial infection. MPT64 is a 23-kD secreted protein restricted to members of the Mycobacterium tuberculosis complex which elicits T cell responses and cutaneous DTH reactions in Myco. tuberculosis-infected animals. Patients with tuberculosis and their tuberculin-positive contacts respond to the protein, but recipients of bacille Calmette-Guérin (BCG) vaccine strains lacking the mpt64 gene do not. In the present study, we describe the development of a unique recombinant mycobacterial vector which secretes the encoded Myco. tuberculosis protein MPT64 at high levels into the culture filtrate, from which the protein is isolated by a single-step affinity chromatographic step. The purified protein was recognized by both polyclonal and monoclonal anti-MPT64 antibodies. The T cell reactivity of the protein was confirmed by its ability to stimulate human anti-rMPB64 T cell lines. The Myco. smegmatis recombinant MPT64 protein was superior to the Escherichia coli rMPB64 protein, which has identical amino acid sequence, in eliciting cutaneous DTH reactions in guinea pigs sensitized with Myco. tuberculosis. Animals sensitized with BCG strains lacking the mpb64 gene failed to respond to MPT64. Similarly, interferon-gamma (IFN-gamma) responses in tuberculosis patients and their contacts were higher to the Myco. smegmatis form of the protein. The potential of this form of the Myco. tuberculosis MPT64 protein as a skin test reagent for tuberculosis is discussed.
跨越分枝杆菌细胞壁分泌的蛋白质是宿主对分枝杆菌感染早期反应中识别的重要抗原。MPT64是一种23-kD的分泌蛋白，仅存在于结核分枝杆菌复合群成员中，可在结核分枝杆菌感染的动物中引发T细胞反应和皮肤迟发型超敏反应（DTH）。结核病患者及其结核菌素阳性接触者对该蛋白有反应，但缺乏mpt64基因的卡介苗（BCG）疫苗株接种者则无反应。在本研究中，我们描述了一种独特的重组分枝杆菌载体的构建，该载体可将编码的结核分枝杆菌蛋白MPT64高水平分泌到培养滤液中，通过一步亲和层析步骤即可从其中分离出该蛋白。纯化后的蛋白可被多克隆和单克隆抗MPT64抗体识别。该蛋白的T细胞反应性通过其刺激人抗rMPB64 T细胞系的能力得到证实。在结核分枝杆菌致敏的豚鼠中，耻垢分枝杆菌重组MPT64蛋白在引发皮肤DTH反应方面优于具有相同氨基酸序列的大肠杆菌rMPB64蛋白。用缺乏mpb64基因的BCG菌株致敏的动物对MPT64无反应。同样，结核病患者及其接触者对耻垢分枝杆菌形式的该蛋白的干扰素-γ（IFN-γ）反应更高。本文讨论了这种形式的结核分枝杆菌MPT64蛋白作为结核病皮肤试验试剂的潜力。
