Prabhakar S, Annapurna P S, Jain N K, Dey A B, Tyagi J S, Prasad H K
Department of Biotechnology, All India Institute of Medical Sciences, New Delhi, India.
Tuber Lung Dis. 1998;79(1):43-53. doi: 10.1054/tuld.1998.0004.
We report the identification of the first histone-like protein of Mycobacterium tuberculosis (MTB) (HLPMt). The T cell blot assay was used to identify antigens of MTB associated with human immune response in healthy contacts. Fraction 21 corresponding to proteins in the molecular weight range of approximately 30 kDa were found to be immunogenic in tuberculin reactors. None of the fractions were found to be immunogenic by this assay in non-reactors to tuberculin. All sera, irrespective of the source, showed reactivity with MTB antigen(s) over a wide molecular weight range (205-->16 kDa). In the present study fraction 21 was processed for the generation of murine polyclonal sera and amino acid sequencing. The sequence of a 16-amino acid long peptide showed a 100% homology with an open reading frame (ORF) in the translated sequence of cosmid cY349 (Sanger Centre, Cambridge, UK). The ORF was predicted to code for a protein of 214 amino acids. Oligonucleotide primers were synthesized based on the nucleotide sequence located at the 5' and 3' regions of the gene. The gene encoding the predicted protein was PCR-amplified, cloned, sequenced and expressed in Escherichia coli as a protein of 28 kDa. The expressed HLPMt protein was shown to react with the polyclonal murine sera originally raised against fraction 21. Human immune response to the recombinant HLPMt protein was demonstrated by its ability to induce lymphoproliferation in peripheral blood derived mononuclear cells, and the presence of anti-HLPMt antibodies in pooled patient sera by immunoblot. The recombinant HLPMt protein elicited a vigorous lymphoproliferative response especially in healthy tuberculin reactors compared to non-reactors and patients of tuberculosis, (P < 0.05). The protein has unique dual domains with homology to both bacterial histone-like proteins (HU) and eukaryotic histone H1. Homology to prokaryotic and eukaryotic deoxyribonucleic acid (DNA)-binding proteins suggested that HLPMt could bind DNA. DNA-binding properties were confirmed by South-Western analysis strongly suggesting an interaction between HLPMt and the MTB chromosome.
我们报告了结核分枝杆菌（MTB）首个类组蛋白（HLPMt）的鉴定结果。采用T细胞印迹分析法来鉴定与健康接触者中人类免疫反应相关的MTB抗原。发现对应于分子量约为30 kDa范围内蛋白质的第21组分在结核菌素反应者中具有免疫原性。通过该检测，在结核菌素非反应者中未发现任何组分具有免疫原性。所有血清，无论来源如何，在广泛的分子量范围（205 kDa至16 kDa）内均显示出与MTB抗原的反应性。在本研究中，对第21组分进行处理以制备鼠多克隆血清并进行氨基酸测序。一段16个氨基酸长的肽段序列与黏粒cY349（英国剑桥桑格中心）翻译序列中的一个开放阅读框（ORF）具有100%的同源性。该ORF预计编码一种214个氨基酸的蛋白质。基于位于该基因5'和3'区域的核苷酸序列合成了寡核苷酸引物。编码预测蛋白质的基因经PCR扩增、克隆、测序，并在大肠杆菌中表达为一种28 kDa的蛋白质。所表达的HLPMt蛋白显示出与最初针对第21组分产生的鼠多克隆血清发生反应。重组HLPMt蛋白能够诱导外周血单个核细胞中的淋巴细胞增殖，并且通过免疫印迹在合并的患者血清中检测到抗HLPMt抗体，从而证明了人类对该重组蛋白的免疫反应。与非反应者和结核病患者相比，重组HLPMt蛋白尤其在健康的结核菌素反应者中引发了强烈的淋巴细胞增殖反应，（P < 0.05）。该蛋白具有独特的双结构域，与细菌类组蛋白（HU）和真核组蛋白H1均具有同源性。与原核和真核脱氧核糖核酸（DNA）结合蛋白的同源性表明HLPMt可能与DNA结合。通过South-Western分析证实了DNA结合特性，强烈提示HLPMt与MTB染色体之间存在相互作用。
