Freer G, Florio W, Dalla Casa B, Bottai D, Batoni G, Maisetta G, Senesi S, Campa M
Dipartimento di Patologia Sperimentale, Biotecnologie Mediche, Infettivologia ed Epidemiologia, Universitá degli Studi di Pisa, Italy.
Res Microbiol. 1998 Apr;149(4):265-75. doi: 10.1016/s0923-2508(98)80302-1.
A novel protein called SA-5K was identified in Mycobacterium bovis BCG (BCG) short-term culture filtrates (CFs) by means of a recently described monoclonal antibody (mAb), L8D8. This protein had an apparent molecular mass (MM) of 5 kDa, as judged by Western blotting after sodium dodecyl sulphate-polyacrylamide gel electrophoresis in reducing conditions, and did not seem to contain any sugar or lipid substituents. In the present work, SA-5K was purified from BCG CFs by affinity chromatography. A protein that could be detected in Western blot but not by standard protein staining techniques was obtained. When SA-5K was subjected to aminoterminal sequencing, the 10 amino acids (aa) found matched the first 10-aa sequence deduced from an open reading frame (ORF) of M. tuberculosis. The ORF encodes a polypeptide, likely to include a signal for secretion, with an estimated MM of 8.3 kDa after signal peptide cleavage. The secretory nature of SA-5K was confirmed by the fact that it could only be detected in CFs, but not in other BCG subcellular fractions. After size exclusion chromatography, reactivity with mAb L8D8 was found to peak in the 45-50- and 14-16-kDa fractions. The latter MM was close to that estimated from the ORF of M. tuberculosis, implying that the 5-kDa antigen detected initially by Western blot in reducing conditions was a portion of SA-5K released after reduction of a disulphide bridge. The presence of the gene for SA-5K in BCG and its identity were confirmed by PCR (polymerase chain reaction) with specific primers and restriction analysis: the PCR product was slightly shorter in BCG than in M. tuberculosis. The gene coding for SA-5K was cloned by PCR from BCG and M. tuberculosis DNA and was expressed in Escherichia coli.
通过一种最近描述的单克隆抗体（mAb）L8D8，在牛分枝杆菌卡介苗（BCG）短期培养滤液（CFs）中鉴定出一种名为SA-5K的新型蛋白质。在还原条件下进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳后，经蛋白质印迹法判断，该蛋白质的表观分子量（MM）为5 kDa，且似乎不含有任何糖或脂质取代基。在本研究中，通过亲和层析从BCG CFs中纯化了SA-5K。获得了一种在蛋白质印迹中可检测到但不能通过标准蛋白质染色技术检测到的蛋白质。对SA-5K进行氨基末端测序时，发现的10个氨基酸（aa）与从结核分枝杆菌开放阅读框（ORF）推导的前10个氨基酸序列匹配。该ORF编码一种多肽，可能包含一个分泌信号，信号肽切割后估计分子量为8.3 kDa。SA-5K的分泌性质通过以下事实得到证实：它只能在CFs中检测到，而在其他BCG亚细胞组分中检测不到。经过尺寸排阻色谱后，发现与mAb L8D8的反应性在45 - 50 kDa和14 - 16 kDa组分中达到峰值。后者的分子量接近从结核分枝杆菌ORF估计的分子量，这意味着在还原条件下最初通过蛋白质印迹检测到的5 kDa抗原是二硫键还原后释放的SA-5K的一部分。通过使用特异性引物的PCR（聚合酶链反应）和限制性分析证实了BCG中SA-5K基因的存在及其同一性：BCG中的PCR产物比结核分枝杆菌中的略短。通过PCR从BCG和结核分枝杆菌DNA中克隆了编码SA-5K的基因，并在大肠杆菌中进行了表达。
